Diego Grando Modolo1 Edislane Barreiros de Souza3 Thatiana Correcirca de Melo1
Diva Denelle Spadacci-Morena4 Roberta Fiusa Magnelli1
Maacutercio Augusto Caldas Rocha de Carvalho1 Paulo Luis de Saacute Juacutenior1
Rodrigo Franco de Carvalho1 Willy Beccedilak1 and Rita de Cassia Stocco1
1Laboratorio de Genetica Instituto Butantan 05503-900 Sao Paulo SP Brazil2Programa de Pos-Graduacao Interunidades em Biotecnologia Instituto de Ciencias Biomedicas (ICB)Universidade de Sao Paulo (USP) 05508-900 Sao Paulo SP Brazil3Laboratorio de Biologia Molecular Genetica e Mutagenese Departamento de Biologia Faculdade de Ciencias e Letras deAssis (FCLA) Universidade Estadual Paulista ldquoJulio de Mesquista Filhordquo (UNESP) 19806-900 Assis SP Brazil4Laboratorio de Fisiopatologia Instituto Butantan 05503-900 Sao Paulo SP Brazil
Correspondence should be addressed to Rita de Cassia Stocco ritastoccobutantangovbr
Received 16 July 2015 Revised 7 October 2015 Accepted 15 October 2015
Academic Editor Janusz Blasiak
Copyright copy 2015 Rodrigo Pinheiro Araldi et alThis is an open access article distributed under the Creative CommonsAttributionLicense which permits unrestricted use distribution and reproduction in anymedium provided the originalwork is properly cited
Bovine papillomavirus (BPV) is considered a usefulmodel to studyHPVoncogenic process BPV interacts with the host chromatinresulting in DNA damage which is attributed to E5 E6 and E7 viral oncoproteins activity However the oncogenic mechanisms ofBPVE6oncoprotein per se remain unknownThis study aimed to evaluate themutagenic potential ofBos taurus papillomavirus type1 (BPV-1) E6 recombinant oncoprotein by the cytokinesis-block micronucleus assay (CBMNA) and comet assay (CA) Peripheralblood samples of five calves were collected Samples were subjected to molecular diagnosis which did not reveal presence of BPVsequences Samples were treated with 1120583gmL of BPV-1 E6 oncoprotein and 50120583gmL of cyclophosphamide (positive control)Negative controls were not submitted to any treatment The samples were submitted to the CBMNA and CA The results showedthat BPV E6 oncoprotein induces clastogenesis per se which is indicative of genomic instability These results allowed betterunderstanding the mechanism of cancer promotion associated with the BPV E6 oncoprotein and revealed that this oncoproteincan induce carcinogenesis per se E6 recombinant oncoprotein has been suggested as a possible vaccine candidate Results pointedout that BPV E6 recombinant oncoprotein modifications are required to use it as vaccine
1 Introduction
Papillomaviruses (PVs) are a group of viruseswith epitheliumand mucous tropism [1 2] PVs can infect all vertebratesincluding rabbits [3ndash5] dogs [6 7] goats [8] humans [9ndash12]and bovines [1 13ndash15] In the last decades an increasing inter-est in studies involving these viruses has been observed [1617] This fact is justified because the PVs are associated withbenign (papillomas) and malignant lesions which can affectboth human [16 18] and animals [1 15 19] In this scenario
Bos taurus papillomavirus is considered the best model foroncogenic process studies associated with PVs [20ndash23]
Bos taurus papillomaviruses also known as bovine papil-lomaviruses (BPVs) have a worldwide distribution [23] Itis estimated that 60 of the Brazilian herd is infected byBPV [1] However this number can be greater once theinfection can be asymptomatic [24] BPVs cause bovine papil-lomatosis an infectious disease characterized by the presenceof multiple papillomas which can regress spontaneously orprogress to malignancy [1] Among the 14 BPVs types already
Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 806361 15 pageshttpdxdoiorg1011552015806361
2 BioMed Research International
Table 1 Studies already published about E6 oncoprotein
Interaction with host cell Virus type Method ReferencesFacilitating virus entry BPV E6 facilitates the BPV entry by clathrin interaction [35]Antiapoptotic effect HPV E6 promotes Bax degradation resulting in an antiapoptotic effect [114 115]Binding to E6AP ubiquitinligase HPV E6 expressed in E coli and insects binds to E6AP ubiquitin ligase [50 95 116 117]
Binding to DNA HPV E6 expressed in E coli binds with DNA [118ndash120]Cell immortalization HPV E6 promotes p53 degradation and cell immortalization [121ndash123]
Epigenetic downregulationof p53
HPV E6 expressed in baculovirus induces hypoacetylation of p53 [124 125]
HPV E6 expressed in E coli interacts with p300CBP reducing p53levels [126]
BPV E6 interacts with p300CBP reducing p53 levels [51]Disruption of mitoticapparatus HPV E6 transfected using LXSN vectors [29 30 127]
Malignant transformationin cell culture HPV Complete genome of HPV-16 transfected in NIH 3T3 cells [45 88 128ndash130]
Interaction with paxillin BPV E6 of BPV-1 interacts with paxillin reducing the focal adhesion [34 69 72 131 132]Repressor activity oftelomerases HPV E6 induces the hTERT expression reducing the telomerase activity [96 133 134]
Immune depletion HPV E6 expressed in yeast reduces the levels of interferon regulatoryfactor-3 [135]
described [25] BPV-1 BPV-2 and BPV-4 have the mostoncogenic potential [21] BPV-1 and BPV-2 are associatedwith urinary bladder cancer [26 27] whereas BPV-4 inducesupper gastrointestinal malignancies [28]
Several studies already pointed out that both HPV [2930] and BPV [1 14 15 24] interact with the host chromatinresulting in DNA and chromosomes damage This damageis induced by the E5 E6 and E7 viral oncoproteins [24]Although the role of these oncoproteins in the oncogenic pro-cess is known [31 32] there are no studies showing the onco-genic mechanisms of E6 oncoprotein of BPV per se to dateTheE6of BPV-1 has 137 amino acids with fourwell conservedCys-X-X-Cysmotifs among all PVs [33ndash35]Moreover the E6oncoprotein has six cysteine residues which turn the onco-protein susceptible to oxidation [36] These characteristicsmake the production purification and obtaining of E6 cor-rect folding difficult [36 37] Due to these difficulties in thelast 30 years different constructions employing expressionvectors in Escherichia coli have been developed in order toproduce large quantities of E6 oncoprotein [36ndash43] Most ofthese studies are directed to HPV-16 and BPV-1 In this sce-nario Mazzuchelli-de-Souza et al [44] successfully purifiedthe BPV-1 E6 recombinant protein A summary of studiesinvolving the E6 oncoprotein is shown in Table 1
Due the low number of studies about the E6 of BPVto date the knowledge of oncogenic mechanisms associatedwith this oncoprotein comes from previous works with E6of HPV [29 30] The first lines of evidence of E6 oncogenicproperties come from studies on human tumors cell lineagesderived from cervical cancer [45] Others studies based onE6 of HPV pointed out that the oncoprotein is able to inducecytogenetic damage resulting in genomic instability [29 30]which is considered a cancer hallmark [46ndash48] Although the
E6 oncoprotein of bothHPV and BPV affects p53 themecha-nisms that induce the reduction levels of this tumor suppres-sor protein are distinct between these PVs types E6 oncopro-tein of HPV binds to the E6AP ubiquitin ligase resulting inp53 ubiquitination [49] This process results in p53 proteaso-mal degradation [50] However the BPV-1 E6 oncoprotein ofBPV does not induce p53 degradation [51] Studies pointedout that BPV-1 E6 oncoprotein interacts with CBPp300promoting the downregulation of p53 [51] These differentmechanisms of E6 action require studies involving the onco-protein of BPV
Mutation is the first step in carcinogenesis process [52]This study aimed to evaluate the mutagenic potential ofE6 recombinant oncoprotein of BPV-1 by the cytokinesis-block micronucleus assay (CBMNA) and comet assay (CA)CBMNA and CA are noninvasive methods recommendedas part of genotoxicity tests battery for drug validation [53]CBMNA consists in evaluating the frequency of micronu-cleus in binucleated lymphocytes obtained through theaddiction of cytochalasin B in lymphocyte culture to preventcytokinesis [54]
Micronuclei (MNs) are acentric fragments expelled fromthe main nucleus at late stages of anaphase [55] MNs canbe formed through two mechanisms chromosomal breaks(clastogenesis) or disruption of the mitotic apparatus (aneu-genesis) [56] These fragments remain not integrated in thenucleus of daughter cell originating the MNs [54 57] TheMNs represent not only chromosomal losses but also theresult of DNA amplification commonly observed in onco-genic process [57]
CA is a simple technique with low cost which can beemployed in any eukaryotic cells [58 59] CA has been usedto study the clastogenic potential of HPV [60] and BPV [24]
BioMed Research International 3
E6
25kDa
15kDa
Figure 1Western blot showing BPV-1 E6 recombinant oncoproteinWeight ladder employed Spectra Multicolor Broad Range ProteinLadder (Fermentas Lithuania)
The CMNA combined with CA allows detecting DNA dam-age as an indication of mutagenesis with high statistical andsensitivity power [53] This is the first study which evaluatedthe mutagenic potential of E6 recombinant oncoprotein ofBPV-1 suggesting that this oncoprotein participates in bothcancer initiation and promotion
2 Material and Methods
21 Expression and Purification of BPV-1 E6 OncoproteinBPV-1 E6 recombinant oncoprotein was expressed and puri-fied according to Mazzuchelli-de-Souza et al [44] usingEscherichia coli BL21 The oncoprotein was subjected to dial-ysis This step was necessary to remove urea and imidazolesubstances used to promote the oncoprotein refolding Thedialysis avoids false-positive results to mutagenesis testswhich could be induced by urea and imidazole Dialysis wasperformed using Slide-A-Lyzer Dialysis Cassette (3 K-12mL)(Thermo Scientific Carlsbad USA) For this step 10mLof sample was dialyzed against two liters of dialysis buffer(20mM Tris-HCl and 500mM NaCl pH 80) for 8 hours at4∘C under constant agitation Figure 1 shows Western blotof BPV-1 E6 recombinant protein used in this study Toconfirm the identity of recombinant oncoprotein the bandobtained in the SDS-PAGE gel was submitted to mass spec-trometry Four peptide sequences were identified using thismethod being (1) KDFHVVIRE (2) KDFHVVIRE (3) RHVLFNEPFCKT and (4) RLWQGVPVTGEEAELLHGKTThese sequences were analyzed in the Swiss-Prot databasewhere score higher than 43 indicates extensive homologyThus mass spectrometry pointed out a score of 94 demon-strating that the recombinant protein shows identity with E6oncoprotein of BPV-1 (access number VE6 BPV1)
22 Peripheral Blood Collection A volume of 5mL of periph-eral bloodwas collected fromfive asymptomatic calves (with-out cutaneous papillomas) (Bos taurus Simmental breed)using theVacutainer systemwith EDTA (molecular diagnosis
of BPV) and heparin (CBMNA and CA) The materialwas collected through venipuncture from jugular vein by aveterinarian The protocols used in this study were approvedby the Ethics Committee onAnimal Use of Butantan Institute(process 103513) The farm of calvesrsquo origin is located in SaoPaulo State (Brazil) This farm was chosen due to the absenceof bracken fern Pteridium aquilinum because studies pointedout that bracken farm has mutagenic and carcinogenic com-pounds such as quercetin and ptaquilosides [61ndash63] whichcould interfere in the studies leading to false-positive results
23 Molecular BPV Identification by PCR Blood DNAextraction the extraction of DNA from peripheral bloodcells was performed using the GenomicPrep Blood MiniKit Illustra Spin (GE Healthcare Buckinghamshire UK)according to the manufacturer The quality of the obtainedDNAwas verified using the polymerase chain reaction (PCR)technique amplifying a bovine 120573-globin gene fragment [64]
Viral identification viral identification was performedusing specific primers for BPV-1 (forward 51015840-GGAGCG-CCTGCTAACTATAGGA-31015840 reverse 51015840-ATCTGTTGT-TTGGGTGGTGAC-31015840) which amplifies the L1 gene result-ing in a 301 bp amplicon BPV-2 (forward 51015840-GTTATACCA-CCCAAAGAAGACCCT-31015840 reverse 51015840-CTGGTTGCA-ACAGCTCTCTTTCTC-31015840) which amplifies the L2 generesulting in a 164 bp amplicon and BPV-4 (forward 51015840-GCTGACCTTCCAGTCTTAAT-31015840 reverse 51015840-CAGTTT-CAATCTCCTCTTCA-31015840) which amplifies the E7 generesulting in a 170 bp amplicon These primers were chosenbecause BPV-1 BPV-2 and BPV-4 are the most frequenttypes in Brazil being associated with oncogenic process [165] Besides these specific primers two degenerate pairs ofprimers were used Delta-Epsilon (forward 51015840-CCAGAY-TAYYTMAAAATGGC-31015840 reverse 51015840-ATAAMKGCTAGC-TTATATTC-31015840) and Xi (forward 51015840-TWYAATAGDCCV-TTTTGGAT-31015840 reverse 51015840-TTMCGCCTACGCTTTGGC-GC-31015840) [66] These primers allow detecting BPVs of generaDelta Epsilonpapillomavirus (Delta-Epsilon) and Xipapillo-mavirus (Xi) [66] Both primers amplify the L1ORF resultingin products with 430 bp (Delta-Epsilon) and 600 bp (Xi)
PCR parameters reactions were performed in a total vol-ume of 500 120583L using 200 ng120583L of DNA template 20120583L offorward primer 20 120583L of reverse primer and 360 120583L ofMas-ter Mix (4G Porto Alegre Brazil) Reactions were done onthermocycler Veriti 96-WellThermal Cycler (Applied Biosys-tems Carlsbad USA) and subjected to the cycles shown inTable 2 Cloned genomes of BPV-1 BPV-2 and BPV-4 inpAT153 vector in Escherichia coliD5H120572were used as positivecontrols BPV-2 viral genome was employed as positivecontrol for Delta-Epsilon primer and BPV-3 for Xi primer
The PCR products were analyzed in 2 agarose gelelectrophoresis stained with GelRed (Biotium USA) in Tris-Acetate-EDTA (TAE) buffer visualized under UV light usingBioDocAnalyze (Biometra Germany)
24 Cytokinesis-Block Micronucleus Assay (CBMNA) UsingPeripheral Blood Mononuclear Cells (PBMCs) For each sam-ple three cultures were established (1) negative control
4 BioMed Research International
Table 2 Frequency of micronucleated lymphocytes Number ofmicronuclei observed perslide (MN) and number of mononucle-ated (119873
1
) binucleated (1198732
) and polynucleated (119873119901
) lymphocytesand anaphase bridges (AB) observed pergroup Based on thesevalues the micronucleated formation frequency (MN
1199030
) and thecytokinesis-block proliferation index (CBPI) and the media (119909) areshown
(not treated with any drug) (2) positive control (treatedwith 50120583gmL of cyclophosphamide) and (3) experimentalgroup (treated with 1 120583gmL of E6 recombinant oncoproteinresuspended in PBS) This concentration of E6 recombinantoncoproteinwas based on previous study involving BPV early(E) protein as vaccine [67] The protocol of CBMNA wasdone according to the technical recommendation proposedby Araldi et al [53] In detail 02mL of peripheral bloodwas transferred to culture flask containing 50mL RPMI1640 medium supplemented with 15 fetal bovine serum01mL L-glutamine and 01mL phytohemagglutinin A Thematerial was incubated at 37∘C for 8 hours After this bothcyclophosphamide and E6 recombinant oncoprotein wereadded to the culture After 44 hours 02mL of cytochalasin Bwas added to block the cytokinesis After 72 hours the culturewas stopped with the addition of 05mL methanol aceticacid fixative (vv) (3 1) for 5 minutes at room temperatureThematerial was centrifuged at 500 g and the supernatantwasdiscardedThepellet was homogenizedwith 5mLfixative andcentrifuged at 500 g The pellet was aspirated and transferredto slides which were stained with a 1 3 Giemsa phosphatebuffer solution pH = 68 for 8 minutes
After staining coverslips were placed on slides withEntellan (Merck Germany) The material was analyzed ina blind test under an Axiophot binocular microscope (Carl
Zeiss Germany) to observe the frequency ofmicronucleated-binucleated lymphocytes in a total of 1000 analyzed cellsaccording to Araldi et al [53] Statistical analysis was per-formed using the Kruskal-Wallis test followed by a post hocDunn test both at a significance level of 5 Statistical testswere done using the BioEstat software [68]
25 Comet Assay Alkaline Method Slides preparation slidesof 26 times 76mm were dipped in a solution of normal meltingpoint agarose (NMP) (Invitrogen Carlsbad USA) dilutedin phosphate-buffered solution (PBS) 15 at 60∘C and oneside of each slide was wiped clean with a paper towel Theconcentration of NMA was based on Araldi et al [53] Theslides were dried in a horizontal position overnight
Peripheral blood incubation with drugs three wholeblood aliquots of 200120583L each were transferred to three15mL polypropylene tubes (1) negative control (2) positivecontrol and (3) experimental group In each tube 200120583Lof RMPI 1640 medium was added The negative control didnot receive any drug and was incubated only in RPMI 1640medium Positive control was incubated with 50 120583gmL ofcyclophosphamide diluted in RPMI 1640 medium Experi-mental group was treated with 1 120583gmL of BPV-1 E6 recombi-nant protein The samples were incubated at 37∘C for 2 hoursunder constant agitation After this time the aliquots werecentrifuged for 1 minute at 500 g and the supernatant wasdiscarded Ten microliters of each obtained pellet was addedto 75120583L low melting point agarose (LMP) A final volume of85 120583L of this suspensionwas immediately transferred toNMPprecoated slides The slides were covered with coverslips andmaintained at 4∘C for 20 minutes The coverslips were gentlyremoved and the slides were placed in a Coplin jar containinglysis solution (25mM NaCl 100mM ethylenediaminete-traacetic acid (EDTA) 10mM Tris-HCl 11 Triton X-100and 112 dimethyl sulfoxide) at 4∘C for 1 hour Subsequentlyall procedures were conducted under dark conditions toprevent the induction of DNA damage
Electrophoresis after lysis slides were washed with PBSand transferred to an electrophoresis tank containing elec-trophoresis buffer (300mM NaOH and 1mM EDTA pH gt13) at 4∘C for 40 minutes to induce unwinding of double-stranded DNA Next the electrophoretic run was performedwith a current of 25V (086Vcm) 300mA for 20 minutesto promote the migration of free DNA fragments toward theanode The slides were transferred to a Coplin jar containingneutralizing buffer (400mM Tris-HCl pH 75) for 5 minutesThe material was fixed in absolute ethanol for 5 minutes
Comet analysis slides were stained with 20 120583L 4 120583gmLpropidium iodide (PI) and analyzed under a fluorescencemicroscope (Carl Zeiss Axio Scope A1 Germany) equippedwith an excitation filter of 510ndash560 nm and barrier of 590 nmThe material was analyzed in a total magnification of 400xOne hundred nucleoids were analyzed per slide which werescored on a scale of 0 (without DNA damage) to 2 (maximumDNAdamage) according to Araldi et al [53]The scores wereobtained by summing the product of the observed numberof nucleoids per class and its respective class value Statisticalanalysis was performedusing theKruskal-Wallis test followedby the post hocDunn test both with a significance level of 5
BioMed Research International 5
Both test and graphical analysis were done using the BioEstatsoftware [68]
26 Cytokinesis-Block Micronucleus Assay (CBMNA) inEpithelial Cells To verify whether the results observed inPBMCs should also be observed in epithelial cells CBMNAwas performed in CRIB cell (commercial epithelial cell lineobtained from bovine kidney) In detail a total of 1 times 105cells were transferred to six-well plate containing a sterilecoverslip of 24 times 24mm with 2mL of MEM mediumsupplementedwith 10 fetal bovine serum and 1 ampicillinand three cultures had been established negative control(not treated with any drug) positive control (treated with50120583gmL of cyclophosphamide) and experimental group(treatedwith 1120583gmLof BPV-1 E6 recombinant oncoprotein)Cyclophosphamide and BPV-1 E6 recombinant oncoproteinwere added together to the cells After 1 hour the threecultures were treated with 6 120583gmL of cytochalasin B (SigmaGermany)Thematerial was incubated for 48 hours the timenecessary to complete two replication cycles once the dupli-cation time of these cells is 24 hours according to our previ-ous study After this time the medium was removed and thecells were washed twice with PBS at 37∘C Cells were stainedwith solution 1 4 Giemsa-PBS for 3 minutes and afterwashed twice with PBS Coverslips containing the biologicalmaterial were mounted on slides using Entelan (Merck Ger-many) Slides were analyzed by Axiophot binocular micro-scope (Carl Zeiss Germany) to observe the frequency ofmicronucleated cells in a total of 1000 analyzed cells accord-ing to Araldi et al [53]
3 Results
31 Molecular BPV Identification by PCR The peripheralblood samples collected from five asymptomatic calves didnot reveal the presence of BPV sequences using both specificand degenerate primers (Figure 2)
32 Cytokinesis-Block Micronucleus Assay (CBMNA) inPBMCs Frequency analysis of micronucleus observed inCBMNA CBMNA showed an elevated number of micronu-cleated lymphocytes as well as anaphase bridges in bothpositive control and the group treated with 1 120583gmL of E6recombinant oncoprotein (Table 2 Figure 3)
Based on the micronucleated lymphocytes numberobserved per group (Table 2) Chi-square (1205942) test andKruskal-Wallis test were done both with 5 significancelevel Chi-square test pointed out statistical differencesbetween positive control and group treated with E6 (Table 3)These results point out that the cyclophosphamide and E6recombinant oncoprotein are able to induce aneugenesis andor clastogenesis However the Chi-square test did not showstatistic difference in negative control (Table 3)
Kruskal-Wallis test revealed statistical significant differ-ences among the groups (119867 = 97297 and 119901 = 00087)Based on this result Dunn post hoc test was performed Thetest pointed out significant difference between negative andpositive control as well as negative control and experimental
Table 3 Chi-square (1205942) test results showing expected value (119864)freedom degree (FD) and probability (119901)
Group 119864 1205942 FD 119901
Negative control 560 2000 4 07358Positive control 430 10698 4 00302Experimental (E6) 456 12202 4 00159
Table 4 Dunn post hoc test results
Groups Rank differences Calculated 119885 Critical 119885 119901
Cminus and C+ 71 25102 2394 lt005Cndash and E6 79 27931 2394 lt005C+ and E6 08 02828 2394 nsns nonsignificant value
Table 5 Student-Newman-Keuls post hoc test results
Groups Rank differences Calculated 119885 Critical 119885 119901
Cminus and C+ 61 21567 2394 00310Cminus and E6 77 27224 2394 00065C+ and E6 16 05657 2394 05716
group (E6) (Table 4) However the test did not show statis-tical difference between positive control and experimentalgroup (Table 4)
Based on the maximum minimum and median valuesof micronucleus observed in the three groups a boxplotwas done (Figure 4) The graph indicates statistically equalmedians between positive control and experimental group(E6) Although the medians between these groups had notshown statistical differences the graph indicates that theexperimental group showed maximum values of binucleatedlymphocytes with micronucleus higher than positive control
Frequency analysis of anaphase bridge observed inCBMNA based on the number of anaphase bridges (AB)observed per group (Table 3) Kruskal-Wallis test was per-formed which indicates significant differences among thegroups (119867 = 83444 119901 = 00154) Student-Newman-Keuls post hoc test revealed differences between negative andpositive control as well as negative control and experimentalgroup (Table 5) The test did not show differences betweenpositive control and experimental group
Analysis of cytokinesis-block proliferation index (CBPI)and cytotoxicity Kruskal-Wallis test based on theCBPI indi-cates statistical differences among the groups (119867 = 93968and 119901 = 00091) Dunn post hoc test revealed significantdifferences between negative and positive control as well asnegative control and experimental group (Table 6) Howeverthe test did not show differences between positive control andexperimental groupThese data indicate that E6 recombinantoncoprotein has CBPI similar to the cyclophosphamide
Besides presenting high CBPI the E6 recombinant onco-protein also showed to induce endoreduplication evidencedby the presence of intracellular cytokinesis suggesting neosis(Figure 5) according to the criteria proposed by Das et al[69] The CBMNA results suggest that E6 recombinant
6 BioMed Research International
301bp
M 0504030201 M M 0504030201BPV1Cminus
(a) (b)
(c) (d)
(e) (f)
164bp170bp
M 0504030201BPV2Cminus M 0504030201BPV4Cminus
430bp
600bp
M 0504030201BPV2Cminus M 0504030201BPV4Cminus
Figure 2 Electrophoresis gel images showing (a) genomicDNA integrity afterDNAextraction using 1 KbDNALadder (Invitrogen CarlsbadUSA) as marker absence of amplicon in samples 01 to 05 using specific primers to BPV-1 (b) BPV-2 (c) and BPV-4 (d) absence of ampliconin samples 01 to 05 using Delta-Epsilon (e) and Xi (f) degenerate primers Images (b)ndash(f) showed amplification only in positive control withthe 100 bp DNA Ladder (Invitrogen Carlsbad USA) being employed as marker
Table 6 Dunn post hoc test results based on the cytokinesis-blockproliferation index
Group Rank differences Calculated 119885 Critical 119885 119901
Cminus and C+ 76 26870 2394 lt005Cminus and E6 74 26163 2394 lt005C+ and E6 02 00707 2394 nsns nonsignificant differences
oncoprotein induces mitotic stress resulting in clastogenesisand neosis
33 Comet Assay Alkaline Method Comet assay results areshown in Table 7 and Figure 6 According to these data
theKruskal-Wallis test was performedwhich pointed out sta-tistical differences among the groups (119867 = 102613 and 119901 =00059) Based on these results the Dunn post hoc test wasperformed which pointed out significant differences betweennegative and positive control as well as negative controland experimental group (Table 8) However the test did notshow differences between positive control and experimentalgroup (Table 8) These results were also verified by Figure 7Comet assay reinforces the CBMNA results indicating themutagenic potential of E6 recombinant oncoprotein
34 Cytokinesis-Block Micronucleus Assay (CBMNA) inPBMCs Results of this test confirmed the previous resultsindicating that the BPV-1 E6 recombinant oncoprotein has
BioMed Research International 7
LB
(a)
LMN
(b)
LMN
LMN
(c)
PA
PA and MN
(d)
Figure 3 (a) Photomicroscopy of binucleated lymphocyte without micronucleus (LB) observed in negative control Image of binucleatedlymphocyte with micronucleus (LMN) observed in positive control (b) and group treated with E6 recombinant oncoprotein (c) Image ofbinucleated lymphocyte with anaphase bridge and micronucleus (PA and MN) observed in group treated with E6 (d) Images obtained withtotal magnification of 1000x
60
50
40
30
20
10
0
Num
ber o
f mic
ronu
clei
GroupCminus C+ E6
Figure 4 Comparative boxplot based on maximum minimumand median values of micronucleus observed per group
a genotoxicity potential being able to induce DNAbreaks in epithelial cells verified by the presence of MNs(Figure 8)
Figure 5 Evidence of neosis Photomicroscopy of lymphocyteshowing endoreduplication (black arrow) suggesting neosis Cellsanalyzed in total magnification of 1000x
4 Discussion
Transforming potential of E6 oncoprotein has been discussedsince the 1980s based on studies of cottontail rabbit papil-lomaviruses (CRPV) [70 71] Although there are lines ofevidence that the E6 oncoprotein can induce transformation
8 BioMed Research International
(a) (b)
(c) (d)
Figure 6 Images of cometsrsquo nucleoids (a) Image of class 0 (without DNA damage) observed in negative control group Images of class 2(maximum DNA damage) observed in positive control (b) and experimental (c) groups (d) Image of hedgehog comet characterized by thehead absence observed in experimental group Images captured in total magnification of 400x
200
150
100
50
0
Com
et sc
ore
GroupsCminus C+ E6
Figure 7 Boxplot of comet score
[72] the mechanisms of initiation and promotion of cancerassociated with the BPV E6 oncoprotein are unknown
Molecular diagnosis of five peripheral blood samplesfrom five calves showed the absence of amplicons for thefive different primers used (Figure 2) However both specific(BPV-1 BPV-2 and BPV-4) and degenerate (Delta-Epsilonand Xi) primers sets amplified the controls genomes of BPV-1 BPV-2 and BPV-4 None of the negative controls revealedthe amplicon presence These results indicate that the fivesampleswere uninfected by BPVMoreover the use of specific
and degenerate primers increases the capacity of BPV iden-tification This occurs because specific primers are moresensitive than degenerates [66] This sensitivity is due tothe absence of degeneration in the 51015840 region which reducesthe primer ability to recognize and to link with DNA targetsequence [73]The absence of BPV infection in the peripheralblood samples is required for the CBMNA and CA allowinginvestigating the aneugenic andor clastogenic action of BPV-1 E6 recombinant oncoprotein This is because the presenceof BPV sequences is associated with the presence of oncopro-teins transcripts [74]Thus studies have shown that the pres-ence of BPVDNA sequences in peripheral blood is associatedwith cytogenetic damage including clastogenesis [1 14 75]Complementary studies show the association of the BPVpresence with cytogenetic damage in both benign lesions(papillomas) [74] and carcinomas [15]
Chi-square test based on the number of micronucleatedcells pointed out significant statistical values in both positivecontrol and group treated with BPV-1 E6 recombinant onco-protein (Table 3) Negative control showed fewer number ofmicronucleated cells This result was expected because thebiological material was transported and processed after threehours of its collection This procedure reduced the influenceof exogenous environmental factors which could induceDNA damage Furthermore the absence of BPV infection
BioMed Research International 9
(a) (b)
8
6
4
2
0
Num
ber o
f mic
ronu
cleat
ed ce
lls
Neg
ativ
e con
trol
Posit
ive c
ontro
l
E6on
copr
otei
n
(c)
Figure 8 Photomicroscope showing CRIB cell not treated with any drug (negative control) indicating absence of micronucleus (a) andtreated with BPV-1 E6 recombinant oncoprotein showing the presence of MN indicated by arrow (b) (c) Number of micronucleated cellsobserved in each group
in the peripheral blood reduces the influence of endogenousenvironmental factors that could interfere in the analysisPositive control showed a high number of micronucle-ated lymphocytes This result was also expected once thisgroup was treated with 50 120583gmL of cyclophosphamide achemotherapeutic drug with cytotoxic and teratogenic effect[76 77] The cells treated with 1120583gmL of BPV-1 E6 recom-binant oncoprotein also showed significant statistical valuesKruskal-Wallis test followed by the Dunn post hoc testwas performed to compare the frequencies of micronucleusamong three groups This test indicated statistical differencebetween negative control and the cells treated with BPV-1 E6 recombinant oncoprotein However the test did notreveal differences between the cells treated with cyclophos-phamide and BPV-1 E6 recombinant oncoprotein (Table 4Figure 4) Similar results were also observed in epithelial cells(Figure 8) These results suggest that the BPV-1 E6 oncopro-tein has an aneugenic andor clastogenic potential similarto or higher than the observed with the cyclophosphamideThis is because the cells treated with the oncoprotein showed
a frequency of micronucleus formation (MN1199030) 14003
higher than those observed in positive controlThe elevated mutagenic potential of the BPV-1 E6 onco-
protein can justify the cytogenetic damage already describedin the literature [1 14 15 24] Similar results were observedin HPV-infected cells [12 78ndash81] The micronucleus induce-ment was already described in cytological samples collectedduring Papanicolaursquos test from healthy cervix infected byHPV [12] For this reason the micronucleus assay has beenproposed as a complementary method for Papanicolaursquos testbeing a suggestive biomarker of lesion degree [78] Howeverthe association between the BPV and the micronucleusinducement was not yet reported
The high frequency of micronucleated lymphocytes insamples infected by HPV has been attributed to the synergicaction of E5 E6 and E7 oncoproteins [29 30 78 79]However this is the first study that pointed out themutagenicaction of BPV-1 E6 protein per se
Student-Newman-Keuls post hoc test based on the fre-quency of anaphase bridges showed significant statistical
10 BioMed Research International
Table 7 Comet assay results showing the number of nucleoidsobserved per class the number of hedgehog comets and cometscore
Sample Class 0 Class 1 Class 2 Hedgehog ScoreNegative control
Groups Rank differences Calculated 119885 Critical 119885 119901
Cminus and C+ 68 24042 2394 lt05Cminus and E6 82 28991 2394 lt05C+ and E6 14 04950 2394 nsns nonsignificant differences
difference between the negative control and the group treatedwith the BPV-1 E6 recombinant oncoprotein (Table 5) How-ever the test did not reveal differences between the cellstreated with cyclophosphamide and BPV-1 E6 recombinantoncoprotein (Table 5)These data reinforce those observed bythe Dunn post hoc test based on the frequency of micronu-cleus indicating the clastogenic action of the E6 oncoproteinMoreover the presence of anaphase bridge was alreadydescribed in cells transfected with recombinant adenoviruscontaining the E6 and E7 ORFs of HPV [29 30] Howeverthere are no studies describing the action of BPVE6 oncopro-tein in the induction of anaphase bridges or studies that showthis same action of E6 oncoprotein of HPV per se Anaphasebridges are indicators of genomic instability [82 83] which isconsidered the first step in carcinogenesis [46 47] Moreoverthe presence of these bridges is an important hallmark ofDNA double strand breaks (DSBs) [84]
Statistical analysis based on the cytokinesis-block prolif-eration index (CBPI) showed that BPV-1 E6 oncoprotein hascytotoxic levels similar to cyclophosphamide (Table 6) Thisresult suggests that the BPV E6 oncoprotein can deregulatethe cell cycle contributing to the cell proliferation andimmortalization To date this mitogenic action is attributedto the E7 oncoprotein [85ndash87] E7 oncoprotein induces
the phosphorylation of pRb resulting in the E2F transcrip-tion factor release [85] E2F when translocated to the nucleusacts as activator and binds to the kinase-dependent cyclinspromoters [85] However the results showed polynucleatedcells in the group treatedwithBPVE6oncoprotein (Figure 5)suggesting that the BPV E6 oncoprotein confers mitogenicstimulation resulting in mitotic stress Moreover studiespointed out that the BPV E6 oncoprotein interacts with theCBPp300 deacetylase promoting p53 downregulation [88]This downregulation increases the expression levels of FoxM1transcription factor (Foxhead box M1) [89] promoting B1cyclin D1 cyclin and cdc25 upregulation [89] The upregu-lation of these genes is associated with increased levels of cellproliferation which is necessary in order to make DNA poly-merases available to virus replication However if this mech-anism guarantees the BPV replication it can contribute to cellimmortalization and cancer progression
The FoxM1 factor also participates in the Wnt120573-cateninsignaling pathway and binds directly to the 120573-catenin [89]This interaction promotes the nuclear 120573-catenin transloca-tion [90] 120573-catenin translocation to nucleus induces thecyclin expression [64] So these data suggest that the BPVE6 oncoprotein not only promotes mitogenic stress but alsocontributes to the epithelial-mesenchymal transition (EMT)Studies based on theHPVE6 oncoprotein have indicated thatthis oncoprotein contributes significantly to the EMT [91]This occurs not only because the E6 oncoprotein induces thetranslocation of 120573-catenin but also due to the proteasomaldegradation of regulatory proteins of apical-basal polarity[92ndash94] Studies also showed that the E6 oncoprotein is ableto bind to Crumbs (Dlg and Patj) proteins resulting in loss ofcell polarity [93 94] Based on these results the E6 oncopro-tein emerges as a possible therapeutic target with biotechno-logical value in cancer treatment
Mitogenic action of E6 oncoprotein can be moreexpressed in cells infected by papillomaviruses once thesecells also expressed E5 and E7 oncoproteins [74] The E7oncoprotein is able to form a complex E7-p600 which pro-motes E6 upregulation [85] Furthermore the E6 oncoproteincan bind to the E6AP ubiquitin ligase forming the E6-E6APcomplex increasing the hTERT levels of expression con-tributing to cell immortalization [95] Moreover E6-E6APinduces NFX1 expression promoting MHC-II downregula-tion [96]Thismechanism reduces the antigenic presentationmediated by CD4+ T lymphocytes contributing to immuneevasion This action guarantees the PV infection for longperiods with or without clinical symptoms [24] Althoughthe BPV infection can be asymptomatic the BPV presence inperipheral blood is associated with DNA damage [1 14 24]
CBMNA analysis pointed out the presence of cells withintracellular cytokinesis (Figure 5) This result suggests theneotic action of BPV E6 oncoprotein Neosis is characterizedby the presence of (1) DNA damage (2) loss of checkpointcontrol (3) repair system failures and (4) endoreduplication[97] In this scenario the E6 oncoprotein attempts all theseneosis criteria Moreover the oncoprotein not only increasesthe cytogenetic damage leading to increasing of micronu-cleus frequency but also induces clastogenesis as shown bythe comet assay and anaphase bridges So statistical analysis
BioMed Research International 11
performed based on the comet scores reinforced the CBMNAresults Comet assay allows detecting both simple and dou-ble DNA breaks being more sensitive than the CBMNAClastogenesis is considered the most serious type of DNAdamage [98] DNA breaks can be repaired by homologous ornonhomologous recombination However studies point outthat the E6 oncoprotein can link to the Holliday junctionduring the homologous repair avoiding the junction reso-lution [99] Moreover E6 oncoprotein promotes the TP53gene deacetylation resulting in p53 downregulation [51]Thisepigenetic effect results in cell cycle deregulation affectingthe checkpoints
Studies have shown that the HPV-induced DNA breaksare required for the virus integration into the host genome[100] Although there are no studies showing the BPV inte-gration to date the results observed in this work in additionto those already published [24] indicate the necessity ofmorestudies to evaluate the virus-host interaction
Studies also showed that the E6 oncoprotein inducesclathrin-transporter adapter protein (AP-1) increasing thelevel of this protein in the plasma membrane [76 77] Theincreasing quantity of AP-1 in membrane can facilitate theBPV virions infection This occurs because the infectionprocess is clathrin dependent In this scenario the E6 onco-protein can contribute to the virus infection [101]
In summary the CBMNA and CA results showed thatthe BPV E6 oncoprotein participates not only in cancerpromotion but also in initiation inducing DNA breaksThese DNA breaks represent mutagenic effects consideredthe first step in the oncogenesis process which are associatedwith genomic instability [47 48 102 103] Thus the E6recombinant oncoprotein has been suggested as a possiblevaccine candidate [104ndash112] due to its immunogenicity [44]However the mutagenic tests such as CBMNA and CA arerequired in drug validation process [113] In this scenariothis study pointed out that the BPV-1 E6 recombinant onco-protein in tested concentration showedmutagenic potentialThis is the first study that reports the mutagenic potential ofa possible therapeutic vaccine candidate On the one handthese results allowed better understanding the mechanism ofcancer promotion associatedwith the BPVE6oncoprotein aswell as revealing that this oncoprotein can induce carcinogen-esis per se on the other hand this data pointed out thatmaybeBPV E6 recombinant oncoprotein requires protein modifica-tions to be used as vaccine
Conflict of Interests
The authors declare that there is no conflict of interests
Acknowledgments
Theauthors thank Fundacao deAmparo a Pesquisa do Estadode Sao Paulo (FAPESP Process 201420617-5) and FundacaoButantan for the financial support
References
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experimental modelrdquo Journal of General Virology vol 79 no 9pp 2127ndash2135 1998
[2] A Antonsson and B G Hansson ldquoHealthy skin ofmany animalspecies harbors papillomaviruses which are closely related totheir human counterpartsrdquo Journal of Virology vol 76 no 24pp 12537ndash12542 2002
[3] P Rous and J W Beard ldquoA virus-induced mammalian growthwith the characters of a tumor (the Shope rabbit papilloma) IThe growth on implantationwithin favorable hostsrdquoThe Journalof Experimental Medicine vol 60 no 6 pp 701ndash722 1934
[4] R Shope and E Hurst ldquoInfectious papillomatosis of rabbitsrdquoThe Journal of Experimental Medicine vol 58 no 1 pp 607ndash624 1933
[5] H Delius M A Van Ranst A B Jenson H Zur Hausen and JP Sundberg ldquoCanine oral papillomavirus genomic sequence aunique 15-kb intervening sequence between the E2 andL2 openreading framesrdquo Virology vol 204 no 1 pp 447ndash452 1994
[6] W A Demonbreun and E Goodpasture ldquoInfectious oral papil-lomatosis of dogsrdquo The American Journal of Pathology vol 8no 1 pp 43ndash56 1932
[7] P K Nicholls B A Klaunberg R A Moore et al ldquoNaturallyoccurring nonregressing canine oral papillomavirus infectionhost immunity virus characterization and experimental infec-tionrdquo Virology vol 265 no 2 pp 365ndash374 1999
[8] A Alberti S Pirino F Pintore et al ldquoOvis aries Papillomavirus3 a prototype of a novel genus in the family Papillomaviridaeassociated with ovine squamous cell carcinomardquo Virology vol407 no 2 pp 352ndash359 2010
[9] H zur Hausen E-M de Villiers and L Gissmann ldquoPapil-lomavirus infections and human genital cancerrdquo GynecologicOncology vol 12 no 2 pp S124ndashS128 1981
[10] C Foresta A Bertoldo A Garolla et al ldquoHuman papillo-mavirus proteins are found in peripheral blood and semenCd20+ and Cd56+ cells during Hpv-16 semen infectionrdquo BMCInfectious Diseases vol 13 article 593 2013
[11] S Ljubojevic andM Skerlev ldquoHPV-associated diseasesrdquo Clinicsin Dermatology vol 32 no 2 pp 227ndash234 2014
[12] A P R Cassel R B Barcellos C M D da Silva S E de MatosAlmeida and M L R Rossetti ldquoAssociation between humanpapillomavirus (HPV) DNA and micronuclei in normal cervi-cal cytologyrdquo Genetics and Molecular Biology vol 37 no 2 pp360ndash363 2014
[13] W Jarrett M Campo B OrsquoNeil H Laird and L Coggins ldquoAnovel bovine papillomavirus (BPV-6) causing true epithelialpapillomas of the mammary gland skin a member of a pro-posed newBPV subgrouprdquoVirology vol 136 no 2 pp 255ndash2641984
[14] T CMelo N Diniz S R C Campos et al ldquoCytogenetic studiesin peripheral blood of bovines afflicted by papillomatosisrdquoVeterinary and Comparative Oncology vol 9 no 4 pp 269ndash2742011
[15] S R C Campos T C Melo S Assaf et al ldquoChromosome aber-rations in cells infected with bovine papillomavirus comparingcutaneous papilloma esophagus papilloma and urinary blad-der lesion cellsrdquo ISRN Oncology vol 2013 Article ID 910849 8pages 2013
[16] H zur Hausen ldquoThe search for infectious causes of humancancers where and whyrdquoVirology vol 392 no 1 pp 1ndash10 2009
[17] R P Araldi R F Carvalho T C Melo et al ldquoBovine papillo-mavirus in beef cattle first description of BPV-12 and putativetype BAPV8 in BrazilrdquoGenetics andMolecular Research vol 13no 3 pp 5644ndash5653 2014
12 BioMed Research International
[18] T Rampias C Sasaki and A Psyrri ldquoMolecular mechanisms ofHPV induced carcinogenesis in head and neckrdquoOral Oncologyvol 50 no 5 pp 356ndash363 2014
[19] S Roperto V Russo L Leonardi et al ldquoBovine papillomavirustype 13 expression in the urothelial bladder tumours of cattlerdquoTransboundary and Emerging Diseases 2015
[20] S RCCampos C TrindadeO P Ferraz et al ldquoCan establishedcultured papilloma cells harbor bovine papillomavirusrdquoGenet-ics and Molecular Research vol 7 no 4 pp 1119ndash1126 2008
[21] M S Campo ldquoAnimalmodels of papilloma virus pathogenesisrdquoVirus Research vol 89 no 2 pp 249ndash261 2002
[22] G Borzacchiello ldquoBovine papillomavirus infections in ani-malsrdquo in Communicating Current Research and EducationalTopics and Trends in AppliedMicrobiology pp 673ndash679 Forma-tex Badajoz Spain 2007
[23] F Bocaneti G Altamura A Corteggio E Velescu F RopertoandG Borzacchiello ldquoBovine papillomavirus new insights intoan old diseaserdquo Transboundary and Emerging Diseases 2014
[24] R P Araldi T C Melo N Diniz et al ldquoBovine papillomavirusclastogenic effect analyzed in comet assayrdquo BioMed ResearchInternational vol 2013 Article ID 630683 7 pages 2013
[25] J Munday N Thomson M Dunowska C Knight R Laurieand S Hills ldquoGenomic characterisation of the feline sarcoid-associated papillomavirus and proposed classification as Bostaurus papillomavirus type 14rdquoVeterinaryMicrobiology vol 177no 3-4 pp 289ndash295 2015
[26] G Borzacchiello V Russo C Spoleto et al ldquoBovine papillo-mavirus type-2 DNA and expression of E5 and E7 oncoproteinsin vascular tumours of the urinary bladder in cattlerdquo CancerLetters vol 250 no 1 pp 82ndash91 2007
[27] P Maiolino A Ozkul A Sepici-Dincel et al ldquoBovine papillo-mavirus type 2 infection and microscopic patterns of urothelialtumors of the urinary bladder in water buffaloesrdquo BioMedResearch International vol 2013 Article ID 937918 6 pages2013
[28] S Roperto G Borzacchiello R Brun et al ldquoMultiple glomustumors of the urinary bladder in a cow associated with bovinepapillomavirus type 2 (BPV-2) infectionrdquo Veterinary Pathologyvol 45 no 1 pp 39ndash42 2008
[29] S Duensing and K Munger ldquoThe human papillomavirus type16 E6 and E7 oncoproteins independently induce numerical andstructural chromosome instabilityrdquoCancer Research vol 62 no23 pp 7075ndash7082 2002
[30] S Duensing L Y Lee A Duensing et al ldquoThe human papil-lomavirus type 16 E6 and E7 oncoproteins cooperate to inducemitotic defects and genomic instability by uncoupling centro-some duplication from the cell division cyclerdquo Proceedings of theNational Academy of Sciences of theUnited States of America vol97 no 18 pp 10002ndash10007 2000
[31] C A Moody and L A Laimins ldquoHuman papillomavirus onco-proteins pathways to transformationrdquo Nature Reviews Cancervol 10 no 8 pp 550ndash560 2010
[32] C Mansur and E Androphy ldquoCellular transformation bypapillomavirus oncoproteinsrdquo Biochimica et Biophysica Actavol 1155 no 3 pp 323ndash345 1993
[33] J J Chen Y Hong and E J Androphy ldquoMutational analysis oftranscriptional activation by the bovine papillomavirus type 1E6rdquo Virology vol 236 no 1 pp 30ndash36 1997
[34] X Tong andPMHowley ldquoThebovine papillomavirus E6 onco-protein interacts with paxillin and disrupts the actin cytoskele-tonrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 94 no 9 pp 4412ndash4417 1997
[35] X TongW Boll T Kirchhausen andPMHowley ldquoInteractionof the bovine papillomavirus E6 protein with the clathrinadaptor complex AP-1rdquo Journal of Virology vol 72 no 1 pp476ndash482 1998
[36] Y Nomine M Masson S Charbonnier et al ldquoStructural andfunctional analysis of E6 oncoprotein insights in the molecularpathways of human papillomavirus-mediated pathogenesisrdquoMolecular Cell vol 21 no 5 pp 665ndash678 2006
[37] T Ristriani MMasson Y Nomine et al ldquoHPV oncoprotein E6is a structure-dependent DNA-binding protein that recognizesfour-way junctionsrdquo Journal of Molecular Biology vol 296 no5 pp 1189ndash1203 2000
[38] Y Liu and J D Baleja ldquoStructure and function of the papil-lomavirus E6 protein and its interacting proteinsrdquo Frontiers inBioscience vol 13 no 1 pp 121ndash134 2008
[39] F Lipari G A McGibbon E Wardrop and M G CordingleyldquoPurification and biophysical characterization of a minimalfunctional domain and of an N-terminal Zn2+-binding frag-ment from the human papillomavirus type 16 E6 proteinrdquoBiochemistry vol 40 no 5 pp 1196ndash1204 2001
[40] Y Nomine T Ristriani C Laurent J-F Lefevre E Weissand G Trave ldquoA strategy for optimizing the monodispersity offusion proteins application to purification of recombinantHPVE6 oncoproteinrdquo Protein Engineering vol 14 no 4 pp 297ndash3052001
[41] C G Ullman P I Haris D A Galloway V C Emery and SJ Perkins ldquoPredicted 120572-helix120573-sheet secondary structures forthe zinc-binding motifs of human papillomavirus E7 and E6proteins by consensus prediction averaging and spectroscopicstudies of E7rdquo Biochemical Journal vol 319 no 1 pp 229ndash2391996
[42] A Sidi K Babah N Brimer et al ldquoStrategies for bacterialexpression of protein-peptide complex application to solubi-lization of papillomavirus E6rdquo Protein Expression and Purifica-tion vol 29 no 6 pp 997ndash1003 2011
[43] K Zanier S Charbonnier A O M O Sidi et al ldquoStructuralbasis for hijacking of cellular LxxLL motifs by papillomavirusE6 oncoproteinsrdquo Science vol 339 no 6120 pp 694ndash698 2013
[44] J Mazzuchelli-de-Souza R F Carvalho R M Ruiz et alldquoExpression and in silico analysis of the recombinant bovinepapillomavirus e6 protein as a model for viral oncoproteinsstudiesrdquo BioMed Research International vol 2013 Article ID421398 9 pages 2013
[45] L Banks P Spence E Androphy et al ldquoIdentification of humanpapillomavirus type 18 E6 polypeptide in cells derived fromhuman cervical carcinomasrdquo Journal of General Virology vol68 no 5 pp 1351ndash1359 1987
[46] D Hanahan and R AWeinberg ldquoHallmarks of cancer the nextgenerationrdquo Cell vol 144 no 5 pp 646ndash674 2011
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[48] S Negrini V G Gorgoulis and T D Halazonetis ldquoGenomicinstabilitymdashan evolving hallmark of cancerrdquo Nature ReviewsMolecular Cell Biology vol 11 no 3 pp 220ndash228 2010
[49] K Zanier S Charbonnier M Baltzinger Y Nomine DAltschuh and G Trave ldquoKinetic analysis of the interactionsof human papillomavirus E6 oncoproteins with the ubiquitinligase E6AP using surface plasmon resonancerdquo Journal ofMolecular Biology vol 349 no 2 pp 401ndash412 2005
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[51] H Zimmermann C-H Koh R Degenkolbe et al ldquoInteractionwith CBPp300 enables the bovine papillomavirus type 1 E6oncoprotein to downregulate CBPp300-mediated transactiva-tion by p53rdquo Journal of General Virology vol 81 no 11 pp 2617ndash2623 2000
[52] M Fenech ldquoThe in vitro micronucleus techniquerdquo MutationResearchmdashFundamental and Molecular Mechanisms of Mutage-nesis vol 455 no 1-2 pp 81ndash95 2000
[53] R P Araldi T C de Melo T B Mendes et al ldquoUsing the cometand micronucleus assays for genotoxicity studies a reviewrdquoBiomedicine amp Pharmacotherapy vol 72 pp 74ndash82 2015
[54] P Thomas and M Fenech ldquoCytokinesis-block micronucleuscytome assay in lymphocytesrdquo Methods in Molecular Biologyvol 682 pp 217ndash234 2011
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[56] S Samanta and P Dey ldquoMicronucleus and its applicationsrdquoDiagnostic Cytopathology vol 40 no 1 pp 84ndash90 2012
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[65] T C Melo R F Carvalho J Mazzucchelli-de-Souza et alldquoPhylogenetic classification and clinical aspects of a new puta-tive Deltapapillomavirus associated with skin lesions in cattlerdquoGenetics and Molecular Research vol 13 no 2 pp 2458ndash24692014
[66] R P Araldi D N S Giovanni T CMelo et al ldquoBovine papillo-mavirus isolation by ultracentrifugationrdquo Journal of VirologicalMethods vol 208 pp 119ndash124 2014
[67] L M Chandrachud B W OrsquoNeil W F H Jarrett G J Grind-lay G M McGarvie and M S Campo ldquoHumoral immuneresponse to the E7 protein of bovine papillomavirus type 4 andidentification of B-cell epitopesrdquoVirology vol 200 no 1 pp 98ndash104 1994
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[70] O Danos E Georges G Orth and M Yaniv ldquoFine structureof the cottontail rabbit papillomavirus mRNAs expressed in thetransplantable VX2 carcinomardquo Journal of Virology vol 53 no3 pp 735ndash741 1985
[71] M Nasseri and F O Wettstein ldquoDifferences exist between viraltranscripts in cottontail rabbit papillomavirus-induced benignand malignant tumors as well as non-virus-producing andvirus-producing tumorsrdquo Journal of Virology vol 51 no 3 pp706ndash712 1984
[72] X Tong R Salgia J-L Li J D Griffin and P M Howley ldquoThebovine papillomavirus E6 protein binds to the LDmotif repeatsof paxillin and blocks its interaction with vinculin and the focaladhesion kinaserdquo Journal of Biological Chemistry vol 272 no52 pp 33373ndash33376 1997
[73] Y Maeda T Shibahara Y Wada et al ldquoAn outbreak of teatpapillomatosis in cattle caused by bovine papilloma virus (BPV)type 6 and unclassified BPVsrdquo Veterinary Microbiology vol 121no 3-4 pp 242ndash248 2007
[74] R P Araldi T C Melo A Neves et al ldquoHyperproliferativeaction of bovine papillomavirus genetic and histopathologicalaspectsrdquo Genetics and Molecular Research vol 14 no 4 pp12942ndash12954 2015
[75] NDiniz T CMelo J F Santos et al ldquoSimultaneous presence ofbovine papillomavirus in blood and in short-term lymphocytecultures from dairy cattle in Pernambuco Brazilrdquo Genetics andMolecular Research vol 8 no 4 pp 1474ndash1480 2009
[76] O M Brito M F Guimaraes and C C Lanna ldquoCiclofosfamidae funcao ovarianardquo Revista Brasileira de Reumatologia vol 48no 1 pp 39ndash45 2008
[77] D Anderson T-W Yu and D B McGregor ldquoComet assayresponses as indicators of carcinogen exposurerdquo Mutagenesisvol 13 no 6 pp 539ndash555 1998
[78] M L Adam C Pini S Tulio J C L L Cantalice R A Torresand M T Dos Santos Correia ldquoAssessment of the associationbetween micronuclei and the degree of uterine lesions and viralload in women with human papillomavirusrdquo Cancer Genomicsamp Proteomics vol 12 no 2 pp 67ndash72 2015
[79] E Cortes-Gutierrez M Davila-Rodrıguez J Vargas-VillarrealF Hernandez-Garza and R Cerda-Flores ldquoAssociationbetween human papilloma virus-type infections with micro-nuclei frequenciesrdquo Prague Medical Report vol 111 no 1 pp35ndash41 2010
[80] R N Chakrabarti and K Dutta ldquoMicronuclei test in routinesmears fromuterine cervixrdquo European Journal of GynaecologicalOncology vol 9 no 5 pp 370ndash372 1988
[81] E M M Cerqueira C L Santoro N F Donozo et al ldquoGeneticdamage in exfoliated cells of the uterine cervix Association
14 BioMed Research International
and interaction between cigarette smoking and progression tomalignant transformationrdquo Acta Cytologica vol 42 no 3 pp639ndash649 1998
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[83] D R Hoffelder L Luo N A Burke S CWatkins S M Gollinand W S Saunders ldquoResolution of anaphase bridges in cancercellsrdquo Chromosoma vol 112 no 8 pp 389ndash397 2004
[84] C Acilan D M Potter and W S Saunders ldquoDNA repairpathways involved in anaphase bridge formationrdquo Genes Chro-mosomes and Cancer vol 46 no 6 pp 522ndash531 2007
[85] J DeMasi M C Chao A S Kumar and P M Howley ldquoBovinepapillomavirus E7 oncoprotein inhibits anoikisrdquo Journal ofVirology vol 81 no 17 pp 9419ndash9425 2007
[86] J DeMasi K-W Huh Y Nakatani K Munger and P MHowley ldquoBovine papillomavirus E7 transformation functioncorrelates with cellular p600 protein bindingrdquo Proceedings of theNational Academy of Sciences of theUnited States of America vol102 no 32 pp 11486ndash11491 2005
[87] E A White and P M Howley ldquoProteomic approaches to thestudy of papillomavirusndashhost interactionsrdquo Virology vol 435no 1 pp 57ndash69 2013
[88] FMantovani and L Banks ldquoThe human papillomavirus E6 pro-tein and its contribution to malignant progressionrdquo Oncogenevol 20 no 54 pp 7874ndash7887 2001
[89] P Chen and H Lee ldquoFOXM1 induced by E6 oncoprotein pro-motes tumor invasion and chemoresistance in HPV-infectedlung cancerrdquo Cancer Cell amp Microenvironment vol 2 no 1 pp6ndash9 2015
[90] N Zhang P Wei A Gong et al ldquoFoxM1 promotes 120573-cateninnuclear localization and controls Wnt target-gene expressionand glioma tumorigenesisrdquo Cancer Cell vol 127 no 4 pp 358ndash366 2012
[91] Z Liu Y Liu Y Hong L Rapp E J Androphy and J JChen ldquoBovine papillomavirus type 1 E6-induced sensitizationto apoptosis is distinct from its transforming activityrdquo Virologyvol 295 no 2 pp 230ndash237 2002
[92] M Thomas P Massimi C Navarro J-P Borg and L BanksldquoThe hScribDlg apico-basal control complex is differentiallytargeted by HPV-16 and HPV-18 E6 proteinsrdquo Oncogene vol24 no 41 pp 6222ndash6230 2005
[93] C H Storrs and S J Silverstein ldquoPATJ a tight junction-associated PDZ protein is a novel degradation target of high-risk human papillomavirus E6 and the alternatively splicedisoform 18 E6rdquo Journal of Virology vol 81 no 8 pp 4080ndash40902007
[94] R Accardi R Rubino M Scalise et al ldquoE6 and E7 from humanpapillomavirus type 16 cooperate to target the PDZ proteinNaH exchange regulatory factor 1rdquo Journal of Virology vol 85no 16 pp 8208ndash8216 2011
[95] S Nakagawa and J M Huibregtse ldquoHuman scribble (Vartul)is targeted for ubiquitin-mediated degradation by the high-risk papillomavirus E6 proteins and the E6APubiquitin-proteinligaserdquoMolecular and Cellular Biology vol 20 no 21 pp 8244ndash8253 2000
[96] L Gewin H Myers T Kiyono and D A Galloway ldquoIdentifi-cation of a novel telomerase repressor that interacts with thehuman papillomavirus type-16 E6E6-AP complexrdquo Genes ampDevelopment vol 18 no 18 pp 2269ndash2282 2004
[97] M Sundaram D L GuernseyMM Rajaraman and R Rajara-man ldquoNeosis a novel type of cell division in cancerrdquo CancerBiology ampTherapy vol 3 no 2 pp 207ndash218 2004
[98] R D Snyder and J W Green ldquoA review of the genotoxicity ofmarketed pharmaceuticalsrdquo Mutation Research vol 488 no 2pp 151ndash169 2001
[99] T Ristriani Y Nomine C Laurent E Weiss and G TraveldquoProtein mutagenesis with monodispersity-based quality prob-ing selective inactivation of p53 degradation and DNA-bindingproperties of HPV E6 oncoproteinrdquo Protein Expression andPurification vol 26 no 3 pp 357ndash367 2002
[100] M Kadaja A Sumerina T Verst M Ojarand E Ustav and MUstav ldquoGenomic instability of the host cell induced by thehuman papillomavirus replicationmachineryrdquoTheEMBO Jour-nal vol 26 no 8 pp 2180ndash2191 2007
[101] PMDay D R Lowy and J T Schiller ldquoPapillomaviruses infectcells via a clathrin-dependent pathwayrdquo Virology vol 307 no 1pp 1ndash11 2003
[102] T D Tlsty A Briot A Gualberto et al ldquoGenomic instabilityand cancerrdquoMutation Research vol 337 no 1 pp 1ndash7 1995
[103] J E Eyfjord and S K Bodvarsdottir ldquoGenomic instabilityand cancer networks involved in response to DNA damagerdquoMutation Research vol 592 no 1-2 pp 18ndash28 2005
[104] Y Yao W Huang X Yang et al ldquoHPV-16 E6 and E7 protein Tcell epitopes prediction analysis based on distributions of HLA-A loci across populations an in silico approachrdquo Vaccine vol31 no 18 pp 2289ndash2294 2013
[105] C-F Hung B Ma A Monie S-W Tsen and T-C Wu ldquoTher-apeutic human papillomavirus vaccines current clinical trialsand future directionsrdquo Expert Opinion on Biological Therapyvol 8 no 4 pp 421ndash439 2008
[106] M J P Welters G G Kenter S J Piersma et al ldquoInductionof tumor-specific CD4+ and CD8+ T-cell immunity in cervicalcancer patients by a human papillomavirus type 16 E6 and E7long peptides vaccinerdquo Clinical Cancer Research vol 14 no 1pp 178ndash187 2008
[107] R Han N M Cladel C A Reed X Peng and N D Chris-tensen ldquoProtection of rabbits from viral challenge by gene gun-based intracutaneous vaccination with a combination of cot-tontail rabbit papillomavirus E1 E2 E6 and E7 genesrdquo Journalof Virology vol 73 no 8 pp 7039ndash7043 1999
[108] Z He A P Wlazlo D W Kowalczyk et al ldquoViral recombinantvaccines to the E6 and E7 antigens of HPV-16rdquo Virology vol270 no 1 pp 146ndash161 2000
[109] A Kaufmann P Stern E Rankin et al ldquoSafety and immuno-genicity of TA-HPV a recombinant vaccinia virus expressingmodified human papillomavirus (HPV)-16 and HPV-18 E6 andE7 genes in women with progressive cervical cancerrdquo ClinicalCancer Research vol 8 no 12 pp 3676ndash3685 2002
[110] L K Borysiewicz A Fiander M Nimako et al ldquoA recombinantvaccinia virus encoding human papillomavirus types 16 and 18E6 and E7 proteins as immunotherapy for cervical cancerrdquoTheLancet vol 347 no 9014 pp 1523ndash1527 1996
[111] A De Jong T OrsquoNeill A Y Khan et al ldquoEnhancement ofhuman papillomavirus (HPV) type 16 E6 and E7-specific T-cellimmunity in healthy volunteers through vaccination with TA-CIN an HPV16 L2E7E6 fusion protein vaccinerdquo Vaccine vol20 no 29-30 pp 3456ndash3464 2002
[112] S Chen C Liao Y Lai et al ldquoDe-oncogenicHPVE6E7 vaccinegets enhanced antigenicity and promotes tumoricidal synergywith cisplatinrdquo Acta Biochimica et Biophysica Sinica vol 46 no1 pp 6ndash14 2014
BioMed Research International 15
[113] J Bonhoeffer E B Imoukhuede G Aldrovandi et al ldquoTemplateprotocol for clinical trials investigating vaccines-focus on safetyelementsrdquo Vaccine vol 31 no 47 pp 5602ndash5620 2013
[114] S Jackson and A Storey ldquoE6 proteins from diverse cutaneonsHPV types inhibit apoptosis in response to UV damagerdquoOncogene vol 19 no 4 pp 592ndash598 2000
[115] M Thomas and L Banks ldquoPDZRN3LNX3 is a novel targetof human papillomavirus type 16 (HPV-16) and HPV-18 E6rdquoJournal of Virology vol 89 no 2 pp 1439ndash1444 2015
[116] P R Daniels C M Sanders P Coulson and N J MaitlandldquoMolecular analysis of the interaction between HPV type 16 E6and human E6-associated proteinrdquo FEBS Letters vol 416 no 1pp 6ndash10 1997
[117] W H Kao S L Beaudenon A L Talis J M Huibregtse andP M Howley ldquoHuman papillomavirus type 16 E6 induces self-ubiquitination of the E6AP ubiquitin-protein ligaserdquo Journal ofVirology vol 74 no 14 pp 6408ndash6417 2000
[118] T Iftner M Elbel B Schopp et al ldquoInterference of papillo-mavirus E6 protein with single-strand break repair by interac-tion with XRCC1rdquo The EMBO Journal vol 21 no 17 pp 4741ndash4748 2002
[119] Y Imai Y Tsunokawa T Sugimura and M Terada ldquoPurifi-cation and DNA-binding properties of human papillomavirustype 16 E6 protein expressed in Escherichia colirdquo Biochemicaland Biophysical Research Communications vol 164 no 3 pp1402ndash1410 1989
[120] M Lusky L Berg H Weiher and M Botchan ldquoBovinepapilloma virus contains an activator of gene expression at thedistal end of the early transcription unitrdquoMolecular andCellularBiology vol 3 no 6 pp 1108ndash1122 1983
[121] S Dalal Q Gao E J Androphy and V Band ldquoMutationalanalysis of human papillomavirus type 16 E6 demonstrates thatp53 degradation is necessary for immortalization of mammaryepithelial cellsrdquo Journal of Virology vol 70 no 2 pp 683ndash6881996
[122] J M Huibregtse M Scheffner and P M Howley ldquoA cellularprotein mediates association of p53 with the E6 oncoprotein ofhuman papillomavirus types 16 or 18rdquo The EMBO Journal vol10 no 13 pp 4129ndash4135 1991
[123] M Scheffner B A Werness J M Huibregtse A J Levine andP M Howley ldquoThe E6 oncoprotein encoded by human papillo-mavirus types 16 and 18 promotes the degradation of p53rdquo Cellvol 63 no 6 pp 1129ndash1136 1990
[124] M Elbel S Carl S Spaderna and T Iftner ldquoA comparativeanalysis of the interactions of the E6 proteins from cutaneousand genital papillomaviruses with p53 and E6AP in correlationto their transforming potentialrdquoVirology vol 239 no 1 pp 132ndash149 1997
[125] M C Thomas and C-M Chiang ldquoE6 oncoprotein repressesp53-dependent gene activation via inhibition of protein acety-lation independently of inducing p53 degradationrdquo MolecularCell vol 17 no 2 pp 251ndash264 2005
[126] H Zimmermann R Degenkolbe H-U Bernard and M JOrsquoConnor ldquoThe human papillomavirus type 16 E6 oncoproteincan down-regulate p53 activity by targeting the transcriptionalcoactivator CBPp300rdquo Journal of Virology vol 73 no 8 pp6209ndash6219 1999
[127] D A Thompson G Belinsky T H-T Chang D L Jones RSchlegel and K Munger ldquoThe human papillomavirus-16 E6oncoprotein decreases the vigilance of mitotic checkpointsrdquoOncogene vol 15 no 25 pp 3025ndash3035 1997
[128] J Bohl K Das B Dasgupta and S B Vande Pol ldquoCompetitivebinding to a charged leucine motif represses transformation bya papillomavirus E6 oncoproteinrdquo Virology vol 271 no 1 pp163ndash170 2000
[129] S Song H C Pitot and P F Lambert ldquoThe human papillo-mavirus type 16 E6 gene alone is sufficient to induce carcinomasin transgenic animalsrdquo Journal of Virology vol 73 no 7 pp5887ndash5893 1999
[130] S Yasumoto A L Burkhardt J Doniger and J A DiPaololdquoHumanpapillomavirus type 16DNA-inducedmalignant trans-formation of NIH 3T3 cellsrdquo Journal of Virology vol 57 no 2pp 572ndash577 1986
[131] S B Vande Pol M C Brown and C E Turner ldquoAssociation ofBovine Papillomavirus Type 1 E6 oncoprotein with the focaladhesion protein paxillin through a conserved protein interac-tion motifrdquo Oncogene vol 16 no 1 pp 43ndash52 1998
[132] R Wade N Brimer and S Vande Pol ldquoTransformation bybovine papillomavirus type 1 E6 requires paxillinrdquo Journal ofVirology vol 82 no 12 pp 5962ndash5966 2008
[133] A J Klingelhutz S A Foster and J KMcDougall ldquoTelomeraseactivation by the E6 gene product of humanpapillomavirus type16rdquo Nature vol 380 no 6569 pp 79ndash82 1996
[134] S T Oh S Kyo and L A Laimins ldquoTelomerase activation byhuman papillomavirus type 16 E6 protein Induction of humantelomerase reverse transcriptase expression through Myc andGC-rich Sp1 binding sitesrdquo Journal of Virology vol 75 no 12pp 5559ndash5566 2001
[135] L V Ronco A Y KarpovaMVidal and PMHowley ldquoHumanpapillomavirus 16 E6 oncoprotein binds to interferon regulatoryfactor-3 and inhibits its transcriptional activityrdquo Genes andDevelopment vol 12 no 13 pp 2061ndash2072 1998
Table 1 Studies already published about E6 oncoprotein
Interaction with host cell Virus type Method ReferencesFacilitating virus entry BPV E6 facilitates the BPV entry by clathrin interaction [35]Antiapoptotic effect HPV E6 promotes Bax degradation resulting in an antiapoptotic effect [114 115]Binding to E6AP ubiquitinligase HPV E6 expressed in E coli and insects binds to E6AP ubiquitin ligase [50 95 116 117]
Binding to DNA HPV E6 expressed in E coli binds with DNA [118ndash120]Cell immortalization HPV E6 promotes p53 degradation and cell immortalization [121ndash123]
Epigenetic downregulationof p53
HPV E6 expressed in baculovirus induces hypoacetylation of p53 [124 125]
HPV E6 expressed in E coli interacts with p300CBP reducing p53levels [126]
BPV E6 interacts with p300CBP reducing p53 levels [51]Disruption of mitoticapparatus HPV E6 transfected using LXSN vectors [29 30 127]
Malignant transformationin cell culture HPV Complete genome of HPV-16 transfected in NIH 3T3 cells [45 88 128ndash130]
Interaction with paxillin BPV E6 of BPV-1 interacts with paxillin reducing the focal adhesion [34 69 72 131 132]Repressor activity oftelomerases HPV E6 induces the hTERT expression reducing the telomerase activity [96 133 134]
Immune depletion HPV E6 expressed in yeast reduces the levels of interferon regulatoryfactor-3 [135]
described [25] BPV-1 BPV-2 and BPV-4 have the mostoncogenic potential [21] BPV-1 and BPV-2 are associatedwith urinary bladder cancer [26 27] whereas BPV-4 inducesupper gastrointestinal malignancies [28]
Several studies already pointed out that both HPV [2930] and BPV [1 14 15 24] interact with the host chromatinresulting in DNA and chromosomes damage This damageis induced by the E5 E6 and E7 viral oncoproteins [24]Although the role of these oncoproteins in the oncogenic pro-cess is known [31 32] there are no studies showing the onco-genic mechanisms of E6 oncoprotein of BPV per se to dateTheE6of BPV-1 has 137 amino acids with fourwell conservedCys-X-X-Cysmotifs among all PVs [33ndash35]Moreover the E6oncoprotein has six cysteine residues which turn the onco-protein susceptible to oxidation [36] These characteristicsmake the production purification and obtaining of E6 cor-rect folding difficult [36 37] Due to these difficulties in thelast 30 years different constructions employing expressionvectors in Escherichia coli have been developed in order toproduce large quantities of E6 oncoprotein [36ndash43] Most ofthese studies are directed to HPV-16 and BPV-1 In this sce-nario Mazzuchelli-de-Souza et al [44] successfully purifiedthe BPV-1 E6 recombinant protein A summary of studiesinvolving the E6 oncoprotein is shown in Table 1
Due the low number of studies about the E6 of BPVto date the knowledge of oncogenic mechanisms associatedwith this oncoprotein comes from previous works with E6of HPV [29 30] The first lines of evidence of E6 oncogenicproperties come from studies on human tumors cell lineagesderived from cervical cancer [45] Others studies based onE6 of HPV pointed out that the oncoprotein is able to inducecytogenetic damage resulting in genomic instability [29 30]which is considered a cancer hallmark [46ndash48] Although the
E6 oncoprotein of bothHPV and BPV affects p53 themecha-nisms that induce the reduction levels of this tumor suppres-sor protein are distinct between these PVs types E6 oncopro-tein of HPV binds to the E6AP ubiquitin ligase resulting inp53 ubiquitination [49] This process results in p53 proteaso-mal degradation [50] However the BPV-1 E6 oncoprotein ofBPV does not induce p53 degradation [51] Studies pointedout that BPV-1 E6 oncoprotein interacts with CBPp300promoting the downregulation of p53 [51] These differentmechanisms of E6 action require studies involving the onco-protein of BPV
Mutation is the first step in carcinogenesis process [52]This study aimed to evaluate the mutagenic potential ofE6 recombinant oncoprotein of BPV-1 by the cytokinesis-block micronucleus assay (CBMNA) and comet assay (CA)CBMNA and CA are noninvasive methods recommendedas part of genotoxicity tests battery for drug validation [53]CBMNA consists in evaluating the frequency of micronu-cleus in binucleated lymphocytes obtained through theaddiction of cytochalasin B in lymphocyte culture to preventcytokinesis [54]
Micronuclei (MNs) are acentric fragments expelled fromthe main nucleus at late stages of anaphase [55] MNs canbe formed through two mechanisms chromosomal breaks(clastogenesis) or disruption of the mitotic apparatus (aneu-genesis) [56] These fragments remain not integrated in thenucleus of daughter cell originating the MNs [54 57] TheMNs represent not only chromosomal losses but also theresult of DNA amplification commonly observed in onco-genic process [57]
CA is a simple technique with low cost which can beemployed in any eukaryotic cells [58 59] CA has been usedto study the clastogenic potential of HPV [60] and BPV [24]
BioMed Research International 3
E6
25kDa
15kDa
Figure 1Western blot showing BPV-1 E6 recombinant oncoproteinWeight ladder employed Spectra Multicolor Broad Range ProteinLadder (Fermentas Lithuania)
The CMNA combined with CA allows detecting DNA dam-age as an indication of mutagenesis with high statistical andsensitivity power [53] This is the first study which evaluatedthe mutagenic potential of E6 recombinant oncoprotein ofBPV-1 suggesting that this oncoprotein participates in bothcancer initiation and promotion
2 Material and Methods
21 Expression and Purification of BPV-1 E6 OncoproteinBPV-1 E6 recombinant oncoprotein was expressed and puri-fied according to Mazzuchelli-de-Souza et al [44] usingEscherichia coli BL21 The oncoprotein was subjected to dial-ysis This step was necessary to remove urea and imidazolesubstances used to promote the oncoprotein refolding Thedialysis avoids false-positive results to mutagenesis testswhich could be induced by urea and imidazole Dialysis wasperformed using Slide-A-Lyzer Dialysis Cassette (3 K-12mL)(Thermo Scientific Carlsbad USA) For this step 10mLof sample was dialyzed against two liters of dialysis buffer(20mM Tris-HCl and 500mM NaCl pH 80) for 8 hours at4∘C under constant agitation Figure 1 shows Western blotof BPV-1 E6 recombinant protein used in this study Toconfirm the identity of recombinant oncoprotein the bandobtained in the SDS-PAGE gel was submitted to mass spec-trometry Four peptide sequences were identified using thismethod being (1) KDFHVVIRE (2) KDFHVVIRE (3) RHVLFNEPFCKT and (4) RLWQGVPVTGEEAELLHGKTThese sequences were analyzed in the Swiss-Prot databasewhere score higher than 43 indicates extensive homologyThus mass spectrometry pointed out a score of 94 demon-strating that the recombinant protein shows identity with E6oncoprotein of BPV-1 (access number VE6 BPV1)
22 Peripheral Blood Collection A volume of 5mL of periph-eral bloodwas collected fromfive asymptomatic calves (with-out cutaneous papillomas) (Bos taurus Simmental breed)using theVacutainer systemwith EDTA (molecular diagnosis
of BPV) and heparin (CBMNA and CA) The materialwas collected through venipuncture from jugular vein by aveterinarian The protocols used in this study were approvedby the Ethics Committee onAnimal Use of Butantan Institute(process 103513) The farm of calvesrsquo origin is located in SaoPaulo State (Brazil) This farm was chosen due to the absenceof bracken fern Pteridium aquilinum because studies pointedout that bracken farm has mutagenic and carcinogenic com-pounds such as quercetin and ptaquilosides [61ndash63] whichcould interfere in the studies leading to false-positive results
23 Molecular BPV Identification by PCR Blood DNAextraction the extraction of DNA from peripheral bloodcells was performed using the GenomicPrep Blood MiniKit Illustra Spin (GE Healthcare Buckinghamshire UK)according to the manufacturer The quality of the obtainedDNAwas verified using the polymerase chain reaction (PCR)technique amplifying a bovine 120573-globin gene fragment [64]
Viral identification viral identification was performedusing specific primers for BPV-1 (forward 51015840-GGAGCG-CCTGCTAACTATAGGA-31015840 reverse 51015840-ATCTGTTGT-TTGGGTGGTGAC-31015840) which amplifies the L1 gene result-ing in a 301 bp amplicon BPV-2 (forward 51015840-GTTATACCA-CCCAAAGAAGACCCT-31015840 reverse 51015840-CTGGTTGCA-ACAGCTCTCTTTCTC-31015840) which amplifies the L2 generesulting in a 164 bp amplicon and BPV-4 (forward 51015840-GCTGACCTTCCAGTCTTAAT-31015840 reverse 51015840-CAGTTT-CAATCTCCTCTTCA-31015840) which amplifies the E7 generesulting in a 170 bp amplicon These primers were chosenbecause BPV-1 BPV-2 and BPV-4 are the most frequenttypes in Brazil being associated with oncogenic process [165] Besides these specific primers two degenerate pairs ofprimers were used Delta-Epsilon (forward 51015840-CCAGAY-TAYYTMAAAATGGC-31015840 reverse 51015840-ATAAMKGCTAGC-TTATATTC-31015840) and Xi (forward 51015840-TWYAATAGDCCV-TTTTGGAT-31015840 reverse 51015840-TTMCGCCTACGCTTTGGC-GC-31015840) [66] These primers allow detecting BPVs of generaDelta Epsilonpapillomavirus (Delta-Epsilon) and Xipapillo-mavirus (Xi) [66] Both primers amplify the L1ORF resultingin products with 430 bp (Delta-Epsilon) and 600 bp (Xi)
PCR parameters reactions were performed in a total vol-ume of 500 120583L using 200 ng120583L of DNA template 20120583L offorward primer 20 120583L of reverse primer and 360 120583L ofMas-ter Mix (4G Porto Alegre Brazil) Reactions were done onthermocycler Veriti 96-WellThermal Cycler (Applied Biosys-tems Carlsbad USA) and subjected to the cycles shown inTable 2 Cloned genomes of BPV-1 BPV-2 and BPV-4 inpAT153 vector in Escherichia coliD5H120572were used as positivecontrols BPV-2 viral genome was employed as positivecontrol for Delta-Epsilon primer and BPV-3 for Xi primer
The PCR products were analyzed in 2 agarose gelelectrophoresis stained with GelRed (Biotium USA) in Tris-Acetate-EDTA (TAE) buffer visualized under UV light usingBioDocAnalyze (Biometra Germany)
24 Cytokinesis-Block Micronucleus Assay (CBMNA) UsingPeripheral Blood Mononuclear Cells (PBMCs) For each sam-ple three cultures were established (1) negative control
4 BioMed Research International
Table 2 Frequency of micronucleated lymphocytes Number ofmicronuclei observed perslide (MN) and number of mononucle-ated (119873
1
) binucleated (1198732
) and polynucleated (119873119901
) lymphocytesand anaphase bridges (AB) observed pergroup Based on thesevalues the micronucleated formation frequency (MN
1199030
) and thecytokinesis-block proliferation index (CBPI) and the media (119909) areshown
(not treated with any drug) (2) positive control (treatedwith 50120583gmL of cyclophosphamide) and (3) experimentalgroup (treated with 1 120583gmL of E6 recombinant oncoproteinresuspended in PBS) This concentration of E6 recombinantoncoproteinwas based on previous study involving BPV early(E) protein as vaccine [67] The protocol of CBMNA wasdone according to the technical recommendation proposedby Araldi et al [53] In detail 02mL of peripheral bloodwas transferred to culture flask containing 50mL RPMI1640 medium supplemented with 15 fetal bovine serum01mL L-glutamine and 01mL phytohemagglutinin A Thematerial was incubated at 37∘C for 8 hours After this bothcyclophosphamide and E6 recombinant oncoprotein wereadded to the culture After 44 hours 02mL of cytochalasin Bwas added to block the cytokinesis After 72 hours the culturewas stopped with the addition of 05mL methanol aceticacid fixative (vv) (3 1) for 5 minutes at room temperatureThematerial was centrifuged at 500 g and the supernatantwasdiscardedThepellet was homogenizedwith 5mLfixative andcentrifuged at 500 g The pellet was aspirated and transferredto slides which were stained with a 1 3 Giemsa phosphatebuffer solution pH = 68 for 8 minutes
After staining coverslips were placed on slides withEntellan (Merck Germany) The material was analyzed ina blind test under an Axiophot binocular microscope (Carl
Zeiss Germany) to observe the frequency ofmicronucleated-binucleated lymphocytes in a total of 1000 analyzed cellsaccording to Araldi et al [53] Statistical analysis was per-formed using the Kruskal-Wallis test followed by a post hocDunn test both at a significance level of 5 Statistical testswere done using the BioEstat software [68]
25 Comet Assay Alkaline Method Slides preparation slidesof 26 times 76mm were dipped in a solution of normal meltingpoint agarose (NMP) (Invitrogen Carlsbad USA) dilutedin phosphate-buffered solution (PBS) 15 at 60∘C and oneside of each slide was wiped clean with a paper towel Theconcentration of NMA was based on Araldi et al [53] Theslides were dried in a horizontal position overnight
Peripheral blood incubation with drugs three wholeblood aliquots of 200120583L each were transferred to three15mL polypropylene tubes (1) negative control (2) positivecontrol and (3) experimental group In each tube 200120583Lof RMPI 1640 medium was added The negative control didnot receive any drug and was incubated only in RPMI 1640medium Positive control was incubated with 50 120583gmL ofcyclophosphamide diluted in RPMI 1640 medium Experi-mental group was treated with 1 120583gmL of BPV-1 E6 recombi-nant protein The samples were incubated at 37∘C for 2 hoursunder constant agitation After this time the aliquots werecentrifuged for 1 minute at 500 g and the supernatant wasdiscarded Ten microliters of each obtained pellet was addedto 75120583L low melting point agarose (LMP) A final volume of85 120583L of this suspensionwas immediately transferred toNMPprecoated slides The slides were covered with coverslips andmaintained at 4∘C for 20 minutes The coverslips were gentlyremoved and the slides were placed in a Coplin jar containinglysis solution (25mM NaCl 100mM ethylenediaminete-traacetic acid (EDTA) 10mM Tris-HCl 11 Triton X-100and 112 dimethyl sulfoxide) at 4∘C for 1 hour Subsequentlyall procedures were conducted under dark conditions toprevent the induction of DNA damage
Electrophoresis after lysis slides were washed with PBSand transferred to an electrophoresis tank containing elec-trophoresis buffer (300mM NaOH and 1mM EDTA pH gt13) at 4∘C for 40 minutes to induce unwinding of double-stranded DNA Next the electrophoretic run was performedwith a current of 25V (086Vcm) 300mA for 20 minutesto promote the migration of free DNA fragments toward theanode The slides were transferred to a Coplin jar containingneutralizing buffer (400mM Tris-HCl pH 75) for 5 minutesThe material was fixed in absolute ethanol for 5 minutes
Comet analysis slides were stained with 20 120583L 4 120583gmLpropidium iodide (PI) and analyzed under a fluorescencemicroscope (Carl Zeiss Axio Scope A1 Germany) equippedwith an excitation filter of 510ndash560 nm and barrier of 590 nmThe material was analyzed in a total magnification of 400xOne hundred nucleoids were analyzed per slide which werescored on a scale of 0 (without DNA damage) to 2 (maximumDNAdamage) according to Araldi et al [53]The scores wereobtained by summing the product of the observed numberof nucleoids per class and its respective class value Statisticalanalysis was performedusing theKruskal-Wallis test followedby the post hocDunn test both with a significance level of 5
BioMed Research International 5
Both test and graphical analysis were done using the BioEstatsoftware [68]
26 Cytokinesis-Block Micronucleus Assay (CBMNA) inEpithelial Cells To verify whether the results observed inPBMCs should also be observed in epithelial cells CBMNAwas performed in CRIB cell (commercial epithelial cell lineobtained from bovine kidney) In detail a total of 1 times 105cells were transferred to six-well plate containing a sterilecoverslip of 24 times 24mm with 2mL of MEM mediumsupplementedwith 10 fetal bovine serum and 1 ampicillinand three cultures had been established negative control(not treated with any drug) positive control (treated with50120583gmL of cyclophosphamide) and experimental group(treatedwith 1120583gmLof BPV-1 E6 recombinant oncoprotein)Cyclophosphamide and BPV-1 E6 recombinant oncoproteinwere added together to the cells After 1 hour the threecultures were treated with 6 120583gmL of cytochalasin B (SigmaGermany)Thematerial was incubated for 48 hours the timenecessary to complete two replication cycles once the dupli-cation time of these cells is 24 hours according to our previ-ous study After this time the medium was removed and thecells were washed twice with PBS at 37∘C Cells were stainedwith solution 1 4 Giemsa-PBS for 3 minutes and afterwashed twice with PBS Coverslips containing the biologicalmaterial were mounted on slides using Entelan (Merck Ger-many) Slides were analyzed by Axiophot binocular micro-scope (Carl Zeiss Germany) to observe the frequency ofmicronucleated cells in a total of 1000 analyzed cells accord-ing to Araldi et al [53]
3 Results
31 Molecular BPV Identification by PCR The peripheralblood samples collected from five asymptomatic calves didnot reveal the presence of BPV sequences using both specificand degenerate primers (Figure 2)
32 Cytokinesis-Block Micronucleus Assay (CBMNA) inPBMCs Frequency analysis of micronucleus observed inCBMNA CBMNA showed an elevated number of micronu-cleated lymphocytes as well as anaphase bridges in bothpositive control and the group treated with 1 120583gmL of E6recombinant oncoprotein (Table 2 Figure 3)
Based on the micronucleated lymphocytes numberobserved per group (Table 2) Chi-square (1205942) test andKruskal-Wallis test were done both with 5 significancelevel Chi-square test pointed out statistical differencesbetween positive control and group treated with E6 (Table 3)These results point out that the cyclophosphamide and E6recombinant oncoprotein are able to induce aneugenesis andor clastogenesis However the Chi-square test did not showstatistic difference in negative control (Table 3)
Kruskal-Wallis test revealed statistical significant differ-ences among the groups (119867 = 97297 and 119901 = 00087)Based on this result Dunn post hoc test was performed Thetest pointed out significant difference between negative andpositive control as well as negative control and experimental
Table 3 Chi-square (1205942) test results showing expected value (119864)freedom degree (FD) and probability (119901)
Group 119864 1205942 FD 119901
Negative control 560 2000 4 07358Positive control 430 10698 4 00302Experimental (E6) 456 12202 4 00159
Table 4 Dunn post hoc test results
Groups Rank differences Calculated 119885 Critical 119885 119901
Cminus and C+ 71 25102 2394 lt005Cndash and E6 79 27931 2394 lt005C+ and E6 08 02828 2394 nsns nonsignificant value
Table 5 Student-Newman-Keuls post hoc test results
Groups Rank differences Calculated 119885 Critical 119885 119901
Cminus and C+ 61 21567 2394 00310Cminus and E6 77 27224 2394 00065C+ and E6 16 05657 2394 05716
group (E6) (Table 4) However the test did not show statis-tical difference between positive control and experimentalgroup (Table 4)
Based on the maximum minimum and median valuesof micronucleus observed in the three groups a boxplotwas done (Figure 4) The graph indicates statistically equalmedians between positive control and experimental group(E6) Although the medians between these groups had notshown statistical differences the graph indicates that theexperimental group showed maximum values of binucleatedlymphocytes with micronucleus higher than positive control
Frequency analysis of anaphase bridge observed inCBMNA based on the number of anaphase bridges (AB)observed per group (Table 3) Kruskal-Wallis test was per-formed which indicates significant differences among thegroups (119867 = 83444 119901 = 00154) Student-Newman-Keuls post hoc test revealed differences between negative andpositive control as well as negative control and experimentalgroup (Table 5) The test did not show differences betweenpositive control and experimental group
Analysis of cytokinesis-block proliferation index (CBPI)and cytotoxicity Kruskal-Wallis test based on theCBPI indi-cates statistical differences among the groups (119867 = 93968and 119901 = 00091) Dunn post hoc test revealed significantdifferences between negative and positive control as well asnegative control and experimental group (Table 6) Howeverthe test did not show differences between positive control andexperimental groupThese data indicate that E6 recombinantoncoprotein has CBPI similar to the cyclophosphamide
Besides presenting high CBPI the E6 recombinant onco-protein also showed to induce endoreduplication evidencedby the presence of intracellular cytokinesis suggesting neosis(Figure 5) according to the criteria proposed by Das et al[69] The CBMNA results suggest that E6 recombinant
6 BioMed Research International
301bp
M 0504030201 M M 0504030201BPV1Cminus
(a) (b)
(c) (d)
(e) (f)
164bp170bp
M 0504030201BPV2Cminus M 0504030201BPV4Cminus
430bp
600bp
M 0504030201BPV2Cminus M 0504030201BPV4Cminus
Figure 2 Electrophoresis gel images showing (a) genomicDNA integrity afterDNAextraction using 1 KbDNALadder (Invitrogen CarlsbadUSA) as marker absence of amplicon in samples 01 to 05 using specific primers to BPV-1 (b) BPV-2 (c) and BPV-4 (d) absence of ampliconin samples 01 to 05 using Delta-Epsilon (e) and Xi (f) degenerate primers Images (b)ndash(f) showed amplification only in positive control withthe 100 bp DNA Ladder (Invitrogen Carlsbad USA) being employed as marker
Table 6 Dunn post hoc test results based on the cytokinesis-blockproliferation index
Group Rank differences Calculated 119885 Critical 119885 119901
Cminus and C+ 76 26870 2394 lt005Cminus and E6 74 26163 2394 lt005C+ and E6 02 00707 2394 nsns nonsignificant differences
oncoprotein induces mitotic stress resulting in clastogenesisand neosis
33 Comet Assay Alkaline Method Comet assay results areshown in Table 7 and Figure 6 According to these data
theKruskal-Wallis test was performedwhich pointed out sta-tistical differences among the groups (119867 = 102613 and 119901 =00059) Based on these results the Dunn post hoc test wasperformed which pointed out significant differences betweennegative and positive control as well as negative controland experimental group (Table 8) However the test did notshow differences between positive control and experimentalgroup (Table 8) These results were also verified by Figure 7Comet assay reinforces the CBMNA results indicating themutagenic potential of E6 recombinant oncoprotein
34 Cytokinesis-Block Micronucleus Assay (CBMNA) inPBMCs Results of this test confirmed the previous resultsindicating that the BPV-1 E6 recombinant oncoprotein has
BioMed Research International 7
LB
(a)
LMN
(b)
LMN
LMN
(c)
PA
PA and MN
(d)
Figure 3 (a) Photomicroscopy of binucleated lymphocyte without micronucleus (LB) observed in negative control Image of binucleatedlymphocyte with micronucleus (LMN) observed in positive control (b) and group treated with E6 recombinant oncoprotein (c) Image ofbinucleated lymphocyte with anaphase bridge and micronucleus (PA and MN) observed in group treated with E6 (d) Images obtained withtotal magnification of 1000x
60
50
40
30
20
10
0
Num
ber o
f mic
ronu
clei
GroupCminus C+ E6
Figure 4 Comparative boxplot based on maximum minimumand median values of micronucleus observed per group
a genotoxicity potential being able to induce DNAbreaks in epithelial cells verified by the presence of MNs(Figure 8)
Figure 5 Evidence of neosis Photomicroscopy of lymphocyteshowing endoreduplication (black arrow) suggesting neosis Cellsanalyzed in total magnification of 1000x
4 Discussion
Transforming potential of E6 oncoprotein has been discussedsince the 1980s based on studies of cottontail rabbit papil-lomaviruses (CRPV) [70 71] Although there are lines ofevidence that the E6 oncoprotein can induce transformation
8 BioMed Research International
(a) (b)
(c) (d)
Figure 6 Images of cometsrsquo nucleoids (a) Image of class 0 (without DNA damage) observed in negative control group Images of class 2(maximum DNA damage) observed in positive control (b) and experimental (c) groups (d) Image of hedgehog comet characterized by thehead absence observed in experimental group Images captured in total magnification of 400x
200
150
100
50
0
Com
et sc
ore
GroupsCminus C+ E6
Figure 7 Boxplot of comet score
[72] the mechanisms of initiation and promotion of cancerassociated with the BPV E6 oncoprotein are unknown
Molecular diagnosis of five peripheral blood samplesfrom five calves showed the absence of amplicons for thefive different primers used (Figure 2) However both specific(BPV-1 BPV-2 and BPV-4) and degenerate (Delta-Epsilonand Xi) primers sets amplified the controls genomes of BPV-1 BPV-2 and BPV-4 None of the negative controls revealedthe amplicon presence These results indicate that the fivesampleswere uninfected by BPVMoreover the use of specific
and degenerate primers increases the capacity of BPV iden-tification This occurs because specific primers are moresensitive than degenerates [66] This sensitivity is due tothe absence of degeneration in the 51015840 region which reducesthe primer ability to recognize and to link with DNA targetsequence [73]The absence of BPV infection in the peripheralblood samples is required for the CBMNA and CA allowinginvestigating the aneugenic andor clastogenic action of BPV-1 E6 recombinant oncoprotein This is because the presenceof BPV sequences is associated with the presence of oncopro-teins transcripts [74]Thus studies have shown that the pres-ence of BPVDNA sequences in peripheral blood is associatedwith cytogenetic damage including clastogenesis [1 14 75]Complementary studies show the association of the BPVpresence with cytogenetic damage in both benign lesions(papillomas) [74] and carcinomas [15]
Chi-square test based on the number of micronucleatedcells pointed out significant statistical values in both positivecontrol and group treated with BPV-1 E6 recombinant onco-protein (Table 3) Negative control showed fewer number ofmicronucleated cells This result was expected because thebiological material was transported and processed after threehours of its collection This procedure reduced the influenceof exogenous environmental factors which could induceDNA damage Furthermore the absence of BPV infection
BioMed Research International 9
(a) (b)
8
6
4
2
0
Num
ber o
f mic
ronu
cleat
ed ce
lls
Neg
ativ
e con
trol
Posit
ive c
ontro
l
E6on
copr
otei
n
(c)
Figure 8 Photomicroscope showing CRIB cell not treated with any drug (negative control) indicating absence of micronucleus (a) andtreated with BPV-1 E6 recombinant oncoprotein showing the presence of MN indicated by arrow (b) (c) Number of micronucleated cellsobserved in each group
in the peripheral blood reduces the influence of endogenousenvironmental factors that could interfere in the analysisPositive control showed a high number of micronucle-ated lymphocytes This result was also expected once thisgroup was treated with 50 120583gmL of cyclophosphamide achemotherapeutic drug with cytotoxic and teratogenic effect[76 77] The cells treated with 1120583gmL of BPV-1 E6 recom-binant oncoprotein also showed significant statistical valuesKruskal-Wallis test followed by the Dunn post hoc testwas performed to compare the frequencies of micronucleusamong three groups This test indicated statistical differencebetween negative control and the cells treated with BPV-1 E6 recombinant oncoprotein However the test did notreveal differences between the cells treated with cyclophos-phamide and BPV-1 E6 recombinant oncoprotein (Table 4Figure 4) Similar results were also observed in epithelial cells(Figure 8) These results suggest that the BPV-1 E6 oncopro-tein has an aneugenic andor clastogenic potential similarto or higher than the observed with the cyclophosphamideThis is because the cells treated with the oncoprotein showed
a frequency of micronucleus formation (MN1199030) 14003
higher than those observed in positive controlThe elevated mutagenic potential of the BPV-1 E6 onco-
protein can justify the cytogenetic damage already describedin the literature [1 14 15 24] Similar results were observedin HPV-infected cells [12 78ndash81] The micronucleus induce-ment was already described in cytological samples collectedduring Papanicolaursquos test from healthy cervix infected byHPV [12] For this reason the micronucleus assay has beenproposed as a complementary method for Papanicolaursquos testbeing a suggestive biomarker of lesion degree [78] Howeverthe association between the BPV and the micronucleusinducement was not yet reported
The high frequency of micronucleated lymphocytes insamples infected by HPV has been attributed to the synergicaction of E5 E6 and E7 oncoproteins [29 30 78 79]However this is the first study that pointed out themutagenicaction of BPV-1 E6 protein per se
Student-Newman-Keuls post hoc test based on the fre-quency of anaphase bridges showed significant statistical
10 BioMed Research International
Table 7 Comet assay results showing the number of nucleoidsobserved per class the number of hedgehog comets and cometscore
Sample Class 0 Class 1 Class 2 Hedgehog ScoreNegative control
Groups Rank differences Calculated 119885 Critical 119885 119901
Cminus and C+ 68 24042 2394 lt05Cminus and E6 82 28991 2394 lt05C+ and E6 14 04950 2394 nsns nonsignificant differences
difference between the negative control and the group treatedwith the BPV-1 E6 recombinant oncoprotein (Table 5) How-ever the test did not reveal differences between the cellstreated with cyclophosphamide and BPV-1 E6 recombinantoncoprotein (Table 5)These data reinforce those observed bythe Dunn post hoc test based on the frequency of micronu-cleus indicating the clastogenic action of the E6 oncoproteinMoreover the presence of anaphase bridge was alreadydescribed in cells transfected with recombinant adenoviruscontaining the E6 and E7 ORFs of HPV [29 30] Howeverthere are no studies describing the action of BPVE6 oncopro-tein in the induction of anaphase bridges or studies that showthis same action of E6 oncoprotein of HPV per se Anaphasebridges are indicators of genomic instability [82 83] which isconsidered the first step in carcinogenesis [46 47] Moreoverthe presence of these bridges is an important hallmark ofDNA double strand breaks (DSBs) [84]
Statistical analysis based on the cytokinesis-block prolif-eration index (CBPI) showed that BPV-1 E6 oncoprotein hascytotoxic levels similar to cyclophosphamide (Table 6) Thisresult suggests that the BPV E6 oncoprotein can deregulatethe cell cycle contributing to the cell proliferation andimmortalization To date this mitogenic action is attributedto the E7 oncoprotein [85ndash87] E7 oncoprotein induces
the phosphorylation of pRb resulting in the E2F transcrip-tion factor release [85] E2F when translocated to the nucleusacts as activator and binds to the kinase-dependent cyclinspromoters [85] However the results showed polynucleatedcells in the group treatedwithBPVE6oncoprotein (Figure 5)suggesting that the BPV E6 oncoprotein confers mitogenicstimulation resulting in mitotic stress Moreover studiespointed out that the BPV E6 oncoprotein interacts with theCBPp300 deacetylase promoting p53 downregulation [88]This downregulation increases the expression levels of FoxM1transcription factor (Foxhead box M1) [89] promoting B1cyclin D1 cyclin and cdc25 upregulation [89] The upregu-lation of these genes is associated with increased levels of cellproliferation which is necessary in order to make DNA poly-merases available to virus replication However if this mech-anism guarantees the BPV replication it can contribute to cellimmortalization and cancer progression
The FoxM1 factor also participates in the Wnt120573-cateninsignaling pathway and binds directly to the 120573-catenin [89]This interaction promotes the nuclear 120573-catenin transloca-tion [90] 120573-catenin translocation to nucleus induces thecyclin expression [64] So these data suggest that the BPVE6 oncoprotein not only promotes mitogenic stress but alsocontributes to the epithelial-mesenchymal transition (EMT)Studies based on theHPVE6 oncoprotein have indicated thatthis oncoprotein contributes significantly to the EMT [91]This occurs not only because the E6 oncoprotein induces thetranslocation of 120573-catenin but also due to the proteasomaldegradation of regulatory proteins of apical-basal polarity[92ndash94] Studies also showed that the E6 oncoprotein is ableto bind to Crumbs (Dlg and Patj) proteins resulting in loss ofcell polarity [93 94] Based on these results the E6 oncopro-tein emerges as a possible therapeutic target with biotechno-logical value in cancer treatment
Mitogenic action of E6 oncoprotein can be moreexpressed in cells infected by papillomaviruses once thesecells also expressed E5 and E7 oncoproteins [74] The E7oncoprotein is able to form a complex E7-p600 which pro-motes E6 upregulation [85] Furthermore the E6 oncoproteincan bind to the E6AP ubiquitin ligase forming the E6-E6APcomplex increasing the hTERT levels of expression con-tributing to cell immortalization [95] Moreover E6-E6APinduces NFX1 expression promoting MHC-II downregula-tion [96]Thismechanism reduces the antigenic presentationmediated by CD4+ T lymphocytes contributing to immuneevasion This action guarantees the PV infection for longperiods with or without clinical symptoms [24] Althoughthe BPV infection can be asymptomatic the BPV presence inperipheral blood is associated with DNA damage [1 14 24]
CBMNA analysis pointed out the presence of cells withintracellular cytokinesis (Figure 5) This result suggests theneotic action of BPV E6 oncoprotein Neosis is characterizedby the presence of (1) DNA damage (2) loss of checkpointcontrol (3) repair system failures and (4) endoreduplication[97] In this scenario the E6 oncoprotein attempts all theseneosis criteria Moreover the oncoprotein not only increasesthe cytogenetic damage leading to increasing of micronu-cleus frequency but also induces clastogenesis as shown bythe comet assay and anaphase bridges So statistical analysis
BioMed Research International 11
performed based on the comet scores reinforced the CBMNAresults Comet assay allows detecting both simple and dou-ble DNA breaks being more sensitive than the CBMNAClastogenesis is considered the most serious type of DNAdamage [98] DNA breaks can be repaired by homologous ornonhomologous recombination However studies point outthat the E6 oncoprotein can link to the Holliday junctionduring the homologous repair avoiding the junction reso-lution [99] Moreover E6 oncoprotein promotes the TP53gene deacetylation resulting in p53 downregulation [51]Thisepigenetic effect results in cell cycle deregulation affectingthe checkpoints
Studies have shown that the HPV-induced DNA breaksare required for the virus integration into the host genome[100] Although there are no studies showing the BPV inte-gration to date the results observed in this work in additionto those already published [24] indicate the necessity ofmorestudies to evaluate the virus-host interaction
Studies also showed that the E6 oncoprotein inducesclathrin-transporter adapter protein (AP-1) increasing thelevel of this protein in the plasma membrane [76 77] Theincreasing quantity of AP-1 in membrane can facilitate theBPV virions infection This occurs because the infectionprocess is clathrin dependent In this scenario the E6 onco-protein can contribute to the virus infection [101]
In summary the CBMNA and CA results showed thatthe BPV E6 oncoprotein participates not only in cancerpromotion but also in initiation inducing DNA breaksThese DNA breaks represent mutagenic effects consideredthe first step in the oncogenesis process which are associatedwith genomic instability [47 48 102 103] Thus the E6recombinant oncoprotein has been suggested as a possiblevaccine candidate [104ndash112] due to its immunogenicity [44]However the mutagenic tests such as CBMNA and CA arerequired in drug validation process [113] In this scenariothis study pointed out that the BPV-1 E6 recombinant onco-protein in tested concentration showedmutagenic potentialThis is the first study that reports the mutagenic potential ofa possible therapeutic vaccine candidate On the one handthese results allowed better understanding the mechanism ofcancer promotion associatedwith the BPVE6oncoprotein aswell as revealing that this oncoprotein can induce carcinogen-esis per se on the other hand this data pointed out thatmaybeBPV E6 recombinant oncoprotein requires protein modifica-tions to be used as vaccine
Conflict of Interests
The authors declare that there is no conflict of interests
Acknowledgments
Theauthors thank Fundacao deAmparo a Pesquisa do Estadode Sao Paulo (FAPESP Process 201420617-5) and FundacaoButantan for the financial support
References
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experimental modelrdquo Journal of General Virology vol 79 no 9pp 2127ndash2135 1998
[2] A Antonsson and B G Hansson ldquoHealthy skin ofmany animalspecies harbors papillomaviruses which are closely related totheir human counterpartsrdquo Journal of Virology vol 76 no 24pp 12537ndash12542 2002
[3] P Rous and J W Beard ldquoA virus-induced mammalian growthwith the characters of a tumor (the Shope rabbit papilloma) IThe growth on implantationwithin favorable hostsrdquoThe Journalof Experimental Medicine vol 60 no 6 pp 701ndash722 1934
[4] R Shope and E Hurst ldquoInfectious papillomatosis of rabbitsrdquoThe Journal of Experimental Medicine vol 58 no 1 pp 607ndash624 1933
[5] H Delius M A Van Ranst A B Jenson H Zur Hausen and JP Sundberg ldquoCanine oral papillomavirus genomic sequence aunique 15-kb intervening sequence between the E2 andL2 openreading framesrdquo Virology vol 204 no 1 pp 447ndash452 1994
[6] W A Demonbreun and E Goodpasture ldquoInfectious oral papil-lomatosis of dogsrdquo The American Journal of Pathology vol 8no 1 pp 43ndash56 1932
[7] P K Nicholls B A Klaunberg R A Moore et al ldquoNaturallyoccurring nonregressing canine oral papillomavirus infectionhost immunity virus characterization and experimental infec-tionrdquo Virology vol 265 no 2 pp 365ndash374 1999
[8] A Alberti S Pirino F Pintore et al ldquoOvis aries Papillomavirus3 a prototype of a novel genus in the family Papillomaviridaeassociated with ovine squamous cell carcinomardquo Virology vol407 no 2 pp 352ndash359 2010
[9] H zur Hausen E-M de Villiers and L Gissmann ldquoPapil-lomavirus infections and human genital cancerrdquo GynecologicOncology vol 12 no 2 pp S124ndashS128 1981
[10] C Foresta A Bertoldo A Garolla et al ldquoHuman papillo-mavirus proteins are found in peripheral blood and semenCd20+ and Cd56+ cells during Hpv-16 semen infectionrdquo BMCInfectious Diseases vol 13 article 593 2013
[11] S Ljubojevic andM Skerlev ldquoHPV-associated diseasesrdquo Clinicsin Dermatology vol 32 no 2 pp 227ndash234 2014
[12] A P R Cassel R B Barcellos C M D da Silva S E de MatosAlmeida and M L R Rossetti ldquoAssociation between humanpapillomavirus (HPV) DNA and micronuclei in normal cervi-cal cytologyrdquo Genetics and Molecular Biology vol 37 no 2 pp360ndash363 2014
[13] W Jarrett M Campo B OrsquoNeil H Laird and L Coggins ldquoAnovel bovine papillomavirus (BPV-6) causing true epithelialpapillomas of the mammary gland skin a member of a pro-posed newBPV subgrouprdquoVirology vol 136 no 2 pp 255ndash2641984
[14] T CMelo N Diniz S R C Campos et al ldquoCytogenetic studiesin peripheral blood of bovines afflicted by papillomatosisrdquoVeterinary and Comparative Oncology vol 9 no 4 pp 269ndash2742011
[15] S R C Campos T C Melo S Assaf et al ldquoChromosome aber-rations in cells infected with bovine papillomavirus comparingcutaneous papilloma esophagus papilloma and urinary blad-der lesion cellsrdquo ISRN Oncology vol 2013 Article ID 910849 8pages 2013
[16] H zur Hausen ldquoThe search for infectious causes of humancancers where and whyrdquoVirology vol 392 no 1 pp 1ndash10 2009
[17] R P Araldi R F Carvalho T C Melo et al ldquoBovine papillo-mavirus in beef cattle first description of BPV-12 and putativetype BAPV8 in BrazilrdquoGenetics andMolecular Research vol 13no 3 pp 5644ndash5653 2014
12 BioMed Research International
[18] T Rampias C Sasaki and A Psyrri ldquoMolecular mechanisms ofHPV induced carcinogenesis in head and neckrdquoOral Oncologyvol 50 no 5 pp 356ndash363 2014
[19] S Roperto V Russo L Leonardi et al ldquoBovine papillomavirustype 13 expression in the urothelial bladder tumours of cattlerdquoTransboundary and Emerging Diseases 2015
[20] S RCCampos C TrindadeO P Ferraz et al ldquoCan establishedcultured papilloma cells harbor bovine papillomavirusrdquoGenet-ics and Molecular Research vol 7 no 4 pp 1119ndash1126 2008
[21] M S Campo ldquoAnimalmodels of papilloma virus pathogenesisrdquoVirus Research vol 89 no 2 pp 249ndash261 2002
[22] G Borzacchiello ldquoBovine papillomavirus infections in ani-malsrdquo in Communicating Current Research and EducationalTopics and Trends in AppliedMicrobiology pp 673ndash679 Forma-tex Badajoz Spain 2007
[23] F Bocaneti G Altamura A Corteggio E Velescu F RopertoandG Borzacchiello ldquoBovine papillomavirus new insights intoan old diseaserdquo Transboundary and Emerging Diseases 2014
[24] R P Araldi T C Melo N Diniz et al ldquoBovine papillomavirusclastogenic effect analyzed in comet assayrdquo BioMed ResearchInternational vol 2013 Article ID 630683 7 pages 2013
[25] J Munday N Thomson M Dunowska C Knight R Laurieand S Hills ldquoGenomic characterisation of the feline sarcoid-associated papillomavirus and proposed classification as Bostaurus papillomavirus type 14rdquoVeterinaryMicrobiology vol 177no 3-4 pp 289ndash295 2015
[26] G Borzacchiello V Russo C Spoleto et al ldquoBovine papillo-mavirus type-2 DNA and expression of E5 and E7 oncoproteinsin vascular tumours of the urinary bladder in cattlerdquo CancerLetters vol 250 no 1 pp 82ndash91 2007
[27] P Maiolino A Ozkul A Sepici-Dincel et al ldquoBovine papillo-mavirus type 2 infection and microscopic patterns of urothelialtumors of the urinary bladder in water buffaloesrdquo BioMedResearch International vol 2013 Article ID 937918 6 pages2013
[28] S Roperto G Borzacchiello R Brun et al ldquoMultiple glomustumors of the urinary bladder in a cow associated with bovinepapillomavirus type 2 (BPV-2) infectionrdquo Veterinary Pathologyvol 45 no 1 pp 39ndash42 2008
[29] S Duensing and K Munger ldquoThe human papillomavirus type16 E6 and E7 oncoproteins independently induce numerical andstructural chromosome instabilityrdquoCancer Research vol 62 no23 pp 7075ndash7082 2002
[30] S Duensing L Y Lee A Duensing et al ldquoThe human papil-lomavirus type 16 E6 and E7 oncoproteins cooperate to inducemitotic defects and genomic instability by uncoupling centro-some duplication from the cell division cyclerdquo Proceedings of theNational Academy of Sciences of theUnited States of America vol97 no 18 pp 10002ndash10007 2000
[31] C A Moody and L A Laimins ldquoHuman papillomavirus onco-proteins pathways to transformationrdquo Nature Reviews Cancervol 10 no 8 pp 550ndash560 2010
[32] C Mansur and E Androphy ldquoCellular transformation bypapillomavirus oncoproteinsrdquo Biochimica et Biophysica Actavol 1155 no 3 pp 323ndash345 1993
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[34] X Tong andPMHowley ldquoThebovine papillomavirus E6 onco-protein interacts with paxillin and disrupts the actin cytoskele-tonrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 94 no 9 pp 4412ndash4417 1997
[35] X TongW Boll T Kirchhausen andPMHowley ldquoInteractionof the bovine papillomavirus E6 protein with the clathrinadaptor complex AP-1rdquo Journal of Virology vol 72 no 1 pp476ndash482 1998
[36] Y Nomine M Masson S Charbonnier et al ldquoStructural andfunctional analysis of E6 oncoprotein insights in the molecularpathways of human papillomavirus-mediated pathogenesisrdquoMolecular Cell vol 21 no 5 pp 665ndash678 2006
[37] T Ristriani MMasson Y Nomine et al ldquoHPV oncoprotein E6is a structure-dependent DNA-binding protein that recognizesfour-way junctionsrdquo Journal of Molecular Biology vol 296 no5 pp 1189ndash1203 2000
[38] Y Liu and J D Baleja ldquoStructure and function of the papil-lomavirus E6 protein and its interacting proteinsrdquo Frontiers inBioscience vol 13 no 1 pp 121ndash134 2008
[39] F Lipari G A McGibbon E Wardrop and M G CordingleyldquoPurification and biophysical characterization of a minimalfunctional domain and of an N-terminal Zn2+-binding frag-ment from the human papillomavirus type 16 E6 proteinrdquoBiochemistry vol 40 no 5 pp 1196ndash1204 2001
[40] Y Nomine T Ristriani C Laurent J-F Lefevre E Weissand G Trave ldquoA strategy for optimizing the monodispersity offusion proteins application to purification of recombinantHPVE6 oncoproteinrdquo Protein Engineering vol 14 no 4 pp 297ndash3052001
[41] C G Ullman P I Haris D A Galloway V C Emery and SJ Perkins ldquoPredicted 120572-helix120573-sheet secondary structures forthe zinc-binding motifs of human papillomavirus E7 and E6proteins by consensus prediction averaging and spectroscopicstudies of E7rdquo Biochemical Journal vol 319 no 1 pp 229ndash2391996
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[43] K Zanier S Charbonnier A O M O Sidi et al ldquoStructuralbasis for hijacking of cellular LxxLL motifs by papillomavirusE6 oncoproteinsrdquo Science vol 339 no 6120 pp 694ndash698 2013
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[49] K Zanier S Charbonnier M Baltzinger Y Nomine DAltschuh and G Trave ldquoKinetic analysis of the interactionsof human papillomavirus E6 oncoproteins with the ubiquitinligase E6AP using surface plasmon resonancerdquo Journal ofMolecular Biology vol 349 no 2 pp 401ndash412 2005
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[54] P Thomas and M Fenech ldquoCytokinesis-block micronucleuscytome assay in lymphocytesrdquo Methods in Molecular Biologyvol 682 pp 217ndash234 2011
[55] M Kirsch-Volders T Sofuni M Aardema et al ldquoReport fromthe in vitro micronucleus assay working grouprdquo MutationResearch Genetic Toxicology and Environmental Mutagenesisvol 540 no 2 pp 153ndash163 2003
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[58] A Azqueta and A R Collins ldquoThe essential comet assay acomprehensive guide to measuring DNA damage and repairrdquoArchives of Toxicology vol 87 no 6 pp 949ndash968 2013
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[62] D M Potter and M S Baird ldquoCarcinogenic effects of ptaquilo-side in bracken fern and related compoundsrdquo British Journal ofCancer vol 83 no 7 pp 914ndash920 2000
[63] R B Lucena D R Rissi G D Kommers et al ldquoA retrospectivestudy of 586 tumours in Brazilian cattlerdquo Journal of ComparativePathology vol 145 no 1 pp 20ndash24 2011
[64] A Yaguiu M L Z Dagli E H Birgel Jr et al ldquoSimultaneouspresence of bovine papillomavirus and bovine leukemia virusin different bovine tissues in situ hybridization and cytogeneticanalysisrdquoGenetics andMolecular Research vol 7 no 2 pp 487ndash497 2008
[65] T C Melo R F Carvalho J Mazzucchelli-de-Souza et alldquoPhylogenetic classification and clinical aspects of a new puta-tive Deltapapillomavirus associated with skin lesions in cattlerdquoGenetics and Molecular Research vol 13 no 2 pp 2458ndash24692014
[66] R P Araldi D N S Giovanni T CMelo et al ldquoBovine papillo-mavirus isolation by ultracentrifugationrdquo Journal of VirologicalMethods vol 208 pp 119ndash124 2014
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[70] O Danos E Georges G Orth and M Yaniv ldquoFine structureof the cottontail rabbit papillomavirus mRNAs expressed in thetransplantable VX2 carcinomardquo Journal of Virology vol 53 no3 pp 735ndash741 1985
[71] M Nasseri and F O Wettstein ldquoDifferences exist between viraltranscripts in cottontail rabbit papillomavirus-induced benignand malignant tumors as well as non-virus-producing andvirus-producing tumorsrdquo Journal of Virology vol 51 no 3 pp706ndash712 1984
[72] X Tong R Salgia J-L Li J D Griffin and P M Howley ldquoThebovine papillomavirus E6 protein binds to the LDmotif repeatsof paxillin and blocks its interaction with vinculin and the focaladhesion kinaserdquo Journal of Biological Chemistry vol 272 no52 pp 33373ndash33376 1997
[73] Y Maeda T Shibahara Y Wada et al ldquoAn outbreak of teatpapillomatosis in cattle caused by bovine papilloma virus (BPV)type 6 and unclassified BPVsrdquo Veterinary Microbiology vol 121no 3-4 pp 242ndash248 2007
[74] R P Araldi T C Melo A Neves et al ldquoHyperproliferativeaction of bovine papillomavirus genetic and histopathologicalaspectsrdquo Genetics and Molecular Research vol 14 no 4 pp12942ndash12954 2015
[75] NDiniz T CMelo J F Santos et al ldquoSimultaneous presence ofbovine papillomavirus in blood and in short-term lymphocytecultures from dairy cattle in Pernambuco Brazilrdquo Genetics andMolecular Research vol 8 no 4 pp 1474ndash1480 2009
[76] O M Brito M F Guimaraes and C C Lanna ldquoCiclofosfamidae funcao ovarianardquo Revista Brasileira de Reumatologia vol 48no 1 pp 39ndash45 2008
[77] D Anderson T-W Yu and D B McGregor ldquoComet assayresponses as indicators of carcinogen exposurerdquo Mutagenesisvol 13 no 6 pp 539ndash555 1998
[78] M L Adam C Pini S Tulio J C L L Cantalice R A Torresand M T Dos Santos Correia ldquoAssessment of the associationbetween micronuclei and the degree of uterine lesions and viralload in women with human papillomavirusrdquo Cancer Genomicsamp Proteomics vol 12 no 2 pp 67ndash72 2015
[79] E Cortes-Gutierrez M Davila-Rodrıguez J Vargas-VillarrealF Hernandez-Garza and R Cerda-Flores ldquoAssociationbetween human papilloma virus-type infections with micro-nuclei frequenciesrdquo Prague Medical Report vol 111 no 1 pp35ndash41 2010
[80] R N Chakrabarti and K Dutta ldquoMicronuclei test in routinesmears fromuterine cervixrdquo European Journal of GynaecologicalOncology vol 9 no 5 pp 370ndash372 1988
[81] E M M Cerqueira C L Santoro N F Donozo et al ldquoGeneticdamage in exfoliated cells of the uterine cervix Association
14 BioMed Research International
and interaction between cigarette smoking and progression tomalignant transformationrdquo Acta Cytologica vol 42 no 3 pp639ndash649 1998
[82] S M Germann V Schramke R T Pedersen et al ldquoTopBP1Dpb11 bindsDNA anaphase bridges to prevent genome instabil-ityrdquoThe Journal of Cell Biology vol 204 no 1 pp 45ndash59 2014
[83] D R Hoffelder L Luo N A Burke S CWatkins S M Gollinand W S Saunders ldquoResolution of anaphase bridges in cancercellsrdquo Chromosoma vol 112 no 8 pp 389ndash397 2004
[84] C Acilan D M Potter and W S Saunders ldquoDNA repairpathways involved in anaphase bridge formationrdquo Genes Chro-mosomes and Cancer vol 46 no 6 pp 522ndash531 2007
[85] J DeMasi M C Chao A S Kumar and P M Howley ldquoBovinepapillomavirus E7 oncoprotein inhibits anoikisrdquo Journal ofVirology vol 81 no 17 pp 9419ndash9425 2007
[86] J DeMasi K-W Huh Y Nakatani K Munger and P MHowley ldquoBovine papillomavirus E7 transformation functioncorrelates with cellular p600 protein bindingrdquo Proceedings of theNational Academy of Sciences of theUnited States of America vol102 no 32 pp 11486ndash11491 2005
[87] E A White and P M Howley ldquoProteomic approaches to thestudy of papillomavirusndashhost interactionsrdquo Virology vol 435no 1 pp 57ndash69 2013
[88] FMantovani and L Banks ldquoThe human papillomavirus E6 pro-tein and its contribution to malignant progressionrdquo Oncogenevol 20 no 54 pp 7874ndash7887 2001
[89] P Chen and H Lee ldquoFOXM1 induced by E6 oncoprotein pro-motes tumor invasion and chemoresistance in HPV-infectedlung cancerrdquo Cancer Cell amp Microenvironment vol 2 no 1 pp6ndash9 2015
[90] N Zhang P Wei A Gong et al ldquoFoxM1 promotes 120573-cateninnuclear localization and controls Wnt target-gene expressionand glioma tumorigenesisrdquo Cancer Cell vol 127 no 4 pp 358ndash366 2012
[91] Z Liu Y Liu Y Hong L Rapp E J Androphy and J JChen ldquoBovine papillomavirus type 1 E6-induced sensitizationto apoptosis is distinct from its transforming activityrdquo Virologyvol 295 no 2 pp 230ndash237 2002
[92] M Thomas P Massimi C Navarro J-P Borg and L BanksldquoThe hScribDlg apico-basal control complex is differentiallytargeted by HPV-16 and HPV-18 E6 proteinsrdquo Oncogene vol24 no 41 pp 6222ndash6230 2005
[93] C H Storrs and S J Silverstein ldquoPATJ a tight junction-associated PDZ protein is a novel degradation target of high-risk human papillomavirus E6 and the alternatively splicedisoform 18 E6rdquo Journal of Virology vol 81 no 8 pp 4080ndash40902007
[94] R Accardi R Rubino M Scalise et al ldquoE6 and E7 from humanpapillomavirus type 16 cooperate to target the PDZ proteinNaH exchange regulatory factor 1rdquo Journal of Virology vol 85no 16 pp 8208ndash8216 2011
[95] S Nakagawa and J M Huibregtse ldquoHuman scribble (Vartul)is targeted for ubiquitin-mediated degradation by the high-risk papillomavirus E6 proteins and the E6APubiquitin-proteinligaserdquoMolecular and Cellular Biology vol 20 no 21 pp 8244ndash8253 2000
[96] L Gewin H Myers T Kiyono and D A Galloway ldquoIdentifi-cation of a novel telomerase repressor that interacts with thehuman papillomavirus type-16 E6E6-AP complexrdquo Genes ampDevelopment vol 18 no 18 pp 2269ndash2282 2004
[97] M Sundaram D L GuernseyMM Rajaraman and R Rajara-man ldquoNeosis a novel type of cell division in cancerrdquo CancerBiology ampTherapy vol 3 no 2 pp 207ndash218 2004
[98] R D Snyder and J W Green ldquoA review of the genotoxicity ofmarketed pharmaceuticalsrdquo Mutation Research vol 488 no 2pp 151ndash169 2001
[99] T Ristriani Y Nomine C Laurent E Weiss and G TraveldquoProtein mutagenesis with monodispersity-based quality prob-ing selective inactivation of p53 degradation and DNA-bindingproperties of HPV E6 oncoproteinrdquo Protein Expression andPurification vol 26 no 3 pp 357ndash367 2002
[100] M Kadaja A Sumerina T Verst M Ojarand E Ustav and MUstav ldquoGenomic instability of the host cell induced by thehuman papillomavirus replicationmachineryrdquoTheEMBO Jour-nal vol 26 no 8 pp 2180ndash2191 2007
[101] PMDay D R Lowy and J T Schiller ldquoPapillomaviruses infectcells via a clathrin-dependent pathwayrdquo Virology vol 307 no 1pp 1ndash11 2003
[102] T D Tlsty A Briot A Gualberto et al ldquoGenomic instabilityand cancerrdquoMutation Research vol 337 no 1 pp 1ndash7 1995
[103] J E Eyfjord and S K Bodvarsdottir ldquoGenomic instabilityand cancer networks involved in response to DNA damagerdquoMutation Research vol 592 no 1-2 pp 18ndash28 2005
[104] Y Yao W Huang X Yang et al ldquoHPV-16 E6 and E7 protein Tcell epitopes prediction analysis based on distributions of HLA-A loci across populations an in silico approachrdquo Vaccine vol31 no 18 pp 2289ndash2294 2013
[105] C-F Hung B Ma A Monie S-W Tsen and T-C Wu ldquoTher-apeutic human papillomavirus vaccines current clinical trialsand future directionsrdquo Expert Opinion on Biological Therapyvol 8 no 4 pp 421ndash439 2008
[106] M J P Welters G G Kenter S J Piersma et al ldquoInductionof tumor-specific CD4+ and CD8+ T-cell immunity in cervicalcancer patients by a human papillomavirus type 16 E6 and E7long peptides vaccinerdquo Clinical Cancer Research vol 14 no 1pp 178ndash187 2008
[107] R Han N M Cladel C A Reed X Peng and N D Chris-tensen ldquoProtection of rabbits from viral challenge by gene gun-based intracutaneous vaccination with a combination of cot-tontail rabbit papillomavirus E1 E2 E6 and E7 genesrdquo Journalof Virology vol 73 no 8 pp 7039ndash7043 1999
[108] Z He A P Wlazlo D W Kowalczyk et al ldquoViral recombinantvaccines to the E6 and E7 antigens of HPV-16rdquo Virology vol270 no 1 pp 146ndash161 2000
[109] A Kaufmann P Stern E Rankin et al ldquoSafety and immuno-genicity of TA-HPV a recombinant vaccinia virus expressingmodified human papillomavirus (HPV)-16 and HPV-18 E6 andE7 genes in women with progressive cervical cancerrdquo ClinicalCancer Research vol 8 no 12 pp 3676ndash3685 2002
[110] L K Borysiewicz A Fiander M Nimako et al ldquoA recombinantvaccinia virus encoding human papillomavirus types 16 and 18E6 and E7 proteins as immunotherapy for cervical cancerrdquoTheLancet vol 347 no 9014 pp 1523ndash1527 1996
[111] A De Jong T OrsquoNeill A Y Khan et al ldquoEnhancement ofhuman papillomavirus (HPV) type 16 E6 and E7-specific T-cellimmunity in healthy volunteers through vaccination with TA-CIN an HPV16 L2E7E6 fusion protein vaccinerdquo Vaccine vol20 no 29-30 pp 3456ndash3464 2002
[112] S Chen C Liao Y Lai et al ldquoDe-oncogenicHPVE6E7 vaccinegets enhanced antigenicity and promotes tumoricidal synergywith cisplatinrdquo Acta Biochimica et Biophysica Sinica vol 46 no1 pp 6ndash14 2014
BioMed Research International 15
[113] J Bonhoeffer E B Imoukhuede G Aldrovandi et al ldquoTemplateprotocol for clinical trials investigating vaccines-focus on safetyelementsrdquo Vaccine vol 31 no 47 pp 5602ndash5620 2013
[114] S Jackson and A Storey ldquoE6 proteins from diverse cutaneonsHPV types inhibit apoptosis in response to UV damagerdquoOncogene vol 19 no 4 pp 592ndash598 2000
[115] M Thomas and L Banks ldquoPDZRN3LNX3 is a novel targetof human papillomavirus type 16 (HPV-16) and HPV-18 E6rdquoJournal of Virology vol 89 no 2 pp 1439ndash1444 2015
[116] P R Daniels C M Sanders P Coulson and N J MaitlandldquoMolecular analysis of the interaction between HPV type 16 E6and human E6-associated proteinrdquo FEBS Letters vol 416 no 1pp 6ndash10 1997
[117] W H Kao S L Beaudenon A L Talis J M Huibregtse andP M Howley ldquoHuman papillomavirus type 16 E6 induces self-ubiquitination of the E6AP ubiquitin-protein ligaserdquo Journal ofVirology vol 74 no 14 pp 6408ndash6417 2000
[118] T Iftner M Elbel B Schopp et al ldquoInterference of papillo-mavirus E6 protein with single-strand break repair by interac-tion with XRCC1rdquo The EMBO Journal vol 21 no 17 pp 4741ndash4748 2002
[119] Y Imai Y Tsunokawa T Sugimura and M Terada ldquoPurifi-cation and DNA-binding properties of human papillomavirustype 16 E6 protein expressed in Escherichia colirdquo Biochemicaland Biophysical Research Communications vol 164 no 3 pp1402ndash1410 1989
[120] M Lusky L Berg H Weiher and M Botchan ldquoBovinepapilloma virus contains an activator of gene expression at thedistal end of the early transcription unitrdquoMolecular andCellularBiology vol 3 no 6 pp 1108ndash1122 1983
[121] S Dalal Q Gao E J Androphy and V Band ldquoMutationalanalysis of human papillomavirus type 16 E6 demonstrates thatp53 degradation is necessary for immortalization of mammaryepithelial cellsrdquo Journal of Virology vol 70 no 2 pp 683ndash6881996
[122] J M Huibregtse M Scheffner and P M Howley ldquoA cellularprotein mediates association of p53 with the E6 oncoprotein ofhuman papillomavirus types 16 or 18rdquo The EMBO Journal vol10 no 13 pp 4129ndash4135 1991
[123] M Scheffner B A Werness J M Huibregtse A J Levine andP M Howley ldquoThe E6 oncoprotein encoded by human papillo-mavirus types 16 and 18 promotes the degradation of p53rdquo Cellvol 63 no 6 pp 1129ndash1136 1990
[124] M Elbel S Carl S Spaderna and T Iftner ldquoA comparativeanalysis of the interactions of the E6 proteins from cutaneousand genital papillomaviruses with p53 and E6AP in correlationto their transforming potentialrdquoVirology vol 239 no 1 pp 132ndash149 1997
[125] M C Thomas and C-M Chiang ldquoE6 oncoprotein repressesp53-dependent gene activation via inhibition of protein acety-lation independently of inducing p53 degradationrdquo MolecularCell vol 17 no 2 pp 251ndash264 2005
[126] H Zimmermann R Degenkolbe H-U Bernard and M JOrsquoConnor ldquoThe human papillomavirus type 16 E6 oncoproteincan down-regulate p53 activity by targeting the transcriptionalcoactivator CBPp300rdquo Journal of Virology vol 73 no 8 pp6209ndash6219 1999
[127] D A Thompson G Belinsky T H-T Chang D L Jones RSchlegel and K Munger ldquoThe human papillomavirus-16 E6oncoprotein decreases the vigilance of mitotic checkpointsrdquoOncogene vol 15 no 25 pp 3025ndash3035 1997
[128] J Bohl K Das B Dasgupta and S B Vande Pol ldquoCompetitivebinding to a charged leucine motif represses transformation bya papillomavirus E6 oncoproteinrdquo Virology vol 271 no 1 pp163ndash170 2000
[129] S Song H C Pitot and P F Lambert ldquoThe human papillo-mavirus type 16 E6 gene alone is sufficient to induce carcinomasin transgenic animalsrdquo Journal of Virology vol 73 no 7 pp5887ndash5893 1999
[130] S Yasumoto A L Burkhardt J Doniger and J A DiPaololdquoHumanpapillomavirus type 16DNA-inducedmalignant trans-formation of NIH 3T3 cellsrdquo Journal of Virology vol 57 no 2pp 572ndash577 1986
[131] S B Vande Pol M C Brown and C E Turner ldquoAssociation ofBovine Papillomavirus Type 1 E6 oncoprotein with the focaladhesion protein paxillin through a conserved protein interac-tion motifrdquo Oncogene vol 16 no 1 pp 43ndash52 1998
[132] R Wade N Brimer and S Vande Pol ldquoTransformation bybovine papillomavirus type 1 E6 requires paxillinrdquo Journal ofVirology vol 82 no 12 pp 5962ndash5966 2008
[133] A J Klingelhutz S A Foster and J KMcDougall ldquoTelomeraseactivation by the E6 gene product of humanpapillomavirus type16rdquo Nature vol 380 no 6569 pp 79ndash82 1996
[134] S T Oh S Kyo and L A Laimins ldquoTelomerase activation byhuman papillomavirus type 16 E6 protein Induction of humantelomerase reverse transcriptase expression through Myc andGC-rich Sp1 binding sitesrdquo Journal of Virology vol 75 no 12pp 5559ndash5566 2001
[135] L V Ronco A Y KarpovaMVidal and PMHowley ldquoHumanpapillomavirus 16 E6 oncoprotein binds to interferon regulatoryfactor-3 and inhibits its transcriptional activityrdquo Genes andDevelopment vol 12 no 13 pp 2061ndash2072 1998
Figure 1Western blot showing BPV-1 E6 recombinant oncoproteinWeight ladder employed Spectra Multicolor Broad Range ProteinLadder (Fermentas Lithuania)
The CMNA combined with CA allows detecting DNA dam-age as an indication of mutagenesis with high statistical andsensitivity power [53] This is the first study which evaluatedthe mutagenic potential of E6 recombinant oncoprotein ofBPV-1 suggesting that this oncoprotein participates in bothcancer initiation and promotion
2 Material and Methods
21 Expression and Purification of BPV-1 E6 OncoproteinBPV-1 E6 recombinant oncoprotein was expressed and puri-fied according to Mazzuchelli-de-Souza et al [44] usingEscherichia coli BL21 The oncoprotein was subjected to dial-ysis This step was necessary to remove urea and imidazolesubstances used to promote the oncoprotein refolding Thedialysis avoids false-positive results to mutagenesis testswhich could be induced by urea and imidazole Dialysis wasperformed using Slide-A-Lyzer Dialysis Cassette (3 K-12mL)(Thermo Scientific Carlsbad USA) For this step 10mLof sample was dialyzed against two liters of dialysis buffer(20mM Tris-HCl and 500mM NaCl pH 80) for 8 hours at4∘C under constant agitation Figure 1 shows Western blotof BPV-1 E6 recombinant protein used in this study Toconfirm the identity of recombinant oncoprotein the bandobtained in the SDS-PAGE gel was submitted to mass spec-trometry Four peptide sequences were identified using thismethod being (1) KDFHVVIRE (2) KDFHVVIRE (3) RHVLFNEPFCKT and (4) RLWQGVPVTGEEAELLHGKTThese sequences were analyzed in the Swiss-Prot databasewhere score higher than 43 indicates extensive homologyThus mass spectrometry pointed out a score of 94 demon-strating that the recombinant protein shows identity with E6oncoprotein of BPV-1 (access number VE6 BPV1)
22 Peripheral Blood Collection A volume of 5mL of periph-eral bloodwas collected fromfive asymptomatic calves (with-out cutaneous papillomas) (Bos taurus Simmental breed)using theVacutainer systemwith EDTA (molecular diagnosis
of BPV) and heparin (CBMNA and CA) The materialwas collected through venipuncture from jugular vein by aveterinarian The protocols used in this study were approvedby the Ethics Committee onAnimal Use of Butantan Institute(process 103513) The farm of calvesrsquo origin is located in SaoPaulo State (Brazil) This farm was chosen due to the absenceof bracken fern Pteridium aquilinum because studies pointedout that bracken farm has mutagenic and carcinogenic com-pounds such as quercetin and ptaquilosides [61ndash63] whichcould interfere in the studies leading to false-positive results
23 Molecular BPV Identification by PCR Blood DNAextraction the extraction of DNA from peripheral bloodcells was performed using the GenomicPrep Blood MiniKit Illustra Spin (GE Healthcare Buckinghamshire UK)according to the manufacturer The quality of the obtainedDNAwas verified using the polymerase chain reaction (PCR)technique amplifying a bovine 120573-globin gene fragment [64]
Viral identification viral identification was performedusing specific primers for BPV-1 (forward 51015840-GGAGCG-CCTGCTAACTATAGGA-31015840 reverse 51015840-ATCTGTTGT-TTGGGTGGTGAC-31015840) which amplifies the L1 gene result-ing in a 301 bp amplicon BPV-2 (forward 51015840-GTTATACCA-CCCAAAGAAGACCCT-31015840 reverse 51015840-CTGGTTGCA-ACAGCTCTCTTTCTC-31015840) which amplifies the L2 generesulting in a 164 bp amplicon and BPV-4 (forward 51015840-GCTGACCTTCCAGTCTTAAT-31015840 reverse 51015840-CAGTTT-CAATCTCCTCTTCA-31015840) which amplifies the E7 generesulting in a 170 bp amplicon These primers were chosenbecause BPV-1 BPV-2 and BPV-4 are the most frequenttypes in Brazil being associated with oncogenic process [165] Besides these specific primers two degenerate pairs ofprimers were used Delta-Epsilon (forward 51015840-CCAGAY-TAYYTMAAAATGGC-31015840 reverse 51015840-ATAAMKGCTAGC-TTATATTC-31015840) and Xi (forward 51015840-TWYAATAGDCCV-TTTTGGAT-31015840 reverse 51015840-TTMCGCCTACGCTTTGGC-GC-31015840) [66] These primers allow detecting BPVs of generaDelta Epsilonpapillomavirus (Delta-Epsilon) and Xipapillo-mavirus (Xi) [66] Both primers amplify the L1ORF resultingin products with 430 bp (Delta-Epsilon) and 600 bp (Xi)
PCR parameters reactions were performed in a total vol-ume of 500 120583L using 200 ng120583L of DNA template 20120583L offorward primer 20 120583L of reverse primer and 360 120583L ofMas-ter Mix (4G Porto Alegre Brazil) Reactions were done onthermocycler Veriti 96-WellThermal Cycler (Applied Biosys-tems Carlsbad USA) and subjected to the cycles shown inTable 2 Cloned genomes of BPV-1 BPV-2 and BPV-4 inpAT153 vector in Escherichia coliD5H120572were used as positivecontrols BPV-2 viral genome was employed as positivecontrol for Delta-Epsilon primer and BPV-3 for Xi primer
The PCR products were analyzed in 2 agarose gelelectrophoresis stained with GelRed (Biotium USA) in Tris-Acetate-EDTA (TAE) buffer visualized under UV light usingBioDocAnalyze (Biometra Germany)
24 Cytokinesis-Block Micronucleus Assay (CBMNA) UsingPeripheral Blood Mononuclear Cells (PBMCs) For each sam-ple three cultures were established (1) negative control
4 BioMed Research International
Table 2 Frequency of micronucleated lymphocytes Number ofmicronuclei observed perslide (MN) and number of mononucle-ated (119873
1
) binucleated (1198732
) and polynucleated (119873119901
) lymphocytesand anaphase bridges (AB) observed pergroup Based on thesevalues the micronucleated formation frequency (MN
1199030
) and thecytokinesis-block proliferation index (CBPI) and the media (119909) areshown
(not treated with any drug) (2) positive control (treatedwith 50120583gmL of cyclophosphamide) and (3) experimentalgroup (treated with 1 120583gmL of E6 recombinant oncoproteinresuspended in PBS) This concentration of E6 recombinantoncoproteinwas based on previous study involving BPV early(E) protein as vaccine [67] The protocol of CBMNA wasdone according to the technical recommendation proposedby Araldi et al [53] In detail 02mL of peripheral bloodwas transferred to culture flask containing 50mL RPMI1640 medium supplemented with 15 fetal bovine serum01mL L-glutamine and 01mL phytohemagglutinin A Thematerial was incubated at 37∘C for 8 hours After this bothcyclophosphamide and E6 recombinant oncoprotein wereadded to the culture After 44 hours 02mL of cytochalasin Bwas added to block the cytokinesis After 72 hours the culturewas stopped with the addition of 05mL methanol aceticacid fixative (vv) (3 1) for 5 minutes at room temperatureThematerial was centrifuged at 500 g and the supernatantwasdiscardedThepellet was homogenizedwith 5mLfixative andcentrifuged at 500 g The pellet was aspirated and transferredto slides which were stained with a 1 3 Giemsa phosphatebuffer solution pH = 68 for 8 minutes
After staining coverslips were placed on slides withEntellan (Merck Germany) The material was analyzed ina blind test under an Axiophot binocular microscope (Carl
Zeiss Germany) to observe the frequency ofmicronucleated-binucleated lymphocytes in a total of 1000 analyzed cellsaccording to Araldi et al [53] Statistical analysis was per-formed using the Kruskal-Wallis test followed by a post hocDunn test both at a significance level of 5 Statistical testswere done using the BioEstat software [68]
25 Comet Assay Alkaline Method Slides preparation slidesof 26 times 76mm were dipped in a solution of normal meltingpoint agarose (NMP) (Invitrogen Carlsbad USA) dilutedin phosphate-buffered solution (PBS) 15 at 60∘C and oneside of each slide was wiped clean with a paper towel Theconcentration of NMA was based on Araldi et al [53] Theslides were dried in a horizontal position overnight
Peripheral blood incubation with drugs three wholeblood aliquots of 200120583L each were transferred to three15mL polypropylene tubes (1) negative control (2) positivecontrol and (3) experimental group In each tube 200120583Lof RMPI 1640 medium was added The negative control didnot receive any drug and was incubated only in RPMI 1640medium Positive control was incubated with 50 120583gmL ofcyclophosphamide diluted in RPMI 1640 medium Experi-mental group was treated with 1 120583gmL of BPV-1 E6 recombi-nant protein The samples were incubated at 37∘C for 2 hoursunder constant agitation After this time the aliquots werecentrifuged for 1 minute at 500 g and the supernatant wasdiscarded Ten microliters of each obtained pellet was addedto 75120583L low melting point agarose (LMP) A final volume of85 120583L of this suspensionwas immediately transferred toNMPprecoated slides The slides were covered with coverslips andmaintained at 4∘C for 20 minutes The coverslips were gentlyremoved and the slides were placed in a Coplin jar containinglysis solution (25mM NaCl 100mM ethylenediaminete-traacetic acid (EDTA) 10mM Tris-HCl 11 Triton X-100and 112 dimethyl sulfoxide) at 4∘C for 1 hour Subsequentlyall procedures were conducted under dark conditions toprevent the induction of DNA damage
Electrophoresis after lysis slides were washed with PBSand transferred to an electrophoresis tank containing elec-trophoresis buffer (300mM NaOH and 1mM EDTA pH gt13) at 4∘C for 40 minutes to induce unwinding of double-stranded DNA Next the electrophoretic run was performedwith a current of 25V (086Vcm) 300mA for 20 minutesto promote the migration of free DNA fragments toward theanode The slides were transferred to a Coplin jar containingneutralizing buffer (400mM Tris-HCl pH 75) for 5 minutesThe material was fixed in absolute ethanol for 5 minutes
Comet analysis slides were stained with 20 120583L 4 120583gmLpropidium iodide (PI) and analyzed under a fluorescencemicroscope (Carl Zeiss Axio Scope A1 Germany) equippedwith an excitation filter of 510ndash560 nm and barrier of 590 nmThe material was analyzed in a total magnification of 400xOne hundred nucleoids were analyzed per slide which werescored on a scale of 0 (without DNA damage) to 2 (maximumDNAdamage) according to Araldi et al [53]The scores wereobtained by summing the product of the observed numberof nucleoids per class and its respective class value Statisticalanalysis was performedusing theKruskal-Wallis test followedby the post hocDunn test both with a significance level of 5
BioMed Research International 5
Both test and graphical analysis were done using the BioEstatsoftware [68]
26 Cytokinesis-Block Micronucleus Assay (CBMNA) inEpithelial Cells To verify whether the results observed inPBMCs should also be observed in epithelial cells CBMNAwas performed in CRIB cell (commercial epithelial cell lineobtained from bovine kidney) In detail a total of 1 times 105cells were transferred to six-well plate containing a sterilecoverslip of 24 times 24mm with 2mL of MEM mediumsupplementedwith 10 fetal bovine serum and 1 ampicillinand three cultures had been established negative control(not treated with any drug) positive control (treated with50120583gmL of cyclophosphamide) and experimental group(treatedwith 1120583gmLof BPV-1 E6 recombinant oncoprotein)Cyclophosphamide and BPV-1 E6 recombinant oncoproteinwere added together to the cells After 1 hour the threecultures were treated with 6 120583gmL of cytochalasin B (SigmaGermany)Thematerial was incubated for 48 hours the timenecessary to complete two replication cycles once the dupli-cation time of these cells is 24 hours according to our previ-ous study After this time the medium was removed and thecells were washed twice with PBS at 37∘C Cells were stainedwith solution 1 4 Giemsa-PBS for 3 minutes and afterwashed twice with PBS Coverslips containing the biologicalmaterial were mounted on slides using Entelan (Merck Ger-many) Slides were analyzed by Axiophot binocular micro-scope (Carl Zeiss Germany) to observe the frequency ofmicronucleated cells in a total of 1000 analyzed cells accord-ing to Araldi et al [53]
3 Results
31 Molecular BPV Identification by PCR The peripheralblood samples collected from five asymptomatic calves didnot reveal the presence of BPV sequences using both specificand degenerate primers (Figure 2)
32 Cytokinesis-Block Micronucleus Assay (CBMNA) inPBMCs Frequency analysis of micronucleus observed inCBMNA CBMNA showed an elevated number of micronu-cleated lymphocytes as well as anaphase bridges in bothpositive control and the group treated with 1 120583gmL of E6recombinant oncoprotein (Table 2 Figure 3)
Based on the micronucleated lymphocytes numberobserved per group (Table 2) Chi-square (1205942) test andKruskal-Wallis test were done both with 5 significancelevel Chi-square test pointed out statistical differencesbetween positive control and group treated with E6 (Table 3)These results point out that the cyclophosphamide and E6recombinant oncoprotein are able to induce aneugenesis andor clastogenesis However the Chi-square test did not showstatistic difference in negative control (Table 3)
Kruskal-Wallis test revealed statistical significant differ-ences among the groups (119867 = 97297 and 119901 = 00087)Based on this result Dunn post hoc test was performed Thetest pointed out significant difference between negative andpositive control as well as negative control and experimental
Table 3 Chi-square (1205942) test results showing expected value (119864)freedom degree (FD) and probability (119901)
Group 119864 1205942 FD 119901
Negative control 560 2000 4 07358Positive control 430 10698 4 00302Experimental (E6) 456 12202 4 00159
Table 4 Dunn post hoc test results
Groups Rank differences Calculated 119885 Critical 119885 119901
Cminus and C+ 71 25102 2394 lt005Cndash and E6 79 27931 2394 lt005C+ and E6 08 02828 2394 nsns nonsignificant value
Table 5 Student-Newman-Keuls post hoc test results
Groups Rank differences Calculated 119885 Critical 119885 119901
Cminus and C+ 61 21567 2394 00310Cminus and E6 77 27224 2394 00065C+ and E6 16 05657 2394 05716
group (E6) (Table 4) However the test did not show statis-tical difference between positive control and experimentalgroup (Table 4)
Based on the maximum minimum and median valuesof micronucleus observed in the three groups a boxplotwas done (Figure 4) The graph indicates statistically equalmedians between positive control and experimental group(E6) Although the medians between these groups had notshown statistical differences the graph indicates that theexperimental group showed maximum values of binucleatedlymphocytes with micronucleus higher than positive control
Frequency analysis of anaphase bridge observed inCBMNA based on the number of anaphase bridges (AB)observed per group (Table 3) Kruskal-Wallis test was per-formed which indicates significant differences among thegroups (119867 = 83444 119901 = 00154) Student-Newman-Keuls post hoc test revealed differences between negative andpositive control as well as negative control and experimentalgroup (Table 5) The test did not show differences betweenpositive control and experimental group
Analysis of cytokinesis-block proliferation index (CBPI)and cytotoxicity Kruskal-Wallis test based on theCBPI indi-cates statistical differences among the groups (119867 = 93968and 119901 = 00091) Dunn post hoc test revealed significantdifferences between negative and positive control as well asnegative control and experimental group (Table 6) Howeverthe test did not show differences between positive control andexperimental groupThese data indicate that E6 recombinantoncoprotein has CBPI similar to the cyclophosphamide
Besides presenting high CBPI the E6 recombinant onco-protein also showed to induce endoreduplication evidencedby the presence of intracellular cytokinesis suggesting neosis(Figure 5) according to the criteria proposed by Das et al[69] The CBMNA results suggest that E6 recombinant
6 BioMed Research International
301bp
M 0504030201 M M 0504030201BPV1Cminus
(a) (b)
(c) (d)
(e) (f)
164bp170bp
M 0504030201BPV2Cminus M 0504030201BPV4Cminus
430bp
600bp
M 0504030201BPV2Cminus M 0504030201BPV4Cminus
Figure 2 Electrophoresis gel images showing (a) genomicDNA integrity afterDNAextraction using 1 KbDNALadder (Invitrogen CarlsbadUSA) as marker absence of amplicon in samples 01 to 05 using specific primers to BPV-1 (b) BPV-2 (c) and BPV-4 (d) absence of ampliconin samples 01 to 05 using Delta-Epsilon (e) and Xi (f) degenerate primers Images (b)ndash(f) showed amplification only in positive control withthe 100 bp DNA Ladder (Invitrogen Carlsbad USA) being employed as marker
Table 6 Dunn post hoc test results based on the cytokinesis-blockproliferation index
Group Rank differences Calculated 119885 Critical 119885 119901
Cminus and C+ 76 26870 2394 lt005Cminus and E6 74 26163 2394 lt005C+ and E6 02 00707 2394 nsns nonsignificant differences
oncoprotein induces mitotic stress resulting in clastogenesisand neosis
33 Comet Assay Alkaline Method Comet assay results areshown in Table 7 and Figure 6 According to these data
theKruskal-Wallis test was performedwhich pointed out sta-tistical differences among the groups (119867 = 102613 and 119901 =00059) Based on these results the Dunn post hoc test wasperformed which pointed out significant differences betweennegative and positive control as well as negative controland experimental group (Table 8) However the test did notshow differences between positive control and experimentalgroup (Table 8) These results were also verified by Figure 7Comet assay reinforces the CBMNA results indicating themutagenic potential of E6 recombinant oncoprotein
34 Cytokinesis-Block Micronucleus Assay (CBMNA) inPBMCs Results of this test confirmed the previous resultsindicating that the BPV-1 E6 recombinant oncoprotein has
BioMed Research International 7
LB
(a)
LMN
(b)
LMN
LMN
(c)
PA
PA and MN
(d)
Figure 3 (a) Photomicroscopy of binucleated lymphocyte without micronucleus (LB) observed in negative control Image of binucleatedlymphocyte with micronucleus (LMN) observed in positive control (b) and group treated with E6 recombinant oncoprotein (c) Image ofbinucleated lymphocyte with anaphase bridge and micronucleus (PA and MN) observed in group treated with E6 (d) Images obtained withtotal magnification of 1000x
60
50
40
30
20
10
0
Num
ber o
f mic
ronu
clei
GroupCminus C+ E6
Figure 4 Comparative boxplot based on maximum minimumand median values of micronucleus observed per group
a genotoxicity potential being able to induce DNAbreaks in epithelial cells verified by the presence of MNs(Figure 8)
Figure 5 Evidence of neosis Photomicroscopy of lymphocyteshowing endoreduplication (black arrow) suggesting neosis Cellsanalyzed in total magnification of 1000x
4 Discussion
Transforming potential of E6 oncoprotein has been discussedsince the 1980s based on studies of cottontail rabbit papil-lomaviruses (CRPV) [70 71] Although there are lines ofevidence that the E6 oncoprotein can induce transformation
8 BioMed Research International
(a) (b)
(c) (d)
Figure 6 Images of cometsrsquo nucleoids (a) Image of class 0 (without DNA damage) observed in negative control group Images of class 2(maximum DNA damage) observed in positive control (b) and experimental (c) groups (d) Image of hedgehog comet characterized by thehead absence observed in experimental group Images captured in total magnification of 400x
200
150
100
50
0
Com
et sc
ore
GroupsCminus C+ E6
Figure 7 Boxplot of comet score
[72] the mechanisms of initiation and promotion of cancerassociated with the BPV E6 oncoprotein are unknown
Molecular diagnosis of five peripheral blood samplesfrom five calves showed the absence of amplicons for thefive different primers used (Figure 2) However both specific(BPV-1 BPV-2 and BPV-4) and degenerate (Delta-Epsilonand Xi) primers sets amplified the controls genomes of BPV-1 BPV-2 and BPV-4 None of the negative controls revealedthe amplicon presence These results indicate that the fivesampleswere uninfected by BPVMoreover the use of specific
and degenerate primers increases the capacity of BPV iden-tification This occurs because specific primers are moresensitive than degenerates [66] This sensitivity is due tothe absence of degeneration in the 51015840 region which reducesthe primer ability to recognize and to link with DNA targetsequence [73]The absence of BPV infection in the peripheralblood samples is required for the CBMNA and CA allowinginvestigating the aneugenic andor clastogenic action of BPV-1 E6 recombinant oncoprotein This is because the presenceof BPV sequences is associated with the presence of oncopro-teins transcripts [74]Thus studies have shown that the pres-ence of BPVDNA sequences in peripheral blood is associatedwith cytogenetic damage including clastogenesis [1 14 75]Complementary studies show the association of the BPVpresence with cytogenetic damage in both benign lesions(papillomas) [74] and carcinomas [15]
Chi-square test based on the number of micronucleatedcells pointed out significant statistical values in both positivecontrol and group treated with BPV-1 E6 recombinant onco-protein (Table 3) Negative control showed fewer number ofmicronucleated cells This result was expected because thebiological material was transported and processed after threehours of its collection This procedure reduced the influenceof exogenous environmental factors which could induceDNA damage Furthermore the absence of BPV infection
BioMed Research International 9
(a) (b)
8
6
4
2
0
Num
ber o
f mic
ronu
cleat
ed ce
lls
Neg
ativ
e con
trol
Posit
ive c
ontro
l
E6on
copr
otei
n
(c)
Figure 8 Photomicroscope showing CRIB cell not treated with any drug (negative control) indicating absence of micronucleus (a) andtreated with BPV-1 E6 recombinant oncoprotein showing the presence of MN indicated by arrow (b) (c) Number of micronucleated cellsobserved in each group
in the peripheral blood reduces the influence of endogenousenvironmental factors that could interfere in the analysisPositive control showed a high number of micronucle-ated lymphocytes This result was also expected once thisgroup was treated with 50 120583gmL of cyclophosphamide achemotherapeutic drug with cytotoxic and teratogenic effect[76 77] The cells treated with 1120583gmL of BPV-1 E6 recom-binant oncoprotein also showed significant statistical valuesKruskal-Wallis test followed by the Dunn post hoc testwas performed to compare the frequencies of micronucleusamong three groups This test indicated statistical differencebetween negative control and the cells treated with BPV-1 E6 recombinant oncoprotein However the test did notreveal differences between the cells treated with cyclophos-phamide and BPV-1 E6 recombinant oncoprotein (Table 4Figure 4) Similar results were also observed in epithelial cells(Figure 8) These results suggest that the BPV-1 E6 oncopro-tein has an aneugenic andor clastogenic potential similarto or higher than the observed with the cyclophosphamideThis is because the cells treated with the oncoprotein showed
a frequency of micronucleus formation (MN1199030) 14003
higher than those observed in positive controlThe elevated mutagenic potential of the BPV-1 E6 onco-
protein can justify the cytogenetic damage already describedin the literature [1 14 15 24] Similar results were observedin HPV-infected cells [12 78ndash81] The micronucleus induce-ment was already described in cytological samples collectedduring Papanicolaursquos test from healthy cervix infected byHPV [12] For this reason the micronucleus assay has beenproposed as a complementary method for Papanicolaursquos testbeing a suggestive biomarker of lesion degree [78] Howeverthe association between the BPV and the micronucleusinducement was not yet reported
The high frequency of micronucleated lymphocytes insamples infected by HPV has been attributed to the synergicaction of E5 E6 and E7 oncoproteins [29 30 78 79]However this is the first study that pointed out themutagenicaction of BPV-1 E6 protein per se
Student-Newman-Keuls post hoc test based on the fre-quency of anaphase bridges showed significant statistical
10 BioMed Research International
Table 7 Comet assay results showing the number of nucleoidsobserved per class the number of hedgehog comets and cometscore
Sample Class 0 Class 1 Class 2 Hedgehog ScoreNegative control
Groups Rank differences Calculated 119885 Critical 119885 119901
Cminus and C+ 68 24042 2394 lt05Cminus and E6 82 28991 2394 lt05C+ and E6 14 04950 2394 nsns nonsignificant differences
difference between the negative control and the group treatedwith the BPV-1 E6 recombinant oncoprotein (Table 5) How-ever the test did not reveal differences between the cellstreated with cyclophosphamide and BPV-1 E6 recombinantoncoprotein (Table 5)These data reinforce those observed bythe Dunn post hoc test based on the frequency of micronu-cleus indicating the clastogenic action of the E6 oncoproteinMoreover the presence of anaphase bridge was alreadydescribed in cells transfected with recombinant adenoviruscontaining the E6 and E7 ORFs of HPV [29 30] Howeverthere are no studies describing the action of BPVE6 oncopro-tein in the induction of anaphase bridges or studies that showthis same action of E6 oncoprotein of HPV per se Anaphasebridges are indicators of genomic instability [82 83] which isconsidered the first step in carcinogenesis [46 47] Moreoverthe presence of these bridges is an important hallmark ofDNA double strand breaks (DSBs) [84]
Statistical analysis based on the cytokinesis-block prolif-eration index (CBPI) showed that BPV-1 E6 oncoprotein hascytotoxic levels similar to cyclophosphamide (Table 6) Thisresult suggests that the BPV E6 oncoprotein can deregulatethe cell cycle contributing to the cell proliferation andimmortalization To date this mitogenic action is attributedto the E7 oncoprotein [85ndash87] E7 oncoprotein induces
the phosphorylation of pRb resulting in the E2F transcrip-tion factor release [85] E2F when translocated to the nucleusacts as activator and binds to the kinase-dependent cyclinspromoters [85] However the results showed polynucleatedcells in the group treatedwithBPVE6oncoprotein (Figure 5)suggesting that the BPV E6 oncoprotein confers mitogenicstimulation resulting in mitotic stress Moreover studiespointed out that the BPV E6 oncoprotein interacts with theCBPp300 deacetylase promoting p53 downregulation [88]This downregulation increases the expression levels of FoxM1transcription factor (Foxhead box M1) [89] promoting B1cyclin D1 cyclin and cdc25 upregulation [89] The upregu-lation of these genes is associated with increased levels of cellproliferation which is necessary in order to make DNA poly-merases available to virus replication However if this mech-anism guarantees the BPV replication it can contribute to cellimmortalization and cancer progression
The FoxM1 factor also participates in the Wnt120573-cateninsignaling pathway and binds directly to the 120573-catenin [89]This interaction promotes the nuclear 120573-catenin transloca-tion [90] 120573-catenin translocation to nucleus induces thecyclin expression [64] So these data suggest that the BPVE6 oncoprotein not only promotes mitogenic stress but alsocontributes to the epithelial-mesenchymal transition (EMT)Studies based on theHPVE6 oncoprotein have indicated thatthis oncoprotein contributes significantly to the EMT [91]This occurs not only because the E6 oncoprotein induces thetranslocation of 120573-catenin but also due to the proteasomaldegradation of regulatory proteins of apical-basal polarity[92ndash94] Studies also showed that the E6 oncoprotein is ableto bind to Crumbs (Dlg and Patj) proteins resulting in loss ofcell polarity [93 94] Based on these results the E6 oncopro-tein emerges as a possible therapeutic target with biotechno-logical value in cancer treatment
Mitogenic action of E6 oncoprotein can be moreexpressed in cells infected by papillomaviruses once thesecells also expressed E5 and E7 oncoproteins [74] The E7oncoprotein is able to form a complex E7-p600 which pro-motes E6 upregulation [85] Furthermore the E6 oncoproteincan bind to the E6AP ubiquitin ligase forming the E6-E6APcomplex increasing the hTERT levels of expression con-tributing to cell immortalization [95] Moreover E6-E6APinduces NFX1 expression promoting MHC-II downregula-tion [96]Thismechanism reduces the antigenic presentationmediated by CD4+ T lymphocytes contributing to immuneevasion This action guarantees the PV infection for longperiods with or without clinical symptoms [24] Althoughthe BPV infection can be asymptomatic the BPV presence inperipheral blood is associated with DNA damage [1 14 24]
CBMNA analysis pointed out the presence of cells withintracellular cytokinesis (Figure 5) This result suggests theneotic action of BPV E6 oncoprotein Neosis is characterizedby the presence of (1) DNA damage (2) loss of checkpointcontrol (3) repair system failures and (4) endoreduplication[97] In this scenario the E6 oncoprotein attempts all theseneosis criteria Moreover the oncoprotein not only increasesthe cytogenetic damage leading to increasing of micronu-cleus frequency but also induces clastogenesis as shown bythe comet assay and anaphase bridges So statistical analysis
BioMed Research International 11
performed based on the comet scores reinforced the CBMNAresults Comet assay allows detecting both simple and dou-ble DNA breaks being more sensitive than the CBMNAClastogenesis is considered the most serious type of DNAdamage [98] DNA breaks can be repaired by homologous ornonhomologous recombination However studies point outthat the E6 oncoprotein can link to the Holliday junctionduring the homologous repair avoiding the junction reso-lution [99] Moreover E6 oncoprotein promotes the TP53gene deacetylation resulting in p53 downregulation [51]Thisepigenetic effect results in cell cycle deregulation affectingthe checkpoints
Studies have shown that the HPV-induced DNA breaksare required for the virus integration into the host genome[100] Although there are no studies showing the BPV inte-gration to date the results observed in this work in additionto those already published [24] indicate the necessity ofmorestudies to evaluate the virus-host interaction
Studies also showed that the E6 oncoprotein inducesclathrin-transporter adapter protein (AP-1) increasing thelevel of this protein in the plasma membrane [76 77] Theincreasing quantity of AP-1 in membrane can facilitate theBPV virions infection This occurs because the infectionprocess is clathrin dependent In this scenario the E6 onco-protein can contribute to the virus infection [101]
In summary the CBMNA and CA results showed thatthe BPV E6 oncoprotein participates not only in cancerpromotion but also in initiation inducing DNA breaksThese DNA breaks represent mutagenic effects consideredthe first step in the oncogenesis process which are associatedwith genomic instability [47 48 102 103] Thus the E6recombinant oncoprotein has been suggested as a possiblevaccine candidate [104ndash112] due to its immunogenicity [44]However the mutagenic tests such as CBMNA and CA arerequired in drug validation process [113] In this scenariothis study pointed out that the BPV-1 E6 recombinant onco-protein in tested concentration showedmutagenic potentialThis is the first study that reports the mutagenic potential ofa possible therapeutic vaccine candidate On the one handthese results allowed better understanding the mechanism ofcancer promotion associatedwith the BPVE6oncoprotein aswell as revealing that this oncoprotein can induce carcinogen-esis per se on the other hand this data pointed out thatmaybeBPV E6 recombinant oncoprotein requires protein modifica-tions to be used as vaccine
Conflict of Interests
The authors declare that there is no conflict of interests
Acknowledgments
Theauthors thank Fundacao deAmparo a Pesquisa do Estadode Sao Paulo (FAPESP Process 201420617-5) and FundacaoButantan for the financial support
References
[1] R C Stocco dos Santos C J Lindsey O P Ferraz et al ldquoBovinepapillomavirus transmission and chromosomal aberrations an
experimental modelrdquo Journal of General Virology vol 79 no 9pp 2127ndash2135 1998
[2] A Antonsson and B G Hansson ldquoHealthy skin ofmany animalspecies harbors papillomaviruses which are closely related totheir human counterpartsrdquo Journal of Virology vol 76 no 24pp 12537ndash12542 2002
[3] P Rous and J W Beard ldquoA virus-induced mammalian growthwith the characters of a tumor (the Shope rabbit papilloma) IThe growth on implantationwithin favorable hostsrdquoThe Journalof Experimental Medicine vol 60 no 6 pp 701ndash722 1934
[4] R Shope and E Hurst ldquoInfectious papillomatosis of rabbitsrdquoThe Journal of Experimental Medicine vol 58 no 1 pp 607ndash624 1933
[5] H Delius M A Van Ranst A B Jenson H Zur Hausen and JP Sundberg ldquoCanine oral papillomavirus genomic sequence aunique 15-kb intervening sequence between the E2 andL2 openreading framesrdquo Virology vol 204 no 1 pp 447ndash452 1994
[6] W A Demonbreun and E Goodpasture ldquoInfectious oral papil-lomatosis of dogsrdquo The American Journal of Pathology vol 8no 1 pp 43ndash56 1932
[7] P K Nicholls B A Klaunberg R A Moore et al ldquoNaturallyoccurring nonregressing canine oral papillomavirus infectionhost immunity virus characterization and experimental infec-tionrdquo Virology vol 265 no 2 pp 365ndash374 1999
[8] A Alberti S Pirino F Pintore et al ldquoOvis aries Papillomavirus3 a prototype of a novel genus in the family Papillomaviridaeassociated with ovine squamous cell carcinomardquo Virology vol407 no 2 pp 352ndash359 2010
[9] H zur Hausen E-M de Villiers and L Gissmann ldquoPapil-lomavirus infections and human genital cancerrdquo GynecologicOncology vol 12 no 2 pp S124ndashS128 1981
[10] C Foresta A Bertoldo A Garolla et al ldquoHuman papillo-mavirus proteins are found in peripheral blood and semenCd20+ and Cd56+ cells during Hpv-16 semen infectionrdquo BMCInfectious Diseases vol 13 article 593 2013
[11] S Ljubojevic andM Skerlev ldquoHPV-associated diseasesrdquo Clinicsin Dermatology vol 32 no 2 pp 227ndash234 2014
[12] A P R Cassel R B Barcellos C M D da Silva S E de MatosAlmeida and M L R Rossetti ldquoAssociation between humanpapillomavirus (HPV) DNA and micronuclei in normal cervi-cal cytologyrdquo Genetics and Molecular Biology vol 37 no 2 pp360ndash363 2014
[13] W Jarrett M Campo B OrsquoNeil H Laird and L Coggins ldquoAnovel bovine papillomavirus (BPV-6) causing true epithelialpapillomas of the mammary gland skin a member of a pro-posed newBPV subgrouprdquoVirology vol 136 no 2 pp 255ndash2641984
[14] T CMelo N Diniz S R C Campos et al ldquoCytogenetic studiesin peripheral blood of bovines afflicted by papillomatosisrdquoVeterinary and Comparative Oncology vol 9 no 4 pp 269ndash2742011
[15] S R C Campos T C Melo S Assaf et al ldquoChromosome aber-rations in cells infected with bovine papillomavirus comparingcutaneous papilloma esophagus papilloma and urinary blad-der lesion cellsrdquo ISRN Oncology vol 2013 Article ID 910849 8pages 2013
[16] H zur Hausen ldquoThe search for infectious causes of humancancers where and whyrdquoVirology vol 392 no 1 pp 1ndash10 2009
[17] R P Araldi R F Carvalho T C Melo et al ldquoBovine papillo-mavirus in beef cattle first description of BPV-12 and putativetype BAPV8 in BrazilrdquoGenetics andMolecular Research vol 13no 3 pp 5644ndash5653 2014
12 BioMed Research International
[18] T Rampias C Sasaki and A Psyrri ldquoMolecular mechanisms ofHPV induced carcinogenesis in head and neckrdquoOral Oncologyvol 50 no 5 pp 356ndash363 2014
[19] S Roperto V Russo L Leonardi et al ldquoBovine papillomavirustype 13 expression in the urothelial bladder tumours of cattlerdquoTransboundary and Emerging Diseases 2015
[20] S RCCampos C TrindadeO P Ferraz et al ldquoCan establishedcultured papilloma cells harbor bovine papillomavirusrdquoGenet-ics and Molecular Research vol 7 no 4 pp 1119ndash1126 2008
[21] M S Campo ldquoAnimalmodels of papilloma virus pathogenesisrdquoVirus Research vol 89 no 2 pp 249ndash261 2002
[22] G Borzacchiello ldquoBovine papillomavirus infections in ani-malsrdquo in Communicating Current Research and EducationalTopics and Trends in AppliedMicrobiology pp 673ndash679 Forma-tex Badajoz Spain 2007
[23] F Bocaneti G Altamura A Corteggio E Velescu F RopertoandG Borzacchiello ldquoBovine papillomavirus new insights intoan old diseaserdquo Transboundary and Emerging Diseases 2014
[24] R P Araldi T C Melo N Diniz et al ldquoBovine papillomavirusclastogenic effect analyzed in comet assayrdquo BioMed ResearchInternational vol 2013 Article ID 630683 7 pages 2013
[25] J Munday N Thomson M Dunowska C Knight R Laurieand S Hills ldquoGenomic characterisation of the feline sarcoid-associated papillomavirus and proposed classification as Bostaurus papillomavirus type 14rdquoVeterinaryMicrobiology vol 177no 3-4 pp 289ndash295 2015
[26] G Borzacchiello V Russo C Spoleto et al ldquoBovine papillo-mavirus type-2 DNA and expression of E5 and E7 oncoproteinsin vascular tumours of the urinary bladder in cattlerdquo CancerLetters vol 250 no 1 pp 82ndash91 2007
[27] P Maiolino A Ozkul A Sepici-Dincel et al ldquoBovine papillo-mavirus type 2 infection and microscopic patterns of urothelialtumors of the urinary bladder in water buffaloesrdquo BioMedResearch International vol 2013 Article ID 937918 6 pages2013
[28] S Roperto G Borzacchiello R Brun et al ldquoMultiple glomustumors of the urinary bladder in a cow associated with bovinepapillomavirus type 2 (BPV-2) infectionrdquo Veterinary Pathologyvol 45 no 1 pp 39ndash42 2008
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[30] S Duensing L Y Lee A Duensing et al ldquoThe human papil-lomavirus type 16 E6 and E7 oncoproteins cooperate to inducemitotic defects and genomic instability by uncoupling centro-some duplication from the cell division cyclerdquo Proceedings of theNational Academy of Sciences of theUnited States of America vol97 no 18 pp 10002ndash10007 2000
[31] C A Moody and L A Laimins ldquoHuman papillomavirus onco-proteins pathways to transformationrdquo Nature Reviews Cancervol 10 no 8 pp 550ndash560 2010
[32] C Mansur and E Androphy ldquoCellular transformation bypapillomavirus oncoproteinsrdquo Biochimica et Biophysica Actavol 1155 no 3 pp 323ndash345 1993
[33] J J Chen Y Hong and E J Androphy ldquoMutational analysis oftranscriptional activation by the bovine papillomavirus type 1E6rdquo Virology vol 236 no 1 pp 30ndash36 1997
[34] X Tong andPMHowley ldquoThebovine papillomavirus E6 onco-protein interacts with paxillin and disrupts the actin cytoskele-tonrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 94 no 9 pp 4412ndash4417 1997
[35] X TongW Boll T Kirchhausen andPMHowley ldquoInteractionof the bovine papillomavirus E6 protein with the clathrinadaptor complex AP-1rdquo Journal of Virology vol 72 no 1 pp476ndash482 1998
[36] Y Nomine M Masson S Charbonnier et al ldquoStructural andfunctional analysis of E6 oncoprotein insights in the molecularpathways of human papillomavirus-mediated pathogenesisrdquoMolecular Cell vol 21 no 5 pp 665ndash678 2006
[37] T Ristriani MMasson Y Nomine et al ldquoHPV oncoprotein E6is a structure-dependent DNA-binding protein that recognizesfour-way junctionsrdquo Journal of Molecular Biology vol 296 no5 pp 1189ndash1203 2000
[38] Y Liu and J D Baleja ldquoStructure and function of the papil-lomavirus E6 protein and its interacting proteinsrdquo Frontiers inBioscience vol 13 no 1 pp 121ndash134 2008
[39] F Lipari G A McGibbon E Wardrop and M G CordingleyldquoPurification and biophysical characterization of a minimalfunctional domain and of an N-terminal Zn2+-binding frag-ment from the human papillomavirus type 16 E6 proteinrdquoBiochemistry vol 40 no 5 pp 1196ndash1204 2001
[40] Y Nomine T Ristriani C Laurent J-F Lefevre E Weissand G Trave ldquoA strategy for optimizing the monodispersity offusion proteins application to purification of recombinantHPVE6 oncoproteinrdquo Protein Engineering vol 14 no 4 pp 297ndash3052001
[41] C G Ullman P I Haris D A Galloway V C Emery and SJ Perkins ldquoPredicted 120572-helix120573-sheet secondary structures forthe zinc-binding motifs of human papillomavirus E7 and E6proteins by consensus prediction averaging and spectroscopicstudies of E7rdquo Biochemical Journal vol 319 no 1 pp 229ndash2391996
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[43] K Zanier S Charbonnier A O M O Sidi et al ldquoStructuralbasis for hijacking of cellular LxxLL motifs by papillomavirusE6 oncoproteinsrdquo Science vol 339 no 6120 pp 694ndash698 2013
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[49] K Zanier S Charbonnier M Baltzinger Y Nomine DAltschuh and G Trave ldquoKinetic analysis of the interactionsof human papillomavirus E6 oncoproteins with the ubiquitinligase E6AP using surface plasmon resonancerdquo Journal ofMolecular Biology vol 349 no 2 pp 401ndash412 2005
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[70] O Danos E Georges G Orth and M Yaniv ldquoFine structureof the cottontail rabbit papillomavirus mRNAs expressed in thetransplantable VX2 carcinomardquo Journal of Virology vol 53 no3 pp 735ndash741 1985
[71] M Nasseri and F O Wettstein ldquoDifferences exist between viraltranscripts in cottontail rabbit papillomavirus-induced benignand malignant tumors as well as non-virus-producing andvirus-producing tumorsrdquo Journal of Virology vol 51 no 3 pp706ndash712 1984
[72] X Tong R Salgia J-L Li J D Griffin and P M Howley ldquoThebovine papillomavirus E6 protein binds to the LDmotif repeatsof paxillin and blocks its interaction with vinculin and the focaladhesion kinaserdquo Journal of Biological Chemistry vol 272 no52 pp 33373ndash33376 1997
[73] Y Maeda T Shibahara Y Wada et al ldquoAn outbreak of teatpapillomatosis in cattle caused by bovine papilloma virus (BPV)type 6 and unclassified BPVsrdquo Veterinary Microbiology vol 121no 3-4 pp 242ndash248 2007
[74] R P Araldi T C Melo A Neves et al ldquoHyperproliferativeaction of bovine papillomavirus genetic and histopathologicalaspectsrdquo Genetics and Molecular Research vol 14 no 4 pp12942ndash12954 2015
[75] NDiniz T CMelo J F Santos et al ldquoSimultaneous presence ofbovine papillomavirus in blood and in short-term lymphocytecultures from dairy cattle in Pernambuco Brazilrdquo Genetics andMolecular Research vol 8 no 4 pp 1474ndash1480 2009
[76] O M Brito M F Guimaraes and C C Lanna ldquoCiclofosfamidae funcao ovarianardquo Revista Brasileira de Reumatologia vol 48no 1 pp 39ndash45 2008
[77] D Anderson T-W Yu and D B McGregor ldquoComet assayresponses as indicators of carcinogen exposurerdquo Mutagenesisvol 13 no 6 pp 539ndash555 1998
[78] M L Adam C Pini S Tulio J C L L Cantalice R A Torresand M T Dos Santos Correia ldquoAssessment of the associationbetween micronuclei and the degree of uterine lesions and viralload in women with human papillomavirusrdquo Cancer Genomicsamp Proteomics vol 12 no 2 pp 67ndash72 2015
[79] E Cortes-Gutierrez M Davila-Rodrıguez J Vargas-VillarrealF Hernandez-Garza and R Cerda-Flores ldquoAssociationbetween human papilloma virus-type infections with micro-nuclei frequenciesrdquo Prague Medical Report vol 111 no 1 pp35ndash41 2010
[80] R N Chakrabarti and K Dutta ldquoMicronuclei test in routinesmears fromuterine cervixrdquo European Journal of GynaecologicalOncology vol 9 no 5 pp 370ndash372 1988
[81] E M M Cerqueira C L Santoro N F Donozo et al ldquoGeneticdamage in exfoliated cells of the uterine cervix Association
14 BioMed Research International
and interaction between cigarette smoking and progression tomalignant transformationrdquo Acta Cytologica vol 42 no 3 pp639ndash649 1998
[82] S M Germann V Schramke R T Pedersen et al ldquoTopBP1Dpb11 bindsDNA anaphase bridges to prevent genome instabil-ityrdquoThe Journal of Cell Biology vol 204 no 1 pp 45ndash59 2014
[83] D R Hoffelder L Luo N A Burke S CWatkins S M Gollinand W S Saunders ldquoResolution of anaphase bridges in cancercellsrdquo Chromosoma vol 112 no 8 pp 389ndash397 2004
[84] C Acilan D M Potter and W S Saunders ldquoDNA repairpathways involved in anaphase bridge formationrdquo Genes Chro-mosomes and Cancer vol 46 no 6 pp 522ndash531 2007
[85] J DeMasi M C Chao A S Kumar and P M Howley ldquoBovinepapillomavirus E7 oncoprotein inhibits anoikisrdquo Journal ofVirology vol 81 no 17 pp 9419ndash9425 2007
[86] J DeMasi K-W Huh Y Nakatani K Munger and P MHowley ldquoBovine papillomavirus E7 transformation functioncorrelates with cellular p600 protein bindingrdquo Proceedings of theNational Academy of Sciences of theUnited States of America vol102 no 32 pp 11486ndash11491 2005
[87] E A White and P M Howley ldquoProteomic approaches to thestudy of papillomavirusndashhost interactionsrdquo Virology vol 435no 1 pp 57ndash69 2013
[88] FMantovani and L Banks ldquoThe human papillomavirus E6 pro-tein and its contribution to malignant progressionrdquo Oncogenevol 20 no 54 pp 7874ndash7887 2001
[89] P Chen and H Lee ldquoFOXM1 induced by E6 oncoprotein pro-motes tumor invasion and chemoresistance in HPV-infectedlung cancerrdquo Cancer Cell amp Microenvironment vol 2 no 1 pp6ndash9 2015
[90] N Zhang P Wei A Gong et al ldquoFoxM1 promotes 120573-cateninnuclear localization and controls Wnt target-gene expressionand glioma tumorigenesisrdquo Cancer Cell vol 127 no 4 pp 358ndash366 2012
[91] Z Liu Y Liu Y Hong L Rapp E J Androphy and J JChen ldquoBovine papillomavirus type 1 E6-induced sensitizationto apoptosis is distinct from its transforming activityrdquo Virologyvol 295 no 2 pp 230ndash237 2002
[92] M Thomas P Massimi C Navarro J-P Borg and L BanksldquoThe hScribDlg apico-basal control complex is differentiallytargeted by HPV-16 and HPV-18 E6 proteinsrdquo Oncogene vol24 no 41 pp 6222ndash6230 2005
[93] C H Storrs and S J Silverstein ldquoPATJ a tight junction-associated PDZ protein is a novel degradation target of high-risk human papillomavirus E6 and the alternatively splicedisoform 18 E6rdquo Journal of Virology vol 81 no 8 pp 4080ndash40902007
[94] R Accardi R Rubino M Scalise et al ldquoE6 and E7 from humanpapillomavirus type 16 cooperate to target the PDZ proteinNaH exchange regulatory factor 1rdquo Journal of Virology vol 85no 16 pp 8208ndash8216 2011
[95] S Nakagawa and J M Huibregtse ldquoHuman scribble (Vartul)is targeted for ubiquitin-mediated degradation by the high-risk papillomavirus E6 proteins and the E6APubiquitin-proteinligaserdquoMolecular and Cellular Biology vol 20 no 21 pp 8244ndash8253 2000
[96] L Gewin H Myers T Kiyono and D A Galloway ldquoIdentifi-cation of a novel telomerase repressor that interacts with thehuman papillomavirus type-16 E6E6-AP complexrdquo Genes ampDevelopment vol 18 no 18 pp 2269ndash2282 2004
[97] M Sundaram D L GuernseyMM Rajaraman and R Rajara-man ldquoNeosis a novel type of cell division in cancerrdquo CancerBiology ampTherapy vol 3 no 2 pp 207ndash218 2004
[98] R D Snyder and J W Green ldquoA review of the genotoxicity ofmarketed pharmaceuticalsrdquo Mutation Research vol 488 no 2pp 151ndash169 2001
[99] T Ristriani Y Nomine C Laurent E Weiss and G TraveldquoProtein mutagenesis with monodispersity-based quality prob-ing selective inactivation of p53 degradation and DNA-bindingproperties of HPV E6 oncoproteinrdquo Protein Expression andPurification vol 26 no 3 pp 357ndash367 2002
[100] M Kadaja A Sumerina T Verst M Ojarand E Ustav and MUstav ldquoGenomic instability of the host cell induced by thehuman papillomavirus replicationmachineryrdquoTheEMBO Jour-nal vol 26 no 8 pp 2180ndash2191 2007
[101] PMDay D R Lowy and J T Schiller ldquoPapillomaviruses infectcells via a clathrin-dependent pathwayrdquo Virology vol 307 no 1pp 1ndash11 2003
[102] T D Tlsty A Briot A Gualberto et al ldquoGenomic instabilityand cancerrdquoMutation Research vol 337 no 1 pp 1ndash7 1995
[103] J E Eyfjord and S K Bodvarsdottir ldquoGenomic instabilityand cancer networks involved in response to DNA damagerdquoMutation Research vol 592 no 1-2 pp 18ndash28 2005
[104] Y Yao W Huang X Yang et al ldquoHPV-16 E6 and E7 protein Tcell epitopes prediction analysis based on distributions of HLA-A loci across populations an in silico approachrdquo Vaccine vol31 no 18 pp 2289ndash2294 2013
[105] C-F Hung B Ma A Monie S-W Tsen and T-C Wu ldquoTher-apeutic human papillomavirus vaccines current clinical trialsand future directionsrdquo Expert Opinion on Biological Therapyvol 8 no 4 pp 421ndash439 2008
[106] M J P Welters G G Kenter S J Piersma et al ldquoInductionof tumor-specific CD4+ and CD8+ T-cell immunity in cervicalcancer patients by a human papillomavirus type 16 E6 and E7long peptides vaccinerdquo Clinical Cancer Research vol 14 no 1pp 178ndash187 2008
[107] R Han N M Cladel C A Reed X Peng and N D Chris-tensen ldquoProtection of rabbits from viral challenge by gene gun-based intracutaneous vaccination with a combination of cot-tontail rabbit papillomavirus E1 E2 E6 and E7 genesrdquo Journalof Virology vol 73 no 8 pp 7039ndash7043 1999
[108] Z He A P Wlazlo D W Kowalczyk et al ldquoViral recombinantvaccines to the E6 and E7 antigens of HPV-16rdquo Virology vol270 no 1 pp 146ndash161 2000
[109] A Kaufmann P Stern E Rankin et al ldquoSafety and immuno-genicity of TA-HPV a recombinant vaccinia virus expressingmodified human papillomavirus (HPV)-16 and HPV-18 E6 andE7 genes in women with progressive cervical cancerrdquo ClinicalCancer Research vol 8 no 12 pp 3676ndash3685 2002
[110] L K Borysiewicz A Fiander M Nimako et al ldquoA recombinantvaccinia virus encoding human papillomavirus types 16 and 18E6 and E7 proteins as immunotherapy for cervical cancerrdquoTheLancet vol 347 no 9014 pp 1523ndash1527 1996
[111] A De Jong T OrsquoNeill A Y Khan et al ldquoEnhancement ofhuman papillomavirus (HPV) type 16 E6 and E7-specific T-cellimmunity in healthy volunteers through vaccination with TA-CIN an HPV16 L2E7E6 fusion protein vaccinerdquo Vaccine vol20 no 29-30 pp 3456ndash3464 2002
[112] S Chen C Liao Y Lai et al ldquoDe-oncogenicHPVE6E7 vaccinegets enhanced antigenicity and promotes tumoricidal synergywith cisplatinrdquo Acta Biochimica et Biophysica Sinica vol 46 no1 pp 6ndash14 2014
BioMed Research International 15
[113] J Bonhoeffer E B Imoukhuede G Aldrovandi et al ldquoTemplateprotocol for clinical trials investigating vaccines-focus on safetyelementsrdquo Vaccine vol 31 no 47 pp 5602ndash5620 2013
[114] S Jackson and A Storey ldquoE6 proteins from diverse cutaneonsHPV types inhibit apoptosis in response to UV damagerdquoOncogene vol 19 no 4 pp 592ndash598 2000
[115] M Thomas and L Banks ldquoPDZRN3LNX3 is a novel targetof human papillomavirus type 16 (HPV-16) and HPV-18 E6rdquoJournal of Virology vol 89 no 2 pp 1439ndash1444 2015
[116] P R Daniels C M Sanders P Coulson and N J MaitlandldquoMolecular analysis of the interaction between HPV type 16 E6and human E6-associated proteinrdquo FEBS Letters vol 416 no 1pp 6ndash10 1997
[117] W H Kao S L Beaudenon A L Talis J M Huibregtse andP M Howley ldquoHuman papillomavirus type 16 E6 induces self-ubiquitination of the E6AP ubiquitin-protein ligaserdquo Journal ofVirology vol 74 no 14 pp 6408ndash6417 2000
[118] T Iftner M Elbel B Schopp et al ldquoInterference of papillo-mavirus E6 protein with single-strand break repair by interac-tion with XRCC1rdquo The EMBO Journal vol 21 no 17 pp 4741ndash4748 2002
[119] Y Imai Y Tsunokawa T Sugimura and M Terada ldquoPurifi-cation and DNA-binding properties of human papillomavirustype 16 E6 protein expressed in Escherichia colirdquo Biochemicaland Biophysical Research Communications vol 164 no 3 pp1402ndash1410 1989
[120] M Lusky L Berg H Weiher and M Botchan ldquoBovinepapilloma virus contains an activator of gene expression at thedistal end of the early transcription unitrdquoMolecular andCellularBiology vol 3 no 6 pp 1108ndash1122 1983
[121] S Dalal Q Gao E J Androphy and V Band ldquoMutationalanalysis of human papillomavirus type 16 E6 demonstrates thatp53 degradation is necessary for immortalization of mammaryepithelial cellsrdquo Journal of Virology vol 70 no 2 pp 683ndash6881996
[122] J M Huibregtse M Scheffner and P M Howley ldquoA cellularprotein mediates association of p53 with the E6 oncoprotein ofhuman papillomavirus types 16 or 18rdquo The EMBO Journal vol10 no 13 pp 4129ndash4135 1991
[123] M Scheffner B A Werness J M Huibregtse A J Levine andP M Howley ldquoThe E6 oncoprotein encoded by human papillo-mavirus types 16 and 18 promotes the degradation of p53rdquo Cellvol 63 no 6 pp 1129ndash1136 1990
[124] M Elbel S Carl S Spaderna and T Iftner ldquoA comparativeanalysis of the interactions of the E6 proteins from cutaneousand genital papillomaviruses with p53 and E6AP in correlationto their transforming potentialrdquoVirology vol 239 no 1 pp 132ndash149 1997
[125] M C Thomas and C-M Chiang ldquoE6 oncoprotein repressesp53-dependent gene activation via inhibition of protein acety-lation independently of inducing p53 degradationrdquo MolecularCell vol 17 no 2 pp 251ndash264 2005
[126] H Zimmermann R Degenkolbe H-U Bernard and M JOrsquoConnor ldquoThe human papillomavirus type 16 E6 oncoproteincan down-regulate p53 activity by targeting the transcriptionalcoactivator CBPp300rdquo Journal of Virology vol 73 no 8 pp6209ndash6219 1999
[127] D A Thompson G Belinsky T H-T Chang D L Jones RSchlegel and K Munger ldquoThe human papillomavirus-16 E6oncoprotein decreases the vigilance of mitotic checkpointsrdquoOncogene vol 15 no 25 pp 3025ndash3035 1997
[128] J Bohl K Das B Dasgupta and S B Vande Pol ldquoCompetitivebinding to a charged leucine motif represses transformation bya papillomavirus E6 oncoproteinrdquo Virology vol 271 no 1 pp163ndash170 2000
[129] S Song H C Pitot and P F Lambert ldquoThe human papillo-mavirus type 16 E6 gene alone is sufficient to induce carcinomasin transgenic animalsrdquo Journal of Virology vol 73 no 7 pp5887ndash5893 1999
[130] S Yasumoto A L Burkhardt J Doniger and J A DiPaololdquoHumanpapillomavirus type 16DNA-inducedmalignant trans-formation of NIH 3T3 cellsrdquo Journal of Virology vol 57 no 2pp 572ndash577 1986
[131] S B Vande Pol M C Brown and C E Turner ldquoAssociation ofBovine Papillomavirus Type 1 E6 oncoprotein with the focaladhesion protein paxillin through a conserved protein interac-tion motifrdquo Oncogene vol 16 no 1 pp 43ndash52 1998
[132] R Wade N Brimer and S Vande Pol ldquoTransformation bybovine papillomavirus type 1 E6 requires paxillinrdquo Journal ofVirology vol 82 no 12 pp 5962ndash5966 2008
[133] A J Klingelhutz S A Foster and J KMcDougall ldquoTelomeraseactivation by the E6 gene product of humanpapillomavirus type16rdquo Nature vol 380 no 6569 pp 79ndash82 1996
[134] S T Oh S Kyo and L A Laimins ldquoTelomerase activation byhuman papillomavirus type 16 E6 protein Induction of humantelomerase reverse transcriptase expression through Myc andGC-rich Sp1 binding sitesrdquo Journal of Virology vol 75 no 12pp 5559ndash5566 2001
[135] L V Ronco A Y KarpovaMVidal and PMHowley ldquoHumanpapillomavirus 16 E6 oncoprotein binds to interferon regulatoryfactor-3 and inhibits its transcriptional activityrdquo Genes andDevelopment vol 12 no 13 pp 2061ndash2072 1998
(not treated with any drug) (2) positive control (treatedwith 50120583gmL of cyclophosphamide) and (3) experimentalgroup (treated with 1 120583gmL of E6 recombinant oncoproteinresuspended in PBS) This concentration of E6 recombinantoncoproteinwas based on previous study involving BPV early(E) protein as vaccine [67] The protocol of CBMNA wasdone according to the technical recommendation proposedby Araldi et al [53] In detail 02mL of peripheral bloodwas transferred to culture flask containing 50mL RPMI1640 medium supplemented with 15 fetal bovine serum01mL L-glutamine and 01mL phytohemagglutinin A Thematerial was incubated at 37∘C for 8 hours After this bothcyclophosphamide and E6 recombinant oncoprotein wereadded to the culture After 44 hours 02mL of cytochalasin Bwas added to block the cytokinesis After 72 hours the culturewas stopped with the addition of 05mL methanol aceticacid fixative (vv) (3 1) for 5 minutes at room temperatureThematerial was centrifuged at 500 g and the supernatantwasdiscardedThepellet was homogenizedwith 5mLfixative andcentrifuged at 500 g The pellet was aspirated and transferredto slides which were stained with a 1 3 Giemsa phosphatebuffer solution pH = 68 for 8 minutes
After staining coverslips were placed on slides withEntellan (Merck Germany) The material was analyzed ina blind test under an Axiophot binocular microscope (Carl
Zeiss Germany) to observe the frequency ofmicronucleated-binucleated lymphocytes in a total of 1000 analyzed cellsaccording to Araldi et al [53] Statistical analysis was per-formed using the Kruskal-Wallis test followed by a post hocDunn test both at a significance level of 5 Statistical testswere done using the BioEstat software [68]
25 Comet Assay Alkaline Method Slides preparation slidesof 26 times 76mm were dipped in a solution of normal meltingpoint agarose (NMP) (Invitrogen Carlsbad USA) dilutedin phosphate-buffered solution (PBS) 15 at 60∘C and oneside of each slide was wiped clean with a paper towel Theconcentration of NMA was based on Araldi et al [53] Theslides were dried in a horizontal position overnight
Peripheral blood incubation with drugs three wholeblood aliquots of 200120583L each were transferred to three15mL polypropylene tubes (1) negative control (2) positivecontrol and (3) experimental group In each tube 200120583Lof RMPI 1640 medium was added The negative control didnot receive any drug and was incubated only in RPMI 1640medium Positive control was incubated with 50 120583gmL ofcyclophosphamide diluted in RPMI 1640 medium Experi-mental group was treated with 1 120583gmL of BPV-1 E6 recombi-nant protein The samples were incubated at 37∘C for 2 hoursunder constant agitation After this time the aliquots werecentrifuged for 1 minute at 500 g and the supernatant wasdiscarded Ten microliters of each obtained pellet was addedto 75120583L low melting point agarose (LMP) A final volume of85 120583L of this suspensionwas immediately transferred toNMPprecoated slides The slides were covered with coverslips andmaintained at 4∘C for 20 minutes The coverslips were gentlyremoved and the slides were placed in a Coplin jar containinglysis solution (25mM NaCl 100mM ethylenediaminete-traacetic acid (EDTA) 10mM Tris-HCl 11 Triton X-100and 112 dimethyl sulfoxide) at 4∘C for 1 hour Subsequentlyall procedures were conducted under dark conditions toprevent the induction of DNA damage
Electrophoresis after lysis slides were washed with PBSand transferred to an electrophoresis tank containing elec-trophoresis buffer (300mM NaOH and 1mM EDTA pH gt13) at 4∘C for 40 minutes to induce unwinding of double-stranded DNA Next the electrophoretic run was performedwith a current of 25V (086Vcm) 300mA for 20 minutesto promote the migration of free DNA fragments toward theanode The slides were transferred to a Coplin jar containingneutralizing buffer (400mM Tris-HCl pH 75) for 5 minutesThe material was fixed in absolute ethanol for 5 minutes
Comet analysis slides were stained with 20 120583L 4 120583gmLpropidium iodide (PI) and analyzed under a fluorescencemicroscope (Carl Zeiss Axio Scope A1 Germany) equippedwith an excitation filter of 510ndash560 nm and barrier of 590 nmThe material was analyzed in a total magnification of 400xOne hundred nucleoids were analyzed per slide which werescored on a scale of 0 (without DNA damage) to 2 (maximumDNAdamage) according to Araldi et al [53]The scores wereobtained by summing the product of the observed numberof nucleoids per class and its respective class value Statisticalanalysis was performedusing theKruskal-Wallis test followedby the post hocDunn test both with a significance level of 5
BioMed Research International 5
Both test and graphical analysis were done using the BioEstatsoftware [68]
26 Cytokinesis-Block Micronucleus Assay (CBMNA) inEpithelial Cells To verify whether the results observed inPBMCs should also be observed in epithelial cells CBMNAwas performed in CRIB cell (commercial epithelial cell lineobtained from bovine kidney) In detail a total of 1 times 105cells were transferred to six-well plate containing a sterilecoverslip of 24 times 24mm with 2mL of MEM mediumsupplementedwith 10 fetal bovine serum and 1 ampicillinand three cultures had been established negative control(not treated with any drug) positive control (treated with50120583gmL of cyclophosphamide) and experimental group(treatedwith 1120583gmLof BPV-1 E6 recombinant oncoprotein)Cyclophosphamide and BPV-1 E6 recombinant oncoproteinwere added together to the cells After 1 hour the threecultures were treated with 6 120583gmL of cytochalasin B (SigmaGermany)Thematerial was incubated for 48 hours the timenecessary to complete two replication cycles once the dupli-cation time of these cells is 24 hours according to our previ-ous study After this time the medium was removed and thecells were washed twice with PBS at 37∘C Cells were stainedwith solution 1 4 Giemsa-PBS for 3 minutes and afterwashed twice with PBS Coverslips containing the biologicalmaterial were mounted on slides using Entelan (Merck Ger-many) Slides were analyzed by Axiophot binocular micro-scope (Carl Zeiss Germany) to observe the frequency ofmicronucleated cells in a total of 1000 analyzed cells accord-ing to Araldi et al [53]
3 Results
31 Molecular BPV Identification by PCR The peripheralblood samples collected from five asymptomatic calves didnot reveal the presence of BPV sequences using both specificand degenerate primers (Figure 2)
32 Cytokinesis-Block Micronucleus Assay (CBMNA) inPBMCs Frequency analysis of micronucleus observed inCBMNA CBMNA showed an elevated number of micronu-cleated lymphocytes as well as anaphase bridges in bothpositive control and the group treated with 1 120583gmL of E6recombinant oncoprotein (Table 2 Figure 3)
Based on the micronucleated lymphocytes numberobserved per group (Table 2) Chi-square (1205942) test andKruskal-Wallis test were done both with 5 significancelevel Chi-square test pointed out statistical differencesbetween positive control and group treated with E6 (Table 3)These results point out that the cyclophosphamide and E6recombinant oncoprotein are able to induce aneugenesis andor clastogenesis However the Chi-square test did not showstatistic difference in negative control (Table 3)
Kruskal-Wallis test revealed statistical significant differ-ences among the groups (119867 = 97297 and 119901 = 00087)Based on this result Dunn post hoc test was performed Thetest pointed out significant difference between negative andpositive control as well as negative control and experimental
Table 3 Chi-square (1205942) test results showing expected value (119864)freedom degree (FD) and probability (119901)
Group 119864 1205942 FD 119901
Negative control 560 2000 4 07358Positive control 430 10698 4 00302Experimental (E6) 456 12202 4 00159
Table 4 Dunn post hoc test results
Groups Rank differences Calculated 119885 Critical 119885 119901
Cminus and C+ 71 25102 2394 lt005Cndash and E6 79 27931 2394 lt005C+ and E6 08 02828 2394 nsns nonsignificant value
Table 5 Student-Newman-Keuls post hoc test results
Groups Rank differences Calculated 119885 Critical 119885 119901
Cminus and C+ 61 21567 2394 00310Cminus and E6 77 27224 2394 00065C+ and E6 16 05657 2394 05716
group (E6) (Table 4) However the test did not show statis-tical difference between positive control and experimentalgroup (Table 4)
Based on the maximum minimum and median valuesof micronucleus observed in the three groups a boxplotwas done (Figure 4) The graph indicates statistically equalmedians between positive control and experimental group(E6) Although the medians between these groups had notshown statistical differences the graph indicates that theexperimental group showed maximum values of binucleatedlymphocytes with micronucleus higher than positive control
Frequency analysis of anaphase bridge observed inCBMNA based on the number of anaphase bridges (AB)observed per group (Table 3) Kruskal-Wallis test was per-formed which indicates significant differences among thegroups (119867 = 83444 119901 = 00154) Student-Newman-Keuls post hoc test revealed differences between negative andpositive control as well as negative control and experimentalgroup (Table 5) The test did not show differences betweenpositive control and experimental group
Analysis of cytokinesis-block proliferation index (CBPI)and cytotoxicity Kruskal-Wallis test based on theCBPI indi-cates statistical differences among the groups (119867 = 93968and 119901 = 00091) Dunn post hoc test revealed significantdifferences between negative and positive control as well asnegative control and experimental group (Table 6) Howeverthe test did not show differences between positive control andexperimental groupThese data indicate that E6 recombinantoncoprotein has CBPI similar to the cyclophosphamide
Besides presenting high CBPI the E6 recombinant onco-protein also showed to induce endoreduplication evidencedby the presence of intracellular cytokinesis suggesting neosis(Figure 5) according to the criteria proposed by Das et al[69] The CBMNA results suggest that E6 recombinant
6 BioMed Research International
301bp
M 0504030201 M M 0504030201BPV1Cminus
(a) (b)
(c) (d)
(e) (f)
164bp170bp
M 0504030201BPV2Cminus M 0504030201BPV4Cminus
430bp
600bp
M 0504030201BPV2Cminus M 0504030201BPV4Cminus
Figure 2 Electrophoresis gel images showing (a) genomicDNA integrity afterDNAextraction using 1 KbDNALadder (Invitrogen CarlsbadUSA) as marker absence of amplicon in samples 01 to 05 using specific primers to BPV-1 (b) BPV-2 (c) and BPV-4 (d) absence of ampliconin samples 01 to 05 using Delta-Epsilon (e) and Xi (f) degenerate primers Images (b)ndash(f) showed amplification only in positive control withthe 100 bp DNA Ladder (Invitrogen Carlsbad USA) being employed as marker
Table 6 Dunn post hoc test results based on the cytokinesis-blockproliferation index
Group Rank differences Calculated 119885 Critical 119885 119901
Cminus and C+ 76 26870 2394 lt005Cminus and E6 74 26163 2394 lt005C+ and E6 02 00707 2394 nsns nonsignificant differences
oncoprotein induces mitotic stress resulting in clastogenesisand neosis
33 Comet Assay Alkaline Method Comet assay results areshown in Table 7 and Figure 6 According to these data
theKruskal-Wallis test was performedwhich pointed out sta-tistical differences among the groups (119867 = 102613 and 119901 =00059) Based on these results the Dunn post hoc test wasperformed which pointed out significant differences betweennegative and positive control as well as negative controland experimental group (Table 8) However the test did notshow differences between positive control and experimentalgroup (Table 8) These results were also verified by Figure 7Comet assay reinforces the CBMNA results indicating themutagenic potential of E6 recombinant oncoprotein
34 Cytokinesis-Block Micronucleus Assay (CBMNA) inPBMCs Results of this test confirmed the previous resultsindicating that the BPV-1 E6 recombinant oncoprotein has
BioMed Research International 7
LB
(a)
LMN
(b)
LMN
LMN
(c)
PA
PA and MN
(d)
Figure 3 (a) Photomicroscopy of binucleated lymphocyte without micronucleus (LB) observed in negative control Image of binucleatedlymphocyte with micronucleus (LMN) observed in positive control (b) and group treated with E6 recombinant oncoprotein (c) Image ofbinucleated lymphocyte with anaphase bridge and micronucleus (PA and MN) observed in group treated with E6 (d) Images obtained withtotal magnification of 1000x
60
50
40
30
20
10
0
Num
ber o
f mic
ronu
clei
GroupCminus C+ E6
Figure 4 Comparative boxplot based on maximum minimumand median values of micronucleus observed per group
a genotoxicity potential being able to induce DNAbreaks in epithelial cells verified by the presence of MNs(Figure 8)
Figure 5 Evidence of neosis Photomicroscopy of lymphocyteshowing endoreduplication (black arrow) suggesting neosis Cellsanalyzed in total magnification of 1000x
4 Discussion
Transforming potential of E6 oncoprotein has been discussedsince the 1980s based on studies of cottontail rabbit papil-lomaviruses (CRPV) [70 71] Although there are lines ofevidence that the E6 oncoprotein can induce transformation
8 BioMed Research International
(a) (b)
(c) (d)
Figure 6 Images of cometsrsquo nucleoids (a) Image of class 0 (without DNA damage) observed in negative control group Images of class 2(maximum DNA damage) observed in positive control (b) and experimental (c) groups (d) Image of hedgehog comet characterized by thehead absence observed in experimental group Images captured in total magnification of 400x
200
150
100
50
0
Com
et sc
ore
GroupsCminus C+ E6
Figure 7 Boxplot of comet score
[72] the mechanisms of initiation and promotion of cancerassociated with the BPV E6 oncoprotein are unknown
Molecular diagnosis of five peripheral blood samplesfrom five calves showed the absence of amplicons for thefive different primers used (Figure 2) However both specific(BPV-1 BPV-2 and BPV-4) and degenerate (Delta-Epsilonand Xi) primers sets amplified the controls genomes of BPV-1 BPV-2 and BPV-4 None of the negative controls revealedthe amplicon presence These results indicate that the fivesampleswere uninfected by BPVMoreover the use of specific
and degenerate primers increases the capacity of BPV iden-tification This occurs because specific primers are moresensitive than degenerates [66] This sensitivity is due tothe absence of degeneration in the 51015840 region which reducesthe primer ability to recognize and to link with DNA targetsequence [73]The absence of BPV infection in the peripheralblood samples is required for the CBMNA and CA allowinginvestigating the aneugenic andor clastogenic action of BPV-1 E6 recombinant oncoprotein This is because the presenceof BPV sequences is associated with the presence of oncopro-teins transcripts [74]Thus studies have shown that the pres-ence of BPVDNA sequences in peripheral blood is associatedwith cytogenetic damage including clastogenesis [1 14 75]Complementary studies show the association of the BPVpresence with cytogenetic damage in both benign lesions(papillomas) [74] and carcinomas [15]
Chi-square test based on the number of micronucleatedcells pointed out significant statistical values in both positivecontrol and group treated with BPV-1 E6 recombinant onco-protein (Table 3) Negative control showed fewer number ofmicronucleated cells This result was expected because thebiological material was transported and processed after threehours of its collection This procedure reduced the influenceof exogenous environmental factors which could induceDNA damage Furthermore the absence of BPV infection
BioMed Research International 9
(a) (b)
8
6
4
2
0
Num
ber o
f mic
ronu
cleat
ed ce
lls
Neg
ativ
e con
trol
Posit
ive c
ontro
l
E6on
copr
otei
n
(c)
Figure 8 Photomicroscope showing CRIB cell not treated with any drug (negative control) indicating absence of micronucleus (a) andtreated with BPV-1 E6 recombinant oncoprotein showing the presence of MN indicated by arrow (b) (c) Number of micronucleated cellsobserved in each group
in the peripheral blood reduces the influence of endogenousenvironmental factors that could interfere in the analysisPositive control showed a high number of micronucle-ated lymphocytes This result was also expected once thisgroup was treated with 50 120583gmL of cyclophosphamide achemotherapeutic drug with cytotoxic and teratogenic effect[76 77] The cells treated with 1120583gmL of BPV-1 E6 recom-binant oncoprotein also showed significant statistical valuesKruskal-Wallis test followed by the Dunn post hoc testwas performed to compare the frequencies of micronucleusamong three groups This test indicated statistical differencebetween negative control and the cells treated with BPV-1 E6 recombinant oncoprotein However the test did notreveal differences between the cells treated with cyclophos-phamide and BPV-1 E6 recombinant oncoprotein (Table 4Figure 4) Similar results were also observed in epithelial cells(Figure 8) These results suggest that the BPV-1 E6 oncopro-tein has an aneugenic andor clastogenic potential similarto or higher than the observed with the cyclophosphamideThis is because the cells treated with the oncoprotein showed
a frequency of micronucleus formation (MN1199030) 14003
higher than those observed in positive controlThe elevated mutagenic potential of the BPV-1 E6 onco-
protein can justify the cytogenetic damage already describedin the literature [1 14 15 24] Similar results were observedin HPV-infected cells [12 78ndash81] The micronucleus induce-ment was already described in cytological samples collectedduring Papanicolaursquos test from healthy cervix infected byHPV [12] For this reason the micronucleus assay has beenproposed as a complementary method for Papanicolaursquos testbeing a suggestive biomarker of lesion degree [78] Howeverthe association between the BPV and the micronucleusinducement was not yet reported
The high frequency of micronucleated lymphocytes insamples infected by HPV has been attributed to the synergicaction of E5 E6 and E7 oncoproteins [29 30 78 79]However this is the first study that pointed out themutagenicaction of BPV-1 E6 protein per se
Student-Newman-Keuls post hoc test based on the fre-quency of anaphase bridges showed significant statistical
10 BioMed Research International
Table 7 Comet assay results showing the number of nucleoidsobserved per class the number of hedgehog comets and cometscore
Sample Class 0 Class 1 Class 2 Hedgehog ScoreNegative control
Groups Rank differences Calculated 119885 Critical 119885 119901
Cminus and C+ 68 24042 2394 lt05Cminus and E6 82 28991 2394 lt05C+ and E6 14 04950 2394 nsns nonsignificant differences
difference between the negative control and the group treatedwith the BPV-1 E6 recombinant oncoprotein (Table 5) How-ever the test did not reveal differences between the cellstreated with cyclophosphamide and BPV-1 E6 recombinantoncoprotein (Table 5)These data reinforce those observed bythe Dunn post hoc test based on the frequency of micronu-cleus indicating the clastogenic action of the E6 oncoproteinMoreover the presence of anaphase bridge was alreadydescribed in cells transfected with recombinant adenoviruscontaining the E6 and E7 ORFs of HPV [29 30] Howeverthere are no studies describing the action of BPVE6 oncopro-tein in the induction of anaphase bridges or studies that showthis same action of E6 oncoprotein of HPV per se Anaphasebridges are indicators of genomic instability [82 83] which isconsidered the first step in carcinogenesis [46 47] Moreoverthe presence of these bridges is an important hallmark ofDNA double strand breaks (DSBs) [84]
Statistical analysis based on the cytokinesis-block prolif-eration index (CBPI) showed that BPV-1 E6 oncoprotein hascytotoxic levels similar to cyclophosphamide (Table 6) Thisresult suggests that the BPV E6 oncoprotein can deregulatethe cell cycle contributing to the cell proliferation andimmortalization To date this mitogenic action is attributedto the E7 oncoprotein [85ndash87] E7 oncoprotein induces
the phosphorylation of pRb resulting in the E2F transcrip-tion factor release [85] E2F when translocated to the nucleusacts as activator and binds to the kinase-dependent cyclinspromoters [85] However the results showed polynucleatedcells in the group treatedwithBPVE6oncoprotein (Figure 5)suggesting that the BPV E6 oncoprotein confers mitogenicstimulation resulting in mitotic stress Moreover studiespointed out that the BPV E6 oncoprotein interacts with theCBPp300 deacetylase promoting p53 downregulation [88]This downregulation increases the expression levels of FoxM1transcription factor (Foxhead box M1) [89] promoting B1cyclin D1 cyclin and cdc25 upregulation [89] The upregu-lation of these genes is associated with increased levels of cellproliferation which is necessary in order to make DNA poly-merases available to virus replication However if this mech-anism guarantees the BPV replication it can contribute to cellimmortalization and cancer progression
The FoxM1 factor also participates in the Wnt120573-cateninsignaling pathway and binds directly to the 120573-catenin [89]This interaction promotes the nuclear 120573-catenin transloca-tion [90] 120573-catenin translocation to nucleus induces thecyclin expression [64] So these data suggest that the BPVE6 oncoprotein not only promotes mitogenic stress but alsocontributes to the epithelial-mesenchymal transition (EMT)Studies based on theHPVE6 oncoprotein have indicated thatthis oncoprotein contributes significantly to the EMT [91]This occurs not only because the E6 oncoprotein induces thetranslocation of 120573-catenin but also due to the proteasomaldegradation of regulatory proteins of apical-basal polarity[92ndash94] Studies also showed that the E6 oncoprotein is ableto bind to Crumbs (Dlg and Patj) proteins resulting in loss ofcell polarity [93 94] Based on these results the E6 oncopro-tein emerges as a possible therapeutic target with biotechno-logical value in cancer treatment
Mitogenic action of E6 oncoprotein can be moreexpressed in cells infected by papillomaviruses once thesecells also expressed E5 and E7 oncoproteins [74] The E7oncoprotein is able to form a complex E7-p600 which pro-motes E6 upregulation [85] Furthermore the E6 oncoproteincan bind to the E6AP ubiquitin ligase forming the E6-E6APcomplex increasing the hTERT levels of expression con-tributing to cell immortalization [95] Moreover E6-E6APinduces NFX1 expression promoting MHC-II downregula-tion [96]Thismechanism reduces the antigenic presentationmediated by CD4+ T lymphocytes contributing to immuneevasion This action guarantees the PV infection for longperiods with or without clinical symptoms [24] Althoughthe BPV infection can be asymptomatic the BPV presence inperipheral blood is associated with DNA damage [1 14 24]
CBMNA analysis pointed out the presence of cells withintracellular cytokinesis (Figure 5) This result suggests theneotic action of BPV E6 oncoprotein Neosis is characterizedby the presence of (1) DNA damage (2) loss of checkpointcontrol (3) repair system failures and (4) endoreduplication[97] In this scenario the E6 oncoprotein attempts all theseneosis criteria Moreover the oncoprotein not only increasesthe cytogenetic damage leading to increasing of micronu-cleus frequency but also induces clastogenesis as shown bythe comet assay and anaphase bridges So statistical analysis
BioMed Research International 11
performed based on the comet scores reinforced the CBMNAresults Comet assay allows detecting both simple and dou-ble DNA breaks being more sensitive than the CBMNAClastogenesis is considered the most serious type of DNAdamage [98] DNA breaks can be repaired by homologous ornonhomologous recombination However studies point outthat the E6 oncoprotein can link to the Holliday junctionduring the homologous repair avoiding the junction reso-lution [99] Moreover E6 oncoprotein promotes the TP53gene deacetylation resulting in p53 downregulation [51]Thisepigenetic effect results in cell cycle deregulation affectingthe checkpoints
Studies have shown that the HPV-induced DNA breaksare required for the virus integration into the host genome[100] Although there are no studies showing the BPV inte-gration to date the results observed in this work in additionto those already published [24] indicate the necessity ofmorestudies to evaluate the virus-host interaction
Studies also showed that the E6 oncoprotein inducesclathrin-transporter adapter protein (AP-1) increasing thelevel of this protein in the plasma membrane [76 77] Theincreasing quantity of AP-1 in membrane can facilitate theBPV virions infection This occurs because the infectionprocess is clathrin dependent In this scenario the E6 onco-protein can contribute to the virus infection [101]
In summary the CBMNA and CA results showed thatthe BPV E6 oncoprotein participates not only in cancerpromotion but also in initiation inducing DNA breaksThese DNA breaks represent mutagenic effects consideredthe first step in the oncogenesis process which are associatedwith genomic instability [47 48 102 103] Thus the E6recombinant oncoprotein has been suggested as a possiblevaccine candidate [104ndash112] due to its immunogenicity [44]However the mutagenic tests such as CBMNA and CA arerequired in drug validation process [113] In this scenariothis study pointed out that the BPV-1 E6 recombinant onco-protein in tested concentration showedmutagenic potentialThis is the first study that reports the mutagenic potential ofa possible therapeutic vaccine candidate On the one handthese results allowed better understanding the mechanism ofcancer promotion associatedwith the BPVE6oncoprotein aswell as revealing that this oncoprotein can induce carcinogen-esis per se on the other hand this data pointed out thatmaybeBPV E6 recombinant oncoprotein requires protein modifica-tions to be used as vaccine
Conflict of Interests
The authors declare that there is no conflict of interests
Acknowledgments
Theauthors thank Fundacao deAmparo a Pesquisa do Estadode Sao Paulo (FAPESP Process 201420617-5) and FundacaoButantan for the financial support
References
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experimental modelrdquo Journal of General Virology vol 79 no 9pp 2127ndash2135 1998
[2] A Antonsson and B G Hansson ldquoHealthy skin ofmany animalspecies harbors papillomaviruses which are closely related totheir human counterpartsrdquo Journal of Virology vol 76 no 24pp 12537ndash12542 2002
[3] P Rous and J W Beard ldquoA virus-induced mammalian growthwith the characters of a tumor (the Shope rabbit papilloma) IThe growth on implantationwithin favorable hostsrdquoThe Journalof Experimental Medicine vol 60 no 6 pp 701ndash722 1934
[4] R Shope and E Hurst ldquoInfectious papillomatosis of rabbitsrdquoThe Journal of Experimental Medicine vol 58 no 1 pp 607ndash624 1933
[5] H Delius M A Van Ranst A B Jenson H Zur Hausen and JP Sundberg ldquoCanine oral papillomavirus genomic sequence aunique 15-kb intervening sequence between the E2 andL2 openreading framesrdquo Virology vol 204 no 1 pp 447ndash452 1994
[6] W A Demonbreun and E Goodpasture ldquoInfectious oral papil-lomatosis of dogsrdquo The American Journal of Pathology vol 8no 1 pp 43ndash56 1932
[7] P K Nicholls B A Klaunberg R A Moore et al ldquoNaturallyoccurring nonregressing canine oral papillomavirus infectionhost immunity virus characterization and experimental infec-tionrdquo Virology vol 265 no 2 pp 365ndash374 1999
[8] A Alberti S Pirino F Pintore et al ldquoOvis aries Papillomavirus3 a prototype of a novel genus in the family Papillomaviridaeassociated with ovine squamous cell carcinomardquo Virology vol407 no 2 pp 352ndash359 2010
[9] H zur Hausen E-M de Villiers and L Gissmann ldquoPapil-lomavirus infections and human genital cancerrdquo GynecologicOncology vol 12 no 2 pp S124ndashS128 1981
[10] C Foresta A Bertoldo A Garolla et al ldquoHuman papillo-mavirus proteins are found in peripheral blood and semenCd20+ and Cd56+ cells during Hpv-16 semen infectionrdquo BMCInfectious Diseases vol 13 article 593 2013
[11] S Ljubojevic andM Skerlev ldquoHPV-associated diseasesrdquo Clinicsin Dermatology vol 32 no 2 pp 227ndash234 2014
[12] A P R Cassel R B Barcellos C M D da Silva S E de MatosAlmeida and M L R Rossetti ldquoAssociation between humanpapillomavirus (HPV) DNA and micronuclei in normal cervi-cal cytologyrdquo Genetics and Molecular Biology vol 37 no 2 pp360ndash363 2014
[13] W Jarrett M Campo B OrsquoNeil H Laird and L Coggins ldquoAnovel bovine papillomavirus (BPV-6) causing true epithelialpapillomas of the mammary gland skin a member of a pro-posed newBPV subgrouprdquoVirology vol 136 no 2 pp 255ndash2641984
[14] T CMelo N Diniz S R C Campos et al ldquoCytogenetic studiesin peripheral blood of bovines afflicted by papillomatosisrdquoVeterinary and Comparative Oncology vol 9 no 4 pp 269ndash2742011
[15] S R C Campos T C Melo S Assaf et al ldquoChromosome aber-rations in cells infected with bovine papillomavirus comparingcutaneous papilloma esophagus papilloma and urinary blad-der lesion cellsrdquo ISRN Oncology vol 2013 Article ID 910849 8pages 2013
[16] H zur Hausen ldquoThe search for infectious causes of humancancers where and whyrdquoVirology vol 392 no 1 pp 1ndash10 2009
[17] R P Araldi R F Carvalho T C Melo et al ldquoBovine papillo-mavirus in beef cattle first description of BPV-12 and putativetype BAPV8 in BrazilrdquoGenetics andMolecular Research vol 13no 3 pp 5644ndash5653 2014
12 BioMed Research International
[18] T Rampias C Sasaki and A Psyrri ldquoMolecular mechanisms ofHPV induced carcinogenesis in head and neckrdquoOral Oncologyvol 50 no 5 pp 356ndash363 2014
[19] S Roperto V Russo L Leonardi et al ldquoBovine papillomavirustype 13 expression in the urothelial bladder tumours of cattlerdquoTransboundary and Emerging Diseases 2015
[20] S RCCampos C TrindadeO P Ferraz et al ldquoCan establishedcultured papilloma cells harbor bovine papillomavirusrdquoGenet-ics and Molecular Research vol 7 no 4 pp 1119ndash1126 2008
[21] M S Campo ldquoAnimalmodels of papilloma virus pathogenesisrdquoVirus Research vol 89 no 2 pp 249ndash261 2002
[22] G Borzacchiello ldquoBovine papillomavirus infections in ani-malsrdquo in Communicating Current Research and EducationalTopics and Trends in AppliedMicrobiology pp 673ndash679 Forma-tex Badajoz Spain 2007
[23] F Bocaneti G Altamura A Corteggio E Velescu F RopertoandG Borzacchiello ldquoBovine papillomavirus new insights intoan old diseaserdquo Transboundary and Emerging Diseases 2014
[24] R P Araldi T C Melo N Diniz et al ldquoBovine papillomavirusclastogenic effect analyzed in comet assayrdquo BioMed ResearchInternational vol 2013 Article ID 630683 7 pages 2013
[25] J Munday N Thomson M Dunowska C Knight R Laurieand S Hills ldquoGenomic characterisation of the feline sarcoid-associated papillomavirus and proposed classification as Bostaurus papillomavirus type 14rdquoVeterinaryMicrobiology vol 177no 3-4 pp 289ndash295 2015
[26] G Borzacchiello V Russo C Spoleto et al ldquoBovine papillo-mavirus type-2 DNA and expression of E5 and E7 oncoproteinsin vascular tumours of the urinary bladder in cattlerdquo CancerLetters vol 250 no 1 pp 82ndash91 2007
[27] P Maiolino A Ozkul A Sepici-Dincel et al ldquoBovine papillo-mavirus type 2 infection and microscopic patterns of urothelialtumors of the urinary bladder in water buffaloesrdquo BioMedResearch International vol 2013 Article ID 937918 6 pages2013
[28] S Roperto G Borzacchiello R Brun et al ldquoMultiple glomustumors of the urinary bladder in a cow associated with bovinepapillomavirus type 2 (BPV-2) infectionrdquo Veterinary Pathologyvol 45 no 1 pp 39ndash42 2008
[29] S Duensing and K Munger ldquoThe human papillomavirus type16 E6 and E7 oncoproteins independently induce numerical andstructural chromosome instabilityrdquoCancer Research vol 62 no23 pp 7075ndash7082 2002
[30] S Duensing L Y Lee A Duensing et al ldquoThe human papil-lomavirus type 16 E6 and E7 oncoproteins cooperate to inducemitotic defects and genomic instability by uncoupling centro-some duplication from the cell division cyclerdquo Proceedings of theNational Academy of Sciences of theUnited States of America vol97 no 18 pp 10002ndash10007 2000
[31] C A Moody and L A Laimins ldquoHuman papillomavirus onco-proteins pathways to transformationrdquo Nature Reviews Cancervol 10 no 8 pp 550ndash560 2010
[32] C Mansur and E Androphy ldquoCellular transformation bypapillomavirus oncoproteinsrdquo Biochimica et Biophysica Actavol 1155 no 3 pp 323ndash345 1993
[33] J J Chen Y Hong and E J Androphy ldquoMutational analysis oftranscriptional activation by the bovine papillomavirus type 1E6rdquo Virology vol 236 no 1 pp 30ndash36 1997
[34] X Tong andPMHowley ldquoThebovine papillomavirus E6 onco-protein interacts with paxillin and disrupts the actin cytoskele-tonrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 94 no 9 pp 4412ndash4417 1997
[35] X TongW Boll T Kirchhausen andPMHowley ldquoInteractionof the bovine papillomavirus E6 protein with the clathrinadaptor complex AP-1rdquo Journal of Virology vol 72 no 1 pp476ndash482 1998
[36] Y Nomine M Masson S Charbonnier et al ldquoStructural andfunctional analysis of E6 oncoprotein insights in the molecularpathways of human papillomavirus-mediated pathogenesisrdquoMolecular Cell vol 21 no 5 pp 665ndash678 2006
[37] T Ristriani MMasson Y Nomine et al ldquoHPV oncoprotein E6is a structure-dependent DNA-binding protein that recognizesfour-way junctionsrdquo Journal of Molecular Biology vol 296 no5 pp 1189ndash1203 2000
[38] Y Liu and J D Baleja ldquoStructure and function of the papil-lomavirus E6 protein and its interacting proteinsrdquo Frontiers inBioscience vol 13 no 1 pp 121ndash134 2008
[39] F Lipari G A McGibbon E Wardrop and M G CordingleyldquoPurification and biophysical characterization of a minimalfunctional domain and of an N-terminal Zn2+-binding frag-ment from the human papillomavirus type 16 E6 proteinrdquoBiochemistry vol 40 no 5 pp 1196ndash1204 2001
[40] Y Nomine T Ristriani C Laurent J-F Lefevre E Weissand G Trave ldquoA strategy for optimizing the monodispersity offusion proteins application to purification of recombinantHPVE6 oncoproteinrdquo Protein Engineering vol 14 no 4 pp 297ndash3052001
[41] C G Ullman P I Haris D A Galloway V C Emery and SJ Perkins ldquoPredicted 120572-helix120573-sheet secondary structures forthe zinc-binding motifs of human papillomavirus E7 and E6proteins by consensus prediction averaging and spectroscopicstudies of E7rdquo Biochemical Journal vol 319 no 1 pp 229ndash2391996
[42] A Sidi K Babah N Brimer et al ldquoStrategies for bacterialexpression of protein-peptide complex application to solubi-lization of papillomavirus E6rdquo Protein Expression and Purifica-tion vol 29 no 6 pp 997ndash1003 2011
[43] K Zanier S Charbonnier A O M O Sidi et al ldquoStructuralbasis for hijacking of cellular LxxLL motifs by papillomavirusE6 oncoproteinsrdquo Science vol 339 no 6120 pp 694ndash698 2013
[44] J Mazzuchelli-de-Souza R F Carvalho R M Ruiz et alldquoExpression and in silico analysis of the recombinant bovinepapillomavirus e6 protein as a model for viral oncoproteinsstudiesrdquo BioMed Research International vol 2013 Article ID421398 9 pages 2013
[45] L Banks P Spence E Androphy et al ldquoIdentification of humanpapillomavirus type 18 E6 polypeptide in cells derived fromhuman cervical carcinomasrdquo Journal of General Virology vol68 no 5 pp 1351ndash1359 1987
[46] D Hanahan and R AWeinberg ldquoHallmarks of cancer the nextgenerationrdquo Cell vol 144 no 5 pp 646ndash674 2011
[47] G S Charames and B Bapat ldquoGenomic instability and cancerrdquoCurrent Molecular Medicine vol 3 no 7 pp 589ndash596 2003
[48] S Negrini V G Gorgoulis and T D Halazonetis ldquoGenomicinstabilitymdashan evolving hallmark of cancerrdquo Nature ReviewsMolecular Cell Biology vol 11 no 3 pp 220ndash228 2010
[49] K Zanier S Charbonnier M Baltzinger Y Nomine DAltschuh and G Trave ldquoKinetic analysis of the interactionsof human papillomavirus E6 oncoproteins with the ubiquitinligase E6AP using surface plasmon resonancerdquo Journal ofMolecular Biology vol 349 no 2 pp 401ndash412 2005
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[51] H Zimmermann C-H Koh R Degenkolbe et al ldquoInteractionwith CBPp300 enables the bovine papillomavirus type 1 E6oncoprotein to downregulate CBPp300-mediated transactiva-tion by p53rdquo Journal of General Virology vol 81 no 11 pp 2617ndash2623 2000
[52] M Fenech ldquoThe in vitro micronucleus techniquerdquo MutationResearchmdashFundamental and Molecular Mechanisms of Mutage-nesis vol 455 no 1-2 pp 81ndash95 2000
[53] R P Araldi T C de Melo T B Mendes et al ldquoUsing the cometand micronucleus assays for genotoxicity studies a reviewrdquoBiomedicine amp Pharmacotherapy vol 72 pp 74ndash82 2015
[54] P Thomas and M Fenech ldquoCytokinesis-block micronucleuscytome assay in lymphocytesrdquo Methods in Molecular Biologyvol 682 pp 217ndash234 2011
[55] M Kirsch-Volders T Sofuni M Aardema et al ldquoReport fromthe in vitro micronucleus assay working grouprdquo MutationResearch Genetic Toxicology and Environmental Mutagenesisvol 540 no 2 pp 153ndash163 2003
[56] S Samanta and P Dey ldquoMicronucleus and its applicationsrdquoDiagnostic Cytopathology vol 40 no 1 pp 84ndash90 2012
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[58] A Azqueta and A R Collins ldquoThe essential comet assay acomprehensive guide to measuring DNA damage and repairrdquoArchives of Toxicology vol 87 no 6 pp 949ndash968 2013
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[64] A Yaguiu M L Z Dagli E H Birgel Jr et al ldquoSimultaneouspresence of bovine papillomavirus and bovine leukemia virusin different bovine tissues in situ hybridization and cytogeneticanalysisrdquoGenetics andMolecular Research vol 7 no 2 pp 487ndash497 2008
[65] T C Melo R F Carvalho J Mazzucchelli-de-Souza et alldquoPhylogenetic classification and clinical aspects of a new puta-tive Deltapapillomavirus associated with skin lesions in cattlerdquoGenetics and Molecular Research vol 13 no 2 pp 2458ndash24692014
[66] R P Araldi D N S Giovanni T CMelo et al ldquoBovine papillo-mavirus isolation by ultracentrifugationrdquo Journal of VirologicalMethods vol 208 pp 119ndash124 2014
[67] L M Chandrachud B W OrsquoNeil W F H Jarrett G J Grind-lay G M McGarvie and M S Campo ldquoHumoral immuneresponse to the E7 protein of bovine papillomavirus type 4 andidentification of B-cell epitopesrdquoVirology vol 200 no 1 pp 98ndash104 1994
[68] M Ayres M J Ayres D L Ayres and A A Santos BioEstatmdashAplicacoes Estatısticas nas Areas de Ciencias Bio-Medicas Insti-tuto Mamiraua Belem Brazil 2007
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[70] O Danos E Georges G Orth and M Yaniv ldquoFine structureof the cottontail rabbit papillomavirus mRNAs expressed in thetransplantable VX2 carcinomardquo Journal of Virology vol 53 no3 pp 735ndash741 1985
[71] M Nasseri and F O Wettstein ldquoDifferences exist between viraltranscripts in cottontail rabbit papillomavirus-induced benignand malignant tumors as well as non-virus-producing andvirus-producing tumorsrdquo Journal of Virology vol 51 no 3 pp706ndash712 1984
[72] X Tong R Salgia J-L Li J D Griffin and P M Howley ldquoThebovine papillomavirus E6 protein binds to the LDmotif repeatsof paxillin and blocks its interaction with vinculin and the focaladhesion kinaserdquo Journal of Biological Chemistry vol 272 no52 pp 33373ndash33376 1997
[73] Y Maeda T Shibahara Y Wada et al ldquoAn outbreak of teatpapillomatosis in cattle caused by bovine papilloma virus (BPV)type 6 and unclassified BPVsrdquo Veterinary Microbiology vol 121no 3-4 pp 242ndash248 2007
[74] R P Araldi T C Melo A Neves et al ldquoHyperproliferativeaction of bovine papillomavirus genetic and histopathologicalaspectsrdquo Genetics and Molecular Research vol 14 no 4 pp12942ndash12954 2015
[75] NDiniz T CMelo J F Santos et al ldquoSimultaneous presence ofbovine papillomavirus in blood and in short-term lymphocytecultures from dairy cattle in Pernambuco Brazilrdquo Genetics andMolecular Research vol 8 no 4 pp 1474ndash1480 2009
[76] O M Brito M F Guimaraes and C C Lanna ldquoCiclofosfamidae funcao ovarianardquo Revista Brasileira de Reumatologia vol 48no 1 pp 39ndash45 2008
[77] D Anderson T-W Yu and D B McGregor ldquoComet assayresponses as indicators of carcinogen exposurerdquo Mutagenesisvol 13 no 6 pp 539ndash555 1998
[78] M L Adam C Pini S Tulio J C L L Cantalice R A Torresand M T Dos Santos Correia ldquoAssessment of the associationbetween micronuclei and the degree of uterine lesions and viralload in women with human papillomavirusrdquo Cancer Genomicsamp Proteomics vol 12 no 2 pp 67ndash72 2015
[79] E Cortes-Gutierrez M Davila-Rodrıguez J Vargas-VillarrealF Hernandez-Garza and R Cerda-Flores ldquoAssociationbetween human papilloma virus-type infections with micro-nuclei frequenciesrdquo Prague Medical Report vol 111 no 1 pp35ndash41 2010
[80] R N Chakrabarti and K Dutta ldquoMicronuclei test in routinesmears fromuterine cervixrdquo European Journal of GynaecologicalOncology vol 9 no 5 pp 370ndash372 1988
[81] E M M Cerqueira C L Santoro N F Donozo et al ldquoGeneticdamage in exfoliated cells of the uterine cervix Association
14 BioMed Research International
and interaction between cigarette smoking and progression tomalignant transformationrdquo Acta Cytologica vol 42 no 3 pp639ndash649 1998
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[83] D R Hoffelder L Luo N A Burke S CWatkins S M Gollinand W S Saunders ldquoResolution of anaphase bridges in cancercellsrdquo Chromosoma vol 112 no 8 pp 389ndash397 2004
[84] C Acilan D M Potter and W S Saunders ldquoDNA repairpathways involved in anaphase bridge formationrdquo Genes Chro-mosomes and Cancer vol 46 no 6 pp 522ndash531 2007
[85] J DeMasi M C Chao A S Kumar and P M Howley ldquoBovinepapillomavirus E7 oncoprotein inhibits anoikisrdquo Journal ofVirology vol 81 no 17 pp 9419ndash9425 2007
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[87] E A White and P M Howley ldquoProteomic approaches to thestudy of papillomavirusndashhost interactionsrdquo Virology vol 435no 1 pp 57ndash69 2013
[88] FMantovani and L Banks ldquoThe human papillomavirus E6 pro-tein and its contribution to malignant progressionrdquo Oncogenevol 20 no 54 pp 7874ndash7887 2001
[89] P Chen and H Lee ldquoFOXM1 induced by E6 oncoprotein pro-motes tumor invasion and chemoresistance in HPV-infectedlung cancerrdquo Cancer Cell amp Microenvironment vol 2 no 1 pp6ndash9 2015
[90] N Zhang P Wei A Gong et al ldquoFoxM1 promotes 120573-cateninnuclear localization and controls Wnt target-gene expressionand glioma tumorigenesisrdquo Cancer Cell vol 127 no 4 pp 358ndash366 2012
[91] Z Liu Y Liu Y Hong L Rapp E J Androphy and J JChen ldquoBovine papillomavirus type 1 E6-induced sensitizationto apoptosis is distinct from its transforming activityrdquo Virologyvol 295 no 2 pp 230ndash237 2002
[92] M Thomas P Massimi C Navarro J-P Borg and L BanksldquoThe hScribDlg apico-basal control complex is differentiallytargeted by HPV-16 and HPV-18 E6 proteinsrdquo Oncogene vol24 no 41 pp 6222ndash6230 2005
[93] C H Storrs and S J Silverstein ldquoPATJ a tight junction-associated PDZ protein is a novel degradation target of high-risk human papillomavirus E6 and the alternatively splicedisoform 18 E6rdquo Journal of Virology vol 81 no 8 pp 4080ndash40902007
[94] R Accardi R Rubino M Scalise et al ldquoE6 and E7 from humanpapillomavirus type 16 cooperate to target the PDZ proteinNaH exchange regulatory factor 1rdquo Journal of Virology vol 85no 16 pp 8208ndash8216 2011
[95] S Nakagawa and J M Huibregtse ldquoHuman scribble (Vartul)is targeted for ubiquitin-mediated degradation by the high-risk papillomavirus E6 proteins and the E6APubiquitin-proteinligaserdquoMolecular and Cellular Biology vol 20 no 21 pp 8244ndash8253 2000
[96] L Gewin H Myers T Kiyono and D A Galloway ldquoIdentifi-cation of a novel telomerase repressor that interacts with thehuman papillomavirus type-16 E6E6-AP complexrdquo Genes ampDevelopment vol 18 no 18 pp 2269ndash2282 2004
[97] M Sundaram D L GuernseyMM Rajaraman and R Rajara-man ldquoNeosis a novel type of cell division in cancerrdquo CancerBiology ampTherapy vol 3 no 2 pp 207ndash218 2004
[98] R D Snyder and J W Green ldquoA review of the genotoxicity ofmarketed pharmaceuticalsrdquo Mutation Research vol 488 no 2pp 151ndash169 2001
[99] T Ristriani Y Nomine C Laurent E Weiss and G TraveldquoProtein mutagenesis with monodispersity-based quality prob-ing selective inactivation of p53 degradation and DNA-bindingproperties of HPV E6 oncoproteinrdquo Protein Expression andPurification vol 26 no 3 pp 357ndash367 2002
[100] M Kadaja A Sumerina T Verst M Ojarand E Ustav and MUstav ldquoGenomic instability of the host cell induced by thehuman papillomavirus replicationmachineryrdquoTheEMBO Jour-nal vol 26 no 8 pp 2180ndash2191 2007
[101] PMDay D R Lowy and J T Schiller ldquoPapillomaviruses infectcells via a clathrin-dependent pathwayrdquo Virology vol 307 no 1pp 1ndash11 2003
[102] T D Tlsty A Briot A Gualberto et al ldquoGenomic instabilityand cancerrdquoMutation Research vol 337 no 1 pp 1ndash7 1995
[103] J E Eyfjord and S K Bodvarsdottir ldquoGenomic instabilityand cancer networks involved in response to DNA damagerdquoMutation Research vol 592 no 1-2 pp 18ndash28 2005
[104] Y Yao W Huang X Yang et al ldquoHPV-16 E6 and E7 protein Tcell epitopes prediction analysis based on distributions of HLA-A loci across populations an in silico approachrdquo Vaccine vol31 no 18 pp 2289ndash2294 2013
[105] C-F Hung B Ma A Monie S-W Tsen and T-C Wu ldquoTher-apeutic human papillomavirus vaccines current clinical trialsand future directionsrdquo Expert Opinion on Biological Therapyvol 8 no 4 pp 421ndash439 2008
[106] M J P Welters G G Kenter S J Piersma et al ldquoInductionof tumor-specific CD4+ and CD8+ T-cell immunity in cervicalcancer patients by a human papillomavirus type 16 E6 and E7long peptides vaccinerdquo Clinical Cancer Research vol 14 no 1pp 178ndash187 2008
[107] R Han N M Cladel C A Reed X Peng and N D Chris-tensen ldquoProtection of rabbits from viral challenge by gene gun-based intracutaneous vaccination with a combination of cot-tontail rabbit papillomavirus E1 E2 E6 and E7 genesrdquo Journalof Virology vol 73 no 8 pp 7039ndash7043 1999
[108] Z He A P Wlazlo D W Kowalczyk et al ldquoViral recombinantvaccines to the E6 and E7 antigens of HPV-16rdquo Virology vol270 no 1 pp 146ndash161 2000
[109] A Kaufmann P Stern E Rankin et al ldquoSafety and immuno-genicity of TA-HPV a recombinant vaccinia virus expressingmodified human papillomavirus (HPV)-16 and HPV-18 E6 andE7 genes in women with progressive cervical cancerrdquo ClinicalCancer Research vol 8 no 12 pp 3676ndash3685 2002
[110] L K Borysiewicz A Fiander M Nimako et al ldquoA recombinantvaccinia virus encoding human papillomavirus types 16 and 18E6 and E7 proteins as immunotherapy for cervical cancerrdquoTheLancet vol 347 no 9014 pp 1523ndash1527 1996
[111] A De Jong T OrsquoNeill A Y Khan et al ldquoEnhancement ofhuman papillomavirus (HPV) type 16 E6 and E7-specific T-cellimmunity in healthy volunteers through vaccination with TA-CIN an HPV16 L2E7E6 fusion protein vaccinerdquo Vaccine vol20 no 29-30 pp 3456ndash3464 2002
[112] S Chen C Liao Y Lai et al ldquoDe-oncogenicHPVE6E7 vaccinegets enhanced antigenicity and promotes tumoricidal synergywith cisplatinrdquo Acta Biochimica et Biophysica Sinica vol 46 no1 pp 6ndash14 2014
BioMed Research International 15
[113] J Bonhoeffer E B Imoukhuede G Aldrovandi et al ldquoTemplateprotocol for clinical trials investigating vaccines-focus on safetyelementsrdquo Vaccine vol 31 no 47 pp 5602ndash5620 2013
[114] S Jackson and A Storey ldquoE6 proteins from diverse cutaneonsHPV types inhibit apoptosis in response to UV damagerdquoOncogene vol 19 no 4 pp 592ndash598 2000
[115] M Thomas and L Banks ldquoPDZRN3LNX3 is a novel targetof human papillomavirus type 16 (HPV-16) and HPV-18 E6rdquoJournal of Virology vol 89 no 2 pp 1439ndash1444 2015
[116] P R Daniels C M Sanders P Coulson and N J MaitlandldquoMolecular analysis of the interaction between HPV type 16 E6and human E6-associated proteinrdquo FEBS Letters vol 416 no 1pp 6ndash10 1997
[117] W H Kao S L Beaudenon A L Talis J M Huibregtse andP M Howley ldquoHuman papillomavirus type 16 E6 induces self-ubiquitination of the E6AP ubiquitin-protein ligaserdquo Journal ofVirology vol 74 no 14 pp 6408ndash6417 2000
[118] T Iftner M Elbel B Schopp et al ldquoInterference of papillo-mavirus E6 protein with single-strand break repair by interac-tion with XRCC1rdquo The EMBO Journal vol 21 no 17 pp 4741ndash4748 2002
[119] Y Imai Y Tsunokawa T Sugimura and M Terada ldquoPurifi-cation and DNA-binding properties of human papillomavirustype 16 E6 protein expressed in Escherichia colirdquo Biochemicaland Biophysical Research Communications vol 164 no 3 pp1402ndash1410 1989
[120] M Lusky L Berg H Weiher and M Botchan ldquoBovinepapilloma virus contains an activator of gene expression at thedistal end of the early transcription unitrdquoMolecular andCellularBiology vol 3 no 6 pp 1108ndash1122 1983
[121] S Dalal Q Gao E J Androphy and V Band ldquoMutationalanalysis of human papillomavirus type 16 E6 demonstrates thatp53 degradation is necessary for immortalization of mammaryepithelial cellsrdquo Journal of Virology vol 70 no 2 pp 683ndash6881996
[122] J M Huibregtse M Scheffner and P M Howley ldquoA cellularprotein mediates association of p53 with the E6 oncoprotein ofhuman papillomavirus types 16 or 18rdquo The EMBO Journal vol10 no 13 pp 4129ndash4135 1991
[123] M Scheffner B A Werness J M Huibregtse A J Levine andP M Howley ldquoThe E6 oncoprotein encoded by human papillo-mavirus types 16 and 18 promotes the degradation of p53rdquo Cellvol 63 no 6 pp 1129ndash1136 1990
[124] M Elbel S Carl S Spaderna and T Iftner ldquoA comparativeanalysis of the interactions of the E6 proteins from cutaneousand genital papillomaviruses with p53 and E6AP in correlationto their transforming potentialrdquoVirology vol 239 no 1 pp 132ndash149 1997
[125] M C Thomas and C-M Chiang ldquoE6 oncoprotein repressesp53-dependent gene activation via inhibition of protein acety-lation independently of inducing p53 degradationrdquo MolecularCell vol 17 no 2 pp 251ndash264 2005
[126] H Zimmermann R Degenkolbe H-U Bernard and M JOrsquoConnor ldquoThe human papillomavirus type 16 E6 oncoproteincan down-regulate p53 activity by targeting the transcriptionalcoactivator CBPp300rdquo Journal of Virology vol 73 no 8 pp6209ndash6219 1999
[127] D A Thompson G Belinsky T H-T Chang D L Jones RSchlegel and K Munger ldquoThe human papillomavirus-16 E6oncoprotein decreases the vigilance of mitotic checkpointsrdquoOncogene vol 15 no 25 pp 3025ndash3035 1997
[128] J Bohl K Das B Dasgupta and S B Vande Pol ldquoCompetitivebinding to a charged leucine motif represses transformation bya papillomavirus E6 oncoproteinrdquo Virology vol 271 no 1 pp163ndash170 2000
[129] S Song H C Pitot and P F Lambert ldquoThe human papillo-mavirus type 16 E6 gene alone is sufficient to induce carcinomasin transgenic animalsrdquo Journal of Virology vol 73 no 7 pp5887ndash5893 1999
[130] S Yasumoto A L Burkhardt J Doniger and J A DiPaololdquoHumanpapillomavirus type 16DNA-inducedmalignant trans-formation of NIH 3T3 cellsrdquo Journal of Virology vol 57 no 2pp 572ndash577 1986
[131] S B Vande Pol M C Brown and C E Turner ldquoAssociation ofBovine Papillomavirus Type 1 E6 oncoprotein with the focaladhesion protein paxillin through a conserved protein interac-tion motifrdquo Oncogene vol 16 no 1 pp 43ndash52 1998
[132] R Wade N Brimer and S Vande Pol ldquoTransformation bybovine papillomavirus type 1 E6 requires paxillinrdquo Journal ofVirology vol 82 no 12 pp 5962ndash5966 2008
[133] A J Klingelhutz S A Foster and J KMcDougall ldquoTelomeraseactivation by the E6 gene product of humanpapillomavirus type16rdquo Nature vol 380 no 6569 pp 79ndash82 1996
[134] S T Oh S Kyo and L A Laimins ldquoTelomerase activation byhuman papillomavirus type 16 E6 protein Induction of humantelomerase reverse transcriptase expression through Myc andGC-rich Sp1 binding sitesrdquo Journal of Virology vol 75 no 12pp 5559ndash5566 2001
[135] L V Ronco A Y KarpovaMVidal and PMHowley ldquoHumanpapillomavirus 16 E6 oncoprotein binds to interferon regulatoryfactor-3 and inhibits its transcriptional activityrdquo Genes andDevelopment vol 12 no 13 pp 2061ndash2072 1998
Both test and graphical analysis were done using the BioEstatsoftware [68]
26 Cytokinesis-Block Micronucleus Assay (CBMNA) inEpithelial Cells To verify whether the results observed inPBMCs should also be observed in epithelial cells CBMNAwas performed in CRIB cell (commercial epithelial cell lineobtained from bovine kidney) In detail a total of 1 times 105cells were transferred to six-well plate containing a sterilecoverslip of 24 times 24mm with 2mL of MEM mediumsupplementedwith 10 fetal bovine serum and 1 ampicillinand three cultures had been established negative control(not treated with any drug) positive control (treated with50120583gmL of cyclophosphamide) and experimental group(treatedwith 1120583gmLof BPV-1 E6 recombinant oncoprotein)Cyclophosphamide and BPV-1 E6 recombinant oncoproteinwere added together to the cells After 1 hour the threecultures were treated with 6 120583gmL of cytochalasin B (SigmaGermany)Thematerial was incubated for 48 hours the timenecessary to complete two replication cycles once the dupli-cation time of these cells is 24 hours according to our previ-ous study After this time the medium was removed and thecells were washed twice with PBS at 37∘C Cells were stainedwith solution 1 4 Giemsa-PBS for 3 minutes and afterwashed twice with PBS Coverslips containing the biologicalmaterial were mounted on slides using Entelan (Merck Ger-many) Slides were analyzed by Axiophot binocular micro-scope (Carl Zeiss Germany) to observe the frequency ofmicronucleated cells in a total of 1000 analyzed cells accord-ing to Araldi et al [53]
3 Results
31 Molecular BPV Identification by PCR The peripheralblood samples collected from five asymptomatic calves didnot reveal the presence of BPV sequences using both specificand degenerate primers (Figure 2)
32 Cytokinesis-Block Micronucleus Assay (CBMNA) inPBMCs Frequency analysis of micronucleus observed inCBMNA CBMNA showed an elevated number of micronu-cleated lymphocytes as well as anaphase bridges in bothpositive control and the group treated with 1 120583gmL of E6recombinant oncoprotein (Table 2 Figure 3)
Based on the micronucleated lymphocytes numberobserved per group (Table 2) Chi-square (1205942) test andKruskal-Wallis test were done both with 5 significancelevel Chi-square test pointed out statistical differencesbetween positive control and group treated with E6 (Table 3)These results point out that the cyclophosphamide and E6recombinant oncoprotein are able to induce aneugenesis andor clastogenesis However the Chi-square test did not showstatistic difference in negative control (Table 3)
Kruskal-Wallis test revealed statistical significant differ-ences among the groups (119867 = 97297 and 119901 = 00087)Based on this result Dunn post hoc test was performed Thetest pointed out significant difference between negative andpositive control as well as negative control and experimental
Table 3 Chi-square (1205942) test results showing expected value (119864)freedom degree (FD) and probability (119901)
Group 119864 1205942 FD 119901
Negative control 560 2000 4 07358Positive control 430 10698 4 00302Experimental (E6) 456 12202 4 00159
Table 4 Dunn post hoc test results
Groups Rank differences Calculated 119885 Critical 119885 119901
Cminus and C+ 71 25102 2394 lt005Cndash and E6 79 27931 2394 lt005C+ and E6 08 02828 2394 nsns nonsignificant value
Table 5 Student-Newman-Keuls post hoc test results
Groups Rank differences Calculated 119885 Critical 119885 119901
Cminus and C+ 61 21567 2394 00310Cminus and E6 77 27224 2394 00065C+ and E6 16 05657 2394 05716
group (E6) (Table 4) However the test did not show statis-tical difference between positive control and experimentalgroup (Table 4)
Based on the maximum minimum and median valuesof micronucleus observed in the three groups a boxplotwas done (Figure 4) The graph indicates statistically equalmedians between positive control and experimental group(E6) Although the medians between these groups had notshown statistical differences the graph indicates that theexperimental group showed maximum values of binucleatedlymphocytes with micronucleus higher than positive control
Frequency analysis of anaphase bridge observed inCBMNA based on the number of anaphase bridges (AB)observed per group (Table 3) Kruskal-Wallis test was per-formed which indicates significant differences among thegroups (119867 = 83444 119901 = 00154) Student-Newman-Keuls post hoc test revealed differences between negative andpositive control as well as negative control and experimentalgroup (Table 5) The test did not show differences betweenpositive control and experimental group
Analysis of cytokinesis-block proliferation index (CBPI)and cytotoxicity Kruskal-Wallis test based on theCBPI indi-cates statistical differences among the groups (119867 = 93968and 119901 = 00091) Dunn post hoc test revealed significantdifferences between negative and positive control as well asnegative control and experimental group (Table 6) Howeverthe test did not show differences between positive control andexperimental groupThese data indicate that E6 recombinantoncoprotein has CBPI similar to the cyclophosphamide
Besides presenting high CBPI the E6 recombinant onco-protein also showed to induce endoreduplication evidencedby the presence of intracellular cytokinesis suggesting neosis(Figure 5) according to the criteria proposed by Das et al[69] The CBMNA results suggest that E6 recombinant
6 BioMed Research International
301bp
M 0504030201 M M 0504030201BPV1Cminus
(a) (b)
(c) (d)
(e) (f)
164bp170bp
M 0504030201BPV2Cminus M 0504030201BPV4Cminus
430bp
600bp
M 0504030201BPV2Cminus M 0504030201BPV4Cminus
Figure 2 Electrophoresis gel images showing (a) genomicDNA integrity afterDNAextraction using 1 KbDNALadder (Invitrogen CarlsbadUSA) as marker absence of amplicon in samples 01 to 05 using specific primers to BPV-1 (b) BPV-2 (c) and BPV-4 (d) absence of ampliconin samples 01 to 05 using Delta-Epsilon (e) and Xi (f) degenerate primers Images (b)ndash(f) showed amplification only in positive control withthe 100 bp DNA Ladder (Invitrogen Carlsbad USA) being employed as marker
Table 6 Dunn post hoc test results based on the cytokinesis-blockproliferation index
Group Rank differences Calculated 119885 Critical 119885 119901
Cminus and C+ 76 26870 2394 lt005Cminus and E6 74 26163 2394 lt005C+ and E6 02 00707 2394 nsns nonsignificant differences
oncoprotein induces mitotic stress resulting in clastogenesisand neosis
33 Comet Assay Alkaline Method Comet assay results areshown in Table 7 and Figure 6 According to these data
theKruskal-Wallis test was performedwhich pointed out sta-tistical differences among the groups (119867 = 102613 and 119901 =00059) Based on these results the Dunn post hoc test wasperformed which pointed out significant differences betweennegative and positive control as well as negative controland experimental group (Table 8) However the test did notshow differences between positive control and experimentalgroup (Table 8) These results were also verified by Figure 7Comet assay reinforces the CBMNA results indicating themutagenic potential of E6 recombinant oncoprotein
34 Cytokinesis-Block Micronucleus Assay (CBMNA) inPBMCs Results of this test confirmed the previous resultsindicating that the BPV-1 E6 recombinant oncoprotein has
BioMed Research International 7
LB
(a)
LMN
(b)
LMN
LMN
(c)
PA
PA and MN
(d)
Figure 3 (a) Photomicroscopy of binucleated lymphocyte without micronucleus (LB) observed in negative control Image of binucleatedlymphocyte with micronucleus (LMN) observed in positive control (b) and group treated with E6 recombinant oncoprotein (c) Image ofbinucleated lymphocyte with anaphase bridge and micronucleus (PA and MN) observed in group treated with E6 (d) Images obtained withtotal magnification of 1000x
60
50
40
30
20
10
0
Num
ber o
f mic
ronu
clei
GroupCminus C+ E6
Figure 4 Comparative boxplot based on maximum minimumand median values of micronucleus observed per group
a genotoxicity potential being able to induce DNAbreaks in epithelial cells verified by the presence of MNs(Figure 8)
Figure 5 Evidence of neosis Photomicroscopy of lymphocyteshowing endoreduplication (black arrow) suggesting neosis Cellsanalyzed in total magnification of 1000x
4 Discussion
Transforming potential of E6 oncoprotein has been discussedsince the 1980s based on studies of cottontail rabbit papil-lomaviruses (CRPV) [70 71] Although there are lines ofevidence that the E6 oncoprotein can induce transformation
8 BioMed Research International
(a) (b)
(c) (d)
Figure 6 Images of cometsrsquo nucleoids (a) Image of class 0 (without DNA damage) observed in negative control group Images of class 2(maximum DNA damage) observed in positive control (b) and experimental (c) groups (d) Image of hedgehog comet characterized by thehead absence observed in experimental group Images captured in total magnification of 400x
200
150
100
50
0
Com
et sc
ore
GroupsCminus C+ E6
Figure 7 Boxplot of comet score
[72] the mechanisms of initiation and promotion of cancerassociated with the BPV E6 oncoprotein are unknown
Molecular diagnosis of five peripheral blood samplesfrom five calves showed the absence of amplicons for thefive different primers used (Figure 2) However both specific(BPV-1 BPV-2 and BPV-4) and degenerate (Delta-Epsilonand Xi) primers sets amplified the controls genomes of BPV-1 BPV-2 and BPV-4 None of the negative controls revealedthe amplicon presence These results indicate that the fivesampleswere uninfected by BPVMoreover the use of specific
and degenerate primers increases the capacity of BPV iden-tification This occurs because specific primers are moresensitive than degenerates [66] This sensitivity is due tothe absence of degeneration in the 51015840 region which reducesthe primer ability to recognize and to link with DNA targetsequence [73]The absence of BPV infection in the peripheralblood samples is required for the CBMNA and CA allowinginvestigating the aneugenic andor clastogenic action of BPV-1 E6 recombinant oncoprotein This is because the presenceof BPV sequences is associated with the presence of oncopro-teins transcripts [74]Thus studies have shown that the pres-ence of BPVDNA sequences in peripheral blood is associatedwith cytogenetic damage including clastogenesis [1 14 75]Complementary studies show the association of the BPVpresence with cytogenetic damage in both benign lesions(papillomas) [74] and carcinomas [15]
Chi-square test based on the number of micronucleatedcells pointed out significant statistical values in both positivecontrol and group treated with BPV-1 E6 recombinant onco-protein (Table 3) Negative control showed fewer number ofmicronucleated cells This result was expected because thebiological material was transported and processed after threehours of its collection This procedure reduced the influenceof exogenous environmental factors which could induceDNA damage Furthermore the absence of BPV infection
BioMed Research International 9
(a) (b)
8
6
4
2
0
Num
ber o
f mic
ronu
cleat
ed ce
lls
Neg
ativ
e con
trol
Posit
ive c
ontro
l
E6on
copr
otei
n
(c)
Figure 8 Photomicroscope showing CRIB cell not treated with any drug (negative control) indicating absence of micronucleus (a) andtreated with BPV-1 E6 recombinant oncoprotein showing the presence of MN indicated by arrow (b) (c) Number of micronucleated cellsobserved in each group
in the peripheral blood reduces the influence of endogenousenvironmental factors that could interfere in the analysisPositive control showed a high number of micronucle-ated lymphocytes This result was also expected once thisgroup was treated with 50 120583gmL of cyclophosphamide achemotherapeutic drug with cytotoxic and teratogenic effect[76 77] The cells treated with 1120583gmL of BPV-1 E6 recom-binant oncoprotein also showed significant statistical valuesKruskal-Wallis test followed by the Dunn post hoc testwas performed to compare the frequencies of micronucleusamong three groups This test indicated statistical differencebetween negative control and the cells treated with BPV-1 E6 recombinant oncoprotein However the test did notreveal differences between the cells treated with cyclophos-phamide and BPV-1 E6 recombinant oncoprotein (Table 4Figure 4) Similar results were also observed in epithelial cells(Figure 8) These results suggest that the BPV-1 E6 oncopro-tein has an aneugenic andor clastogenic potential similarto or higher than the observed with the cyclophosphamideThis is because the cells treated with the oncoprotein showed
a frequency of micronucleus formation (MN1199030) 14003
higher than those observed in positive controlThe elevated mutagenic potential of the BPV-1 E6 onco-
protein can justify the cytogenetic damage already describedin the literature [1 14 15 24] Similar results were observedin HPV-infected cells [12 78ndash81] The micronucleus induce-ment was already described in cytological samples collectedduring Papanicolaursquos test from healthy cervix infected byHPV [12] For this reason the micronucleus assay has beenproposed as a complementary method for Papanicolaursquos testbeing a suggestive biomarker of lesion degree [78] Howeverthe association between the BPV and the micronucleusinducement was not yet reported
The high frequency of micronucleated lymphocytes insamples infected by HPV has been attributed to the synergicaction of E5 E6 and E7 oncoproteins [29 30 78 79]However this is the first study that pointed out themutagenicaction of BPV-1 E6 protein per se
Student-Newman-Keuls post hoc test based on the fre-quency of anaphase bridges showed significant statistical
10 BioMed Research International
Table 7 Comet assay results showing the number of nucleoidsobserved per class the number of hedgehog comets and cometscore
Sample Class 0 Class 1 Class 2 Hedgehog ScoreNegative control
Groups Rank differences Calculated 119885 Critical 119885 119901
Cminus and C+ 68 24042 2394 lt05Cminus and E6 82 28991 2394 lt05C+ and E6 14 04950 2394 nsns nonsignificant differences
difference between the negative control and the group treatedwith the BPV-1 E6 recombinant oncoprotein (Table 5) How-ever the test did not reveal differences between the cellstreated with cyclophosphamide and BPV-1 E6 recombinantoncoprotein (Table 5)These data reinforce those observed bythe Dunn post hoc test based on the frequency of micronu-cleus indicating the clastogenic action of the E6 oncoproteinMoreover the presence of anaphase bridge was alreadydescribed in cells transfected with recombinant adenoviruscontaining the E6 and E7 ORFs of HPV [29 30] Howeverthere are no studies describing the action of BPVE6 oncopro-tein in the induction of anaphase bridges or studies that showthis same action of E6 oncoprotein of HPV per se Anaphasebridges are indicators of genomic instability [82 83] which isconsidered the first step in carcinogenesis [46 47] Moreoverthe presence of these bridges is an important hallmark ofDNA double strand breaks (DSBs) [84]
Statistical analysis based on the cytokinesis-block prolif-eration index (CBPI) showed that BPV-1 E6 oncoprotein hascytotoxic levels similar to cyclophosphamide (Table 6) Thisresult suggests that the BPV E6 oncoprotein can deregulatethe cell cycle contributing to the cell proliferation andimmortalization To date this mitogenic action is attributedto the E7 oncoprotein [85ndash87] E7 oncoprotein induces
the phosphorylation of pRb resulting in the E2F transcrip-tion factor release [85] E2F when translocated to the nucleusacts as activator and binds to the kinase-dependent cyclinspromoters [85] However the results showed polynucleatedcells in the group treatedwithBPVE6oncoprotein (Figure 5)suggesting that the BPV E6 oncoprotein confers mitogenicstimulation resulting in mitotic stress Moreover studiespointed out that the BPV E6 oncoprotein interacts with theCBPp300 deacetylase promoting p53 downregulation [88]This downregulation increases the expression levels of FoxM1transcription factor (Foxhead box M1) [89] promoting B1cyclin D1 cyclin and cdc25 upregulation [89] The upregu-lation of these genes is associated with increased levels of cellproliferation which is necessary in order to make DNA poly-merases available to virus replication However if this mech-anism guarantees the BPV replication it can contribute to cellimmortalization and cancer progression
The FoxM1 factor also participates in the Wnt120573-cateninsignaling pathway and binds directly to the 120573-catenin [89]This interaction promotes the nuclear 120573-catenin transloca-tion [90] 120573-catenin translocation to nucleus induces thecyclin expression [64] So these data suggest that the BPVE6 oncoprotein not only promotes mitogenic stress but alsocontributes to the epithelial-mesenchymal transition (EMT)Studies based on theHPVE6 oncoprotein have indicated thatthis oncoprotein contributes significantly to the EMT [91]This occurs not only because the E6 oncoprotein induces thetranslocation of 120573-catenin but also due to the proteasomaldegradation of regulatory proteins of apical-basal polarity[92ndash94] Studies also showed that the E6 oncoprotein is ableto bind to Crumbs (Dlg and Patj) proteins resulting in loss ofcell polarity [93 94] Based on these results the E6 oncopro-tein emerges as a possible therapeutic target with biotechno-logical value in cancer treatment
Mitogenic action of E6 oncoprotein can be moreexpressed in cells infected by papillomaviruses once thesecells also expressed E5 and E7 oncoproteins [74] The E7oncoprotein is able to form a complex E7-p600 which pro-motes E6 upregulation [85] Furthermore the E6 oncoproteincan bind to the E6AP ubiquitin ligase forming the E6-E6APcomplex increasing the hTERT levels of expression con-tributing to cell immortalization [95] Moreover E6-E6APinduces NFX1 expression promoting MHC-II downregula-tion [96]Thismechanism reduces the antigenic presentationmediated by CD4+ T lymphocytes contributing to immuneevasion This action guarantees the PV infection for longperiods with or without clinical symptoms [24] Althoughthe BPV infection can be asymptomatic the BPV presence inperipheral blood is associated with DNA damage [1 14 24]
CBMNA analysis pointed out the presence of cells withintracellular cytokinesis (Figure 5) This result suggests theneotic action of BPV E6 oncoprotein Neosis is characterizedby the presence of (1) DNA damage (2) loss of checkpointcontrol (3) repair system failures and (4) endoreduplication[97] In this scenario the E6 oncoprotein attempts all theseneosis criteria Moreover the oncoprotein not only increasesthe cytogenetic damage leading to increasing of micronu-cleus frequency but also induces clastogenesis as shown bythe comet assay and anaphase bridges So statistical analysis
BioMed Research International 11
performed based on the comet scores reinforced the CBMNAresults Comet assay allows detecting both simple and dou-ble DNA breaks being more sensitive than the CBMNAClastogenesis is considered the most serious type of DNAdamage [98] DNA breaks can be repaired by homologous ornonhomologous recombination However studies point outthat the E6 oncoprotein can link to the Holliday junctionduring the homologous repair avoiding the junction reso-lution [99] Moreover E6 oncoprotein promotes the TP53gene deacetylation resulting in p53 downregulation [51]Thisepigenetic effect results in cell cycle deregulation affectingthe checkpoints
Studies have shown that the HPV-induced DNA breaksare required for the virus integration into the host genome[100] Although there are no studies showing the BPV inte-gration to date the results observed in this work in additionto those already published [24] indicate the necessity ofmorestudies to evaluate the virus-host interaction
Studies also showed that the E6 oncoprotein inducesclathrin-transporter adapter protein (AP-1) increasing thelevel of this protein in the plasma membrane [76 77] Theincreasing quantity of AP-1 in membrane can facilitate theBPV virions infection This occurs because the infectionprocess is clathrin dependent In this scenario the E6 onco-protein can contribute to the virus infection [101]
In summary the CBMNA and CA results showed thatthe BPV E6 oncoprotein participates not only in cancerpromotion but also in initiation inducing DNA breaksThese DNA breaks represent mutagenic effects consideredthe first step in the oncogenesis process which are associatedwith genomic instability [47 48 102 103] Thus the E6recombinant oncoprotein has been suggested as a possiblevaccine candidate [104ndash112] due to its immunogenicity [44]However the mutagenic tests such as CBMNA and CA arerequired in drug validation process [113] In this scenariothis study pointed out that the BPV-1 E6 recombinant onco-protein in tested concentration showedmutagenic potentialThis is the first study that reports the mutagenic potential ofa possible therapeutic vaccine candidate On the one handthese results allowed better understanding the mechanism ofcancer promotion associatedwith the BPVE6oncoprotein aswell as revealing that this oncoprotein can induce carcinogen-esis per se on the other hand this data pointed out thatmaybeBPV E6 recombinant oncoprotein requires protein modifica-tions to be used as vaccine
Conflict of Interests
The authors declare that there is no conflict of interests
Acknowledgments
Theauthors thank Fundacao deAmparo a Pesquisa do Estadode Sao Paulo (FAPESP Process 201420617-5) and FundacaoButantan for the financial support
References
[1] R C Stocco dos Santos C J Lindsey O P Ferraz et al ldquoBovinepapillomavirus transmission and chromosomal aberrations an
experimental modelrdquo Journal of General Virology vol 79 no 9pp 2127ndash2135 1998
[2] A Antonsson and B G Hansson ldquoHealthy skin ofmany animalspecies harbors papillomaviruses which are closely related totheir human counterpartsrdquo Journal of Virology vol 76 no 24pp 12537ndash12542 2002
[3] P Rous and J W Beard ldquoA virus-induced mammalian growthwith the characters of a tumor (the Shope rabbit papilloma) IThe growth on implantationwithin favorable hostsrdquoThe Journalof Experimental Medicine vol 60 no 6 pp 701ndash722 1934
[4] R Shope and E Hurst ldquoInfectious papillomatosis of rabbitsrdquoThe Journal of Experimental Medicine vol 58 no 1 pp 607ndash624 1933
[5] H Delius M A Van Ranst A B Jenson H Zur Hausen and JP Sundberg ldquoCanine oral papillomavirus genomic sequence aunique 15-kb intervening sequence between the E2 andL2 openreading framesrdquo Virology vol 204 no 1 pp 447ndash452 1994
[6] W A Demonbreun and E Goodpasture ldquoInfectious oral papil-lomatosis of dogsrdquo The American Journal of Pathology vol 8no 1 pp 43ndash56 1932
[7] P K Nicholls B A Klaunberg R A Moore et al ldquoNaturallyoccurring nonregressing canine oral papillomavirus infectionhost immunity virus characterization and experimental infec-tionrdquo Virology vol 265 no 2 pp 365ndash374 1999
[8] A Alberti S Pirino F Pintore et al ldquoOvis aries Papillomavirus3 a prototype of a novel genus in the family Papillomaviridaeassociated with ovine squamous cell carcinomardquo Virology vol407 no 2 pp 352ndash359 2010
[9] H zur Hausen E-M de Villiers and L Gissmann ldquoPapil-lomavirus infections and human genital cancerrdquo GynecologicOncology vol 12 no 2 pp S124ndashS128 1981
[10] C Foresta A Bertoldo A Garolla et al ldquoHuman papillo-mavirus proteins are found in peripheral blood and semenCd20+ and Cd56+ cells during Hpv-16 semen infectionrdquo BMCInfectious Diseases vol 13 article 593 2013
[11] S Ljubojevic andM Skerlev ldquoHPV-associated diseasesrdquo Clinicsin Dermatology vol 32 no 2 pp 227ndash234 2014
[12] A P R Cassel R B Barcellos C M D da Silva S E de MatosAlmeida and M L R Rossetti ldquoAssociation between humanpapillomavirus (HPV) DNA and micronuclei in normal cervi-cal cytologyrdquo Genetics and Molecular Biology vol 37 no 2 pp360ndash363 2014
[13] W Jarrett M Campo B OrsquoNeil H Laird and L Coggins ldquoAnovel bovine papillomavirus (BPV-6) causing true epithelialpapillomas of the mammary gland skin a member of a pro-posed newBPV subgrouprdquoVirology vol 136 no 2 pp 255ndash2641984
[14] T CMelo N Diniz S R C Campos et al ldquoCytogenetic studiesin peripheral blood of bovines afflicted by papillomatosisrdquoVeterinary and Comparative Oncology vol 9 no 4 pp 269ndash2742011
[15] S R C Campos T C Melo S Assaf et al ldquoChromosome aber-rations in cells infected with bovine papillomavirus comparingcutaneous papilloma esophagus papilloma and urinary blad-der lesion cellsrdquo ISRN Oncology vol 2013 Article ID 910849 8pages 2013
[16] H zur Hausen ldquoThe search for infectious causes of humancancers where and whyrdquoVirology vol 392 no 1 pp 1ndash10 2009
[17] R P Araldi R F Carvalho T C Melo et al ldquoBovine papillo-mavirus in beef cattle first description of BPV-12 and putativetype BAPV8 in BrazilrdquoGenetics andMolecular Research vol 13no 3 pp 5644ndash5653 2014
12 BioMed Research International
[18] T Rampias C Sasaki and A Psyrri ldquoMolecular mechanisms ofHPV induced carcinogenesis in head and neckrdquoOral Oncologyvol 50 no 5 pp 356ndash363 2014
[19] S Roperto V Russo L Leonardi et al ldquoBovine papillomavirustype 13 expression in the urothelial bladder tumours of cattlerdquoTransboundary and Emerging Diseases 2015
[20] S RCCampos C TrindadeO P Ferraz et al ldquoCan establishedcultured papilloma cells harbor bovine papillomavirusrdquoGenet-ics and Molecular Research vol 7 no 4 pp 1119ndash1126 2008
[21] M S Campo ldquoAnimalmodels of papilloma virus pathogenesisrdquoVirus Research vol 89 no 2 pp 249ndash261 2002
[22] G Borzacchiello ldquoBovine papillomavirus infections in ani-malsrdquo in Communicating Current Research and EducationalTopics and Trends in AppliedMicrobiology pp 673ndash679 Forma-tex Badajoz Spain 2007
[23] F Bocaneti G Altamura A Corteggio E Velescu F RopertoandG Borzacchiello ldquoBovine papillomavirus new insights intoan old diseaserdquo Transboundary and Emerging Diseases 2014
[24] R P Araldi T C Melo N Diniz et al ldquoBovine papillomavirusclastogenic effect analyzed in comet assayrdquo BioMed ResearchInternational vol 2013 Article ID 630683 7 pages 2013
[25] J Munday N Thomson M Dunowska C Knight R Laurieand S Hills ldquoGenomic characterisation of the feline sarcoid-associated papillomavirus and proposed classification as Bostaurus papillomavirus type 14rdquoVeterinaryMicrobiology vol 177no 3-4 pp 289ndash295 2015
[26] G Borzacchiello V Russo C Spoleto et al ldquoBovine papillo-mavirus type-2 DNA and expression of E5 and E7 oncoproteinsin vascular tumours of the urinary bladder in cattlerdquo CancerLetters vol 250 no 1 pp 82ndash91 2007
[27] P Maiolino A Ozkul A Sepici-Dincel et al ldquoBovine papillo-mavirus type 2 infection and microscopic patterns of urothelialtumors of the urinary bladder in water buffaloesrdquo BioMedResearch International vol 2013 Article ID 937918 6 pages2013
[28] S Roperto G Borzacchiello R Brun et al ldquoMultiple glomustumors of the urinary bladder in a cow associated with bovinepapillomavirus type 2 (BPV-2) infectionrdquo Veterinary Pathologyvol 45 no 1 pp 39ndash42 2008
[29] S Duensing and K Munger ldquoThe human papillomavirus type16 E6 and E7 oncoproteins independently induce numerical andstructural chromosome instabilityrdquoCancer Research vol 62 no23 pp 7075ndash7082 2002
[30] S Duensing L Y Lee A Duensing et al ldquoThe human papil-lomavirus type 16 E6 and E7 oncoproteins cooperate to inducemitotic defects and genomic instability by uncoupling centro-some duplication from the cell division cyclerdquo Proceedings of theNational Academy of Sciences of theUnited States of America vol97 no 18 pp 10002ndash10007 2000
[31] C A Moody and L A Laimins ldquoHuman papillomavirus onco-proteins pathways to transformationrdquo Nature Reviews Cancervol 10 no 8 pp 550ndash560 2010
[32] C Mansur and E Androphy ldquoCellular transformation bypapillomavirus oncoproteinsrdquo Biochimica et Biophysica Actavol 1155 no 3 pp 323ndash345 1993
[33] J J Chen Y Hong and E J Androphy ldquoMutational analysis oftranscriptional activation by the bovine papillomavirus type 1E6rdquo Virology vol 236 no 1 pp 30ndash36 1997
[34] X Tong andPMHowley ldquoThebovine papillomavirus E6 onco-protein interacts with paxillin and disrupts the actin cytoskele-tonrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 94 no 9 pp 4412ndash4417 1997
[35] X TongW Boll T Kirchhausen andPMHowley ldquoInteractionof the bovine papillomavirus E6 protein with the clathrinadaptor complex AP-1rdquo Journal of Virology vol 72 no 1 pp476ndash482 1998
[36] Y Nomine M Masson S Charbonnier et al ldquoStructural andfunctional analysis of E6 oncoprotein insights in the molecularpathways of human papillomavirus-mediated pathogenesisrdquoMolecular Cell vol 21 no 5 pp 665ndash678 2006
[37] T Ristriani MMasson Y Nomine et al ldquoHPV oncoprotein E6is a structure-dependent DNA-binding protein that recognizesfour-way junctionsrdquo Journal of Molecular Biology vol 296 no5 pp 1189ndash1203 2000
[38] Y Liu and J D Baleja ldquoStructure and function of the papil-lomavirus E6 protein and its interacting proteinsrdquo Frontiers inBioscience vol 13 no 1 pp 121ndash134 2008
[39] F Lipari G A McGibbon E Wardrop and M G CordingleyldquoPurification and biophysical characterization of a minimalfunctional domain and of an N-terminal Zn2+-binding frag-ment from the human papillomavirus type 16 E6 proteinrdquoBiochemistry vol 40 no 5 pp 1196ndash1204 2001
[40] Y Nomine T Ristriani C Laurent J-F Lefevre E Weissand G Trave ldquoA strategy for optimizing the monodispersity offusion proteins application to purification of recombinantHPVE6 oncoproteinrdquo Protein Engineering vol 14 no 4 pp 297ndash3052001
[41] C G Ullman P I Haris D A Galloway V C Emery and SJ Perkins ldquoPredicted 120572-helix120573-sheet secondary structures forthe zinc-binding motifs of human papillomavirus E7 and E6proteins by consensus prediction averaging and spectroscopicstudies of E7rdquo Biochemical Journal vol 319 no 1 pp 229ndash2391996
[42] A Sidi K Babah N Brimer et al ldquoStrategies for bacterialexpression of protein-peptide complex application to solubi-lization of papillomavirus E6rdquo Protein Expression and Purifica-tion vol 29 no 6 pp 997ndash1003 2011
[43] K Zanier S Charbonnier A O M O Sidi et al ldquoStructuralbasis for hijacking of cellular LxxLL motifs by papillomavirusE6 oncoproteinsrdquo Science vol 339 no 6120 pp 694ndash698 2013
[44] J Mazzuchelli-de-Souza R F Carvalho R M Ruiz et alldquoExpression and in silico analysis of the recombinant bovinepapillomavirus e6 protein as a model for viral oncoproteinsstudiesrdquo BioMed Research International vol 2013 Article ID421398 9 pages 2013
[45] L Banks P Spence E Androphy et al ldquoIdentification of humanpapillomavirus type 18 E6 polypeptide in cells derived fromhuman cervical carcinomasrdquo Journal of General Virology vol68 no 5 pp 1351ndash1359 1987
[46] D Hanahan and R AWeinberg ldquoHallmarks of cancer the nextgenerationrdquo Cell vol 144 no 5 pp 646ndash674 2011
[47] G S Charames and B Bapat ldquoGenomic instability and cancerrdquoCurrent Molecular Medicine vol 3 no 7 pp 589ndash596 2003
[48] S Negrini V G Gorgoulis and T D Halazonetis ldquoGenomicinstabilitymdashan evolving hallmark of cancerrdquo Nature ReviewsMolecular Cell Biology vol 11 no 3 pp 220ndash228 2010
[49] K Zanier S Charbonnier M Baltzinger Y Nomine DAltschuh and G Trave ldquoKinetic analysis of the interactionsof human papillomavirus E6 oncoproteins with the ubiquitinligase E6AP using surface plasmon resonancerdquo Journal ofMolecular Biology vol 349 no 2 pp 401ndash412 2005
BioMed Research International 13
[50] M Scheffner J M Huibregtse R D Vierstra and PM HowleyldquoThe HPV-16 E6 and E6-AP complex functions as a ubiquitin-protein ligase in the ubiquitination of p53rdquo Cell vol 75 no 3pp 495ndash505 1993
[51] H Zimmermann C-H Koh R Degenkolbe et al ldquoInteractionwith CBPp300 enables the bovine papillomavirus type 1 E6oncoprotein to downregulate CBPp300-mediated transactiva-tion by p53rdquo Journal of General Virology vol 81 no 11 pp 2617ndash2623 2000
[52] M Fenech ldquoThe in vitro micronucleus techniquerdquo MutationResearchmdashFundamental and Molecular Mechanisms of Mutage-nesis vol 455 no 1-2 pp 81ndash95 2000
[53] R P Araldi T C de Melo T B Mendes et al ldquoUsing the cometand micronucleus assays for genotoxicity studies a reviewrdquoBiomedicine amp Pharmacotherapy vol 72 pp 74ndash82 2015
[54] P Thomas and M Fenech ldquoCytokinesis-block micronucleuscytome assay in lymphocytesrdquo Methods in Molecular Biologyvol 682 pp 217ndash234 2011
[55] M Kirsch-Volders T Sofuni M Aardema et al ldquoReport fromthe in vitro micronucleus assay working grouprdquo MutationResearch Genetic Toxicology and Environmental Mutagenesisvol 540 no 2 pp 153ndash163 2003
[56] S Samanta and P Dey ldquoMicronucleus and its applicationsrdquoDiagnostic Cytopathology vol 40 no 1 pp 84ndash90 2012
[57] M Hayashi J T MacGregor D G Gatehouse et al ldquoIn vivoerythrocyte micronucleus assay III Validation and regulatoryacceptance of automated scoring and the use of rat periph-eral blood reticulocytes with discussion of non-hematopoietictarget cells and a single dose-level limit testrdquo MutationResearchGenetic Toxicology and Environmental Mutagenesisvol 627 no 1 pp 10ndash30 2007
[58] A Azqueta and A R Collins ldquoThe essential comet assay acomprehensive guide to measuring DNA damage and repairrdquoArchives of Toxicology vol 87 no 6 pp 949ndash968 2013
[59] N P Singh M T McCoy R R Tice and E L Schneider ldquoAsimple technique for quantitation of low levels of DNA damagein individual cellsrdquo Experimental Cell Research vol 175 no 1pp 184ndash191 1988
[60] J H Calinisan S R Chan A King and P J Chan ldquoHumanpapillomavirus and blastocyst apoptosisrdquo Journal of AssistedReproduction and Genetics vol 19 no 3 pp 132ndash136 2002
[61] R Oliveira Studies on the biopathological actions of Pteridiumaquilinum [PhD thesis] nstituto de Ciencias Biomedicas AbelSalazar 2012
[62] D M Potter and M S Baird ldquoCarcinogenic effects of ptaquilo-side in bracken fern and related compoundsrdquo British Journal ofCancer vol 83 no 7 pp 914ndash920 2000
[63] R B Lucena D R Rissi G D Kommers et al ldquoA retrospectivestudy of 586 tumours in Brazilian cattlerdquo Journal of ComparativePathology vol 145 no 1 pp 20ndash24 2011
[64] A Yaguiu M L Z Dagli E H Birgel Jr et al ldquoSimultaneouspresence of bovine papillomavirus and bovine leukemia virusin different bovine tissues in situ hybridization and cytogeneticanalysisrdquoGenetics andMolecular Research vol 7 no 2 pp 487ndash497 2008
[65] T C Melo R F Carvalho J Mazzucchelli-de-Souza et alldquoPhylogenetic classification and clinical aspects of a new puta-tive Deltapapillomavirus associated with skin lesions in cattlerdquoGenetics and Molecular Research vol 13 no 2 pp 2458ndash24692014
[66] R P Araldi D N S Giovanni T CMelo et al ldquoBovine papillo-mavirus isolation by ultracentrifugationrdquo Journal of VirologicalMethods vol 208 pp 119ndash124 2014
[67] L M Chandrachud B W OrsquoNeil W F H Jarrett G J Grind-lay G M McGarvie and M S Campo ldquoHumoral immuneresponse to the E7 protein of bovine papillomavirus type 4 andidentification of B-cell epitopesrdquoVirology vol 200 no 1 pp 98ndash104 1994
[68] M Ayres M J Ayres D L Ayres and A A Santos BioEstatmdashAplicacoes Estatısticas nas Areas de Ciencias Bio-Medicas Insti-tuto Mamiraua Belem Brazil 2007
[69] K Das J Bohl and S B Vande Pol ldquoIdentification of a secondtransforming function in bovine papillomavirus type 1 E6 andthe role of E6 interactions with paxillin E6BP and E6APrdquoJournal of Virology vol 74 no 2 pp 812ndash816 2000
[70] O Danos E Georges G Orth and M Yaniv ldquoFine structureof the cottontail rabbit papillomavirus mRNAs expressed in thetransplantable VX2 carcinomardquo Journal of Virology vol 53 no3 pp 735ndash741 1985
[71] M Nasseri and F O Wettstein ldquoDifferences exist between viraltranscripts in cottontail rabbit papillomavirus-induced benignand malignant tumors as well as non-virus-producing andvirus-producing tumorsrdquo Journal of Virology vol 51 no 3 pp706ndash712 1984
[72] X Tong R Salgia J-L Li J D Griffin and P M Howley ldquoThebovine papillomavirus E6 protein binds to the LDmotif repeatsof paxillin and blocks its interaction with vinculin and the focaladhesion kinaserdquo Journal of Biological Chemistry vol 272 no52 pp 33373ndash33376 1997
[73] Y Maeda T Shibahara Y Wada et al ldquoAn outbreak of teatpapillomatosis in cattle caused by bovine papilloma virus (BPV)type 6 and unclassified BPVsrdquo Veterinary Microbiology vol 121no 3-4 pp 242ndash248 2007
[74] R P Araldi T C Melo A Neves et al ldquoHyperproliferativeaction of bovine papillomavirus genetic and histopathologicalaspectsrdquo Genetics and Molecular Research vol 14 no 4 pp12942ndash12954 2015
[75] NDiniz T CMelo J F Santos et al ldquoSimultaneous presence ofbovine papillomavirus in blood and in short-term lymphocytecultures from dairy cattle in Pernambuco Brazilrdquo Genetics andMolecular Research vol 8 no 4 pp 1474ndash1480 2009
[76] O M Brito M F Guimaraes and C C Lanna ldquoCiclofosfamidae funcao ovarianardquo Revista Brasileira de Reumatologia vol 48no 1 pp 39ndash45 2008
[77] D Anderson T-W Yu and D B McGregor ldquoComet assayresponses as indicators of carcinogen exposurerdquo Mutagenesisvol 13 no 6 pp 539ndash555 1998
[78] M L Adam C Pini S Tulio J C L L Cantalice R A Torresand M T Dos Santos Correia ldquoAssessment of the associationbetween micronuclei and the degree of uterine lesions and viralload in women with human papillomavirusrdquo Cancer Genomicsamp Proteomics vol 12 no 2 pp 67ndash72 2015
[79] E Cortes-Gutierrez M Davila-Rodrıguez J Vargas-VillarrealF Hernandez-Garza and R Cerda-Flores ldquoAssociationbetween human papilloma virus-type infections with micro-nuclei frequenciesrdquo Prague Medical Report vol 111 no 1 pp35ndash41 2010
[80] R N Chakrabarti and K Dutta ldquoMicronuclei test in routinesmears fromuterine cervixrdquo European Journal of GynaecologicalOncology vol 9 no 5 pp 370ndash372 1988
[81] E M M Cerqueira C L Santoro N F Donozo et al ldquoGeneticdamage in exfoliated cells of the uterine cervix Association
14 BioMed Research International
and interaction between cigarette smoking and progression tomalignant transformationrdquo Acta Cytologica vol 42 no 3 pp639ndash649 1998
[82] S M Germann V Schramke R T Pedersen et al ldquoTopBP1Dpb11 bindsDNA anaphase bridges to prevent genome instabil-ityrdquoThe Journal of Cell Biology vol 204 no 1 pp 45ndash59 2014
[83] D R Hoffelder L Luo N A Burke S CWatkins S M Gollinand W S Saunders ldquoResolution of anaphase bridges in cancercellsrdquo Chromosoma vol 112 no 8 pp 389ndash397 2004
[84] C Acilan D M Potter and W S Saunders ldquoDNA repairpathways involved in anaphase bridge formationrdquo Genes Chro-mosomes and Cancer vol 46 no 6 pp 522ndash531 2007
[85] J DeMasi M C Chao A S Kumar and P M Howley ldquoBovinepapillomavirus E7 oncoprotein inhibits anoikisrdquo Journal ofVirology vol 81 no 17 pp 9419ndash9425 2007
[86] J DeMasi K-W Huh Y Nakatani K Munger and P MHowley ldquoBovine papillomavirus E7 transformation functioncorrelates with cellular p600 protein bindingrdquo Proceedings of theNational Academy of Sciences of theUnited States of America vol102 no 32 pp 11486ndash11491 2005
[87] E A White and P M Howley ldquoProteomic approaches to thestudy of papillomavirusndashhost interactionsrdquo Virology vol 435no 1 pp 57ndash69 2013
[88] FMantovani and L Banks ldquoThe human papillomavirus E6 pro-tein and its contribution to malignant progressionrdquo Oncogenevol 20 no 54 pp 7874ndash7887 2001
[89] P Chen and H Lee ldquoFOXM1 induced by E6 oncoprotein pro-motes tumor invasion and chemoresistance in HPV-infectedlung cancerrdquo Cancer Cell amp Microenvironment vol 2 no 1 pp6ndash9 2015
[90] N Zhang P Wei A Gong et al ldquoFoxM1 promotes 120573-cateninnuclear localization and controls Wnt target-gene expressionand glioma tumorigenesisrdquo Cancer Cell vol 127 no 4 pp 358ndash366 2012
[91] Z Liu Y Liu Y Hong L Rapp E J Androphy and J JChen ldquoBovine papillomavirus type 1 E6-induced sensitizationto apoptosis is distinct from its transforming activityrdquo Virologyvol 295 no 2 pp 230ndash237 2002
[92] M Thomas P Massimi C Navarro J-P Borg and L BanksldquoThe hScribDlg apico-basal control complex is differentiallytargeted by HPV-16 and HPV-18 E6 proteinsrdquo Oncogene vol24 no 41 pp 6222ndash6230 2005
[93] C H Storrs and S J Silverstein ldquoPATJ a tight junction-associated PDZ protein is a novel degradation target of high-risk human papillomavirus E6 and the alternatively splicedisoform 18 E6rdquo Journal of Virology vol 81 no 8 pp 4080ndash40902007
[94] R Accardi R Rubino M Scalise et al ldquoE6 and E7 from humanpapillomavirus type 16 cooperate to target the PDZ proteinNaH exchange regulatory factor 1rdquo Journal of Virology vol 85no 16 pp 8208ndash8216 2011
[95] S Nakagawa and J M Huibregtse ldquoHuman scribble (Vartul)is targeted for ubiquitin-mediated degradation by the high-risk papillomavirus E6 proteins and the E6APubiquitin-proteinligaserdquoMolecular and Cellular Biology vol 20 no 21 pp 8244ndash8253 2000
[96] L Gewin H Myers T Kiyono and D A Galloway ldquoIdentifi-cation of a novel telomerase repressor that interacts with thehuman papillomavirus type-16 E6E6-AP complexrdquo Genes ampDevelopment vol 18 no 18 pp 2269ndash2282 2004
[97] M Sundaram D L GuernseyMM Rajaraman and R Rajara-man ldquoNeosis a novel type of cell division in cancerrdquo CancerBiology ampTherapy vol 3 no 2 pp 207ndash218 2004
[98] R D Snyder and J W Green ldquoA review of the genotoxicity ofmarketed pharmaceuticalsrdquo Mutation Research vol 488 no 2pp 151ndash169 2001
[99] T Ristriani Y Nomine C Laurent E Weiss and G TraveldquoProtein mutagenesis with monodispersity-based quality prob-ing selective inactivation of p53 degradation and DNA-bindingproperties of HPV E6 oncoproteinrdquo Protein Expression andPurification vol 26 no 3 pp 357ndash367 2002
[100] M Kadaja A Sumerina T Verst M Ojarand E Ustav and MUstav ldquoGenomic instability of the host cell induced by thehuman papillomavirus replicationmachineryrdquoTheEMBO Jour-nal vol 26 no 8 pp 2180ndash2191 2007
[101] PMDay D R Lowy and J T Schiller ldquoPapillomaviruses infectcells via a clathrin-dependent pathwayrdquo Virology vol 307 no 1pp 1ndash11 2003
[102] T D Tlsty A Briot A Gualberto et al ldquoGenomic instabilityand cancerrdquoMutation Research vol 337 no 1 pp 1ndash7 1995
[103] J E Eyfjord and S K Bodvarsdottir ldquoGenomic instabilityand cancer networks involved in response to DNA damagerdquoMutation Research vol 592 no 1-2 pp 18ndash28 2005
[104] Y Yao W Huang X Yang et al ldquoHPV-16 E6 and E7 protein Tcell epitopes prediction analysis based on distributions of HLA-A loci across populations an in silico approachrdquo Vaccine vol31 no 18 pp 2289ndash2294 2013
[105] C-F Hung B Ma A Monie S-W Tsen and T-C Wu ldquoTher-apeutic human papillomavirus vaccines current clinical trialsand future directionsrdquo Expert Opinion on Biological Therapyvol 8 no 4 pp 421ndash439 2008
[106] M J P Welters G G Kenter S J Piersma et al ldquoInductionof tumor-specific CD4+ and CD8+ T-cell immunity in cervicalcancer patients by a human papillomavirus type 16 E6 and E7long peptides vaccinerdquo Clinical Cancer Research vol 14 no 1pp 178ndash187 2008
[107] R Han N M Cladel C A Reed X Peng and N D Chris-tensen ldquoProtection of rabbits from viral challenge by gene gun-based intracutaneous vaccination with a combination of cot-tontail rabbit papillomavirus E1 E2 E6 and E7 genesrdquo Journalof Virology vol 73 no 8 pp 7039ndash7043 1999
[108] Z He A P Wlazlo D W Kowalczyk et al ldquoViral recombinantvaccines to the E6 and E7 antigens of HPV-16rdquo Virology vol270 no 1 pp 146ndash161 2000
[109] A Kaufmann P Stern E Rankin et al ldquoSafety and immuno-genicity of TA-HPV a recombinant vaccinia virus expressingmodified human papillomavirus (HPV)-16 and HPV-18 E6 andE7 genes in women with progressive cervical cancerrdquo ClinicalCancer Research vol 8 no 12 pp 3676ndash3685 2002
[110] L K Borysiewicz A Fiander M Nimako et al ldquoA recombinantvaccinia virus encoding human papillomavirus types 16 and 18E6 and E7 proteins as immunotherapy for cervical cancerrdquoTheLancet vol 347 no 9014 pp 1523ndash1527 1996
[111] A De Jong T OrsquoNeill A Y Khan et al ldquoEnhancement ofhuman papillomavirus (HPV) type 16 E6 and E7-specific T-cellimmunity in healthy volunteers through vaccination with TA-CIN an HPV16 L2E7E6 fusion protein vaccinerdquo Vaccine vol20 no 29-30 pp 3456ndash3464 2002
[112] S Chen C Liao Y Lai et al ldquoDe-oncogenicHPVE6E7 vaccinegets enhanced antigenicity and promotes tumoricidal synergywith cisplatinrdquo Acta Biochimica et Biophysica Sinica vol 46 no1 pp 6ndash14 2014
BioMed Research International 15
[113] J Bonhoeffer E B Imoukhuede G Aldrovandi et al ldquoTemplateprotocol for clinical trials investigating vaccines-focus on safetyelementsrdquo Vaccine vol 31 no 47 pp 5602ndash5620 2013
[114] S Jackson and A Storey ldquoE6 proteins from diverse cutaneonsHPV types inhibit apoptosis in response to UV damagerdquoOncogene vol 19 no 4 pp 592ndash598 2000
[115] M Thomas and L Banks ldquoPDZRN3LNX3 is a novel targetof human papillomavirus type 16 (HPV-16) and HPV-18 E6rdquoJournal of Virology vol 89 no 2 pp 1439ndash1444 2015
[116] P R Daniels C M Sanders P Coulson and N J MaitlandldquoMolecular analysis of the interaction between HPV type 16 E6and human E6-associated proteinrdquo FEBS Letters vol 416 no 1pp 6ndash10 1997
[117] W H Kao S L Beaudenon A L Talis J M Huibregtse andP M Howley ldquoHuman papillomavirus type 16 E6 induces self-ubiquitination of the E6AP ubiquitin-protein ligaserdquo Journal ofVirology vol 74 no 14 pp 6408ndash6417 2000
[118] T Iftner M Elbel B Schopp et al ldquoInterference of papillo-mavirus E6 protein with single-strand break repair by interac-tion with XRCC1rdquo The EMBO Journal vol 21 no 17 pp 4741ndash4748 2002
[119] Y Imai Y Tsunokawa T Sugimura and M Terada ldquoPurifi-cation and DNA-binding properties of human papillomavirustype 16 E6 protein expressed in Escherichia colirdquo Biochemicaland Biophysical Research Communications vol 164 no 3 pp1402ndash1410 1989
[120] M Lusky L Berg H Weiher and M Botchan ldquoBovinepapilloma virus contains an activator of gene expression at thedistal end of the early transcription unitrdquoMolecular andCellularBiology vol 3 no 6 pp 1108ndash1122 1983
[121] S Dalal Q Gao E J Androphy and V Band ldquoMutationalanalysis of human papillomavirus type 16 E6 demonstrates thatp53 degradation is necessary for immortalization of mammaryepithelial cellsrdquo Journal of Virology vol 70 no 2 pp 683ndash6881996
[122] J M Huibregtse M Scheffner and P M Howley ldquoA cellularprotein mediates association of p53 with the E6 oncoprotein ofhuman papillomavirus types 16 or 18rdquo The EMBO Journal vol10 no 13 pp 4129ndash4135 1991
[123] M Scheffner B A Werness J M Huibregtse A J Levine andP M Howley ldquoThe E6 oncoprotein encoded by human papillo-mavirus types 16 and 18 promotes the degradation of p53rdquo Cellvol 63 no 6 pp 1129ndash1136 1990
[124] M Elbel S Carl S Spaderna and T Iftner ldquoA comparativeanalysis of the interactions of the E6 proteins from cutaneousand genital papillomaviruses with p53 and E6AP in correlationto their transforming potentialrdquoVirology vol 239 no 1 pp 132ndash149 1997
[125] M C Thomas and C-M Chiang ldquoE6 oncoprotein repressesp53-dependent gene activation via inhibition of protein acety-lation independently of inducing p53 degradationrdquo MolecularCell vol 17 no 2 pp 251ndash264 2005
[126] H Zimmermann R Degenkolbe H-U Bernard and M JOrsquoConnor ldquoThe human papillomavirus type 16 E6 oncoproteincan down-regulate p53 activity by targeting the transcriptionalcoactivator CBPp300rdquo Journal of Virology vol 73 no 8 pp6209ndash6219 1999
[127] D A Thompson G Belinsky T H-T Chang D L Jones RSchlegel and K Munger ldquoThe human papillomavirus-16 E6oncoprotein decreases the vigilance of mitotic checkpointsrdquoOncogene vol 15 no 25 pp 3025ndash3035 1997
[128] J Bohl K Das B Dasgupta and S B Vande Pol ldquoCompetitivebinding to a charged leucine motif represses transformation bya papillomavirus E6 oncoproteinrdquo Virology vol 271 no 1 pp163ndash170 2000
[129] S Song H C Pitot and P F Lambert ldquoThe human papillo-mavirus type 16 E6 gene alone is sufficient to induce carcinomasin transgenic animalsrdquo Journal of Virology vol 73 no 7 pp5887ndash5893 1999
[130] S Yasumoto A L Burkhardt J Doniger and J A DiPaololdquoHumanpapillomavirus type 16DNA-inducedmalignant trans-formation of NIH 3T3 cellsrdquo Journal of Virology vol 57 no 2pp 572ndash577 1986
[131] S B Vande Pol M C Brown and C E Turner ldquoAssociation ofBovine Papillomavirus Type 1 E6 oncoprotein with the focaladhesion protein paxillin through a conserved protein interac-tion motifrdquo Oncogene vol 16 no 1 pp 43ndash52 1998
[132] R Wade N Brimer and S Vande Pol ldquoTransformation bybovine papillomavirus type 1 E6 requires paxillinrdquo Journal ofVirology vol 82 no 12 pp 5962ndash5966 2008
[133] A J Klingelhutz S A Foster and J KMcDougall ldquoTelomeraseactivation by the E6 gene product of humanpapillomavirus type16rdquo Nature vol 380 no 6569 pp 79ndash82 1996
[134] S T Oh S Kyo and L A Laimins ldquoTelomerase activation byhuman papillomavirus type 16 E6 protein Induction of humantelomerase reverse transcriptase expression through Myc andGC-rich Sp1 binding sitesrdquo Journal of Virology vol 75 no 12pp 5559ndash5566 2001
[135] L V Ronco A Y KarpovaMVidal and PMHowley ldquoHumanpapillomavirus 16 E6 oncoprotein binds to interferon regulatoryfactor-3 and inhibits its transcriptional activityrdquo Genes andDevelopment vol 12 no 13 pp 2061ndash2072 1998
Figure 2 Electrophoresis gel images showing (a) genomicDNA integrity afterDNAextraction using 1 KbDNALadder (Invitrogen CarlsbadUSA) as marker absence of amplicon in samples 01 to 05 using specific primers to BPV-1 (b) BPV-2 (c) and BPV-4 (d) absence of ampliconin samples 01 to 05 using Delta-Epsilon (e) and Xi (f) degenerate primers Images (b)ndash(f) showed amplification only in positive control withthe 100 bp DNA Ladder (Invitrogen Carlsbad USA) being employed as marker
Table 6 Dunn post hoc test results based on the cytokinesis-blockproliferation index
Group Rank differences Calculated 119885 Critical 119885 119901
Cminus and C+ 76 26870 2394 lt005Cminus and E6 74 26163 2394 lt005C+ and E6 02 00707 2394 nsns nonsignificant differences
oncoprotein induces mitotic stress resulting in clastogenesisand neosis
33 Comet Assay Alkaline Method Comet assay results areshown in Table 7 and Figure 6 According to these data
theKruskal-Wallis test was performedwhich pointed out sta-tistical differences among the groups (119867 = 102613 and 119901 =00059) Based on these results the Dunn post hoc test wasperformed which pointed out significant differences betweennegative and positive control as well as negative controland experimental group (Table 8) However the test did notshow differences between positive control and experimentalgroup (Table 8) These results were also verified by Figure 7Comet assay reinforces the CBMNA results indicating themutagenic potential of E6 recombinant oncoprotein
34 Cytokinesis-Block Micronucleus Assay (CBMNA) inPBMCs Results of this test confirmed the previous resultsindicating that the BPV-1 E6 recombinant oncoprotein has
BioMed Research International 7
LB
(a)
LMN
(b)
LMN
LMN
(c)
PA
PA and MN
(d)
Figure 3 (a) Photomicroscopy of binucleated lymphocyte without micronucleus (LB) observed in negative control Image of binucleatedlymphocyte with micronucleus (LMN) observed in positive control (b) and group treated with E6 recombinant oncoprotein (c) Image ofbinucleated lymphocyte with anaphase bridge and micronucleus (PA and MN) observed in group treated with E6 (d) Images obtained withtotal magnification of 1000x
60
50
40
30
20
10
0
Num
ber o
f mic
ronu
clei
GroupCminus C+ E6
Figure 4 Comparative boxplot based on maximum minimumand median values of micronucleus observed per group
a genotoxicity potential being able to induce DNAbreaks in epithelial cells verified by the presence of MNs(Figure 8)
Figure 5 Evidence of neosis Photomicroscopy of lymphocyteshowing endoreduplication (black arrow) suggesting neosis Cellsanalyzed in total magnification of 1000x
4 Discussion
Transforming potential of E6 oncoprotein has been discussedsince the 1980s based on studies of cottontail rabbit papil-lomaviruses (CRPV) [70 71] Although there are lines ofevidence that the E6 oncoprotein can induce transformation
8 BioMed Research International
(a) (b)
(c) (d)
Figure 6 Images of cometsrsquo nucleoids (a) Image of class 0 (without DNA damage) observed in negative control group Images of class 2(maximum DNA damage) observed in positive control (b) and experimental (c) groups (d) Image of hedgehog comet characterized by thehead absence observed in experimental group Images captured in total magnification of 400x
200
150
100
50
0
Com
et sc
ore
GroupsCminus C+ E6
Figure 7 Boxplot of comet score
[72] the mechanisms of initiation and promotion of cancerassociated with the BPV E6 oncoprotein are unknown
Molecular diagnosis of five peripheral blood samplesfrom five calves showed the absence of amplicons for thefive different primers used (Figure 2) However both specific(BPV-1 BPV-2 and BPV-4) and degenerate (Delta-Epsilonand Xi) primers sets amplified the controls genomes of BPV-1 BPV-2 and BPV-4 None of the negative controls revealedthe amplicon presence These results indicate that the fivesampleswere uninfected by BPVMoreover the use of specific
and degenerate primers increases the capacity of BPV iden-tification This occurs because specific primers are moresensitive than degenerates [66] This sensitivity is due tothe absence of degeneration in the 51015840 region which reducesthe primer ability to recognize and to link with DNA targetsequence [73]The absence of BPV infection in the peripheralblood samples is required for the CBMNA and CA allowinginvestigating the aneugenic andor clastogenic action of BPV-1 E6 recombinant oncoprotein This is because the presenceof BPV sequences is associated with the presence of oncopro-teins transcripts [74]Thus studies have shown that the pres-ence of BPVDNA sequences in peripheral blood is associatedwith cytogenetic damage including clastogenesis [1 14 75]Complementary studies show the association of the BPVpresence with cytogenetic damage in both benign lesions(papillomas) [74] and carcinomas [15]
Chi-square test based on the number of micronucleatedcells pointed out significant statistical values in both positivecontrol and group treated with BPV-1 E6 recombinant onco-protein (Table 3) Negative control showed fewer number ofmicronucleated cells This result was expected because thebiological material was transported and processed after threehours of its collection This procedure reduced the influenceof exogenous environmental factors which could induceDNA damage Furthermore the absence of BPV infection
BioMed Research International 9
(a) (b)
8
6
4
2
0
Num
ber o
f mic
ronu
cleat
ed ce
lls
Neg
ativ
e con
trol
Posit
ive c
ontro
l
E6on
copr
otei
n
(c)
Figure 8 Photomicroscope showing CRIB cell not treated with any drug (negative control) indicating absence of micronucleus (a) andtreated with BPV-1 E6 recombinant oncoprotein showing the presence of MN indicated by arrow (b) (c) Number of micronucleated cellsobserved in each group
in the peripheral blood reduces the influence of endogenousenvironmental factors that could interfere in the analysisPositive control showed a high number of micronucle-ated lymphocytes This result was also expected once thisgroup was treated with 50 120583gmL of cyclophosphamide achemotherapeutic drug with cytotoxic and teratogenic effect[76 77] The cells treated with 1120583gmL of BPV-1 E6 recom-binant oncoprotein also showed significant statistical valuesKruskal-Wallis test followed by the Dunn post hoc testwas performed to compare the frequencies of micronucleusamong three groups This test indicated statistical differencebetween negative control and the cells treated with BPV-1 E6 recombinant oncoprotein However the test did notreveal differences between the cells treated with cyclophos-phamide and BPV-1 E6 recombinant oncoprotein (Table 4Figure 4) Similar results were also observed in epithelial cells(Figure 8) These results suggest that the BPV-1 E6 oncopro-tein has an aneugenic andor clastogenic potential similarto or higher than the observed with the cyclophosphamideThis is because the cells treated with the oncoprotein showed
a frequency of micronucleus formation (MN1199030) 14003
higher than those observed in positive controlThe elevated mutagenic potential of the BPV-1 E6 onco-
protein can justify the cytogenetic damage already describedin the literature [1 14 15 24] Similar results were observedin HPV-infected cells [12 78ndash81] The micronucleus induce-ment was already described in cytological samples collectedduring Papanicolaursquos test from healthy cervix infected byHPV [12] For this reason the micronucleus assay has beenproposed as a complementary method for Papanicolaursquos testbeing a suggestive biomarker of lesion degree [78] Howeverthe association between the BPV and the micronucleusinducement was not yet reported
The high frequency of micronucleated lymphocytes insamples infected by HPV has been attributed to the synergicaction of E5 E6 and E7 oncoproteins [29 30 78 79]However this is the first study that pointed out themutagenicaction of BPV-1 E6 protein per se
Student-Newman-Keuls post hoc test based on the fre-quency of anaphase bridges showed significant statistical
10 BioMed Research International
Table 7 Comet assay results showing the number of nucleoidsobserved per class the number of hedgehog comets and cometscore
Sample Class 0 Class 1 Class 2 Hedgehog ScoreNegative control
Groups Rank differences Calculated 119885 Critical 119885 119901
Cminus and C+ 68 24042 2394 lt05Cminus and E6 82 28991 2394 lt05C+ and E6 14 04950 2394 nsns nonsignificant differences
difference between the negative control and the group treatedwith the BPV-1 E6 recombinant oncoprotein (Table 5) How-ever the test did not reveal differences between the cellstreated with cyclophosphamide and BPV-1 E6 recombinantoncoprotein (Table 5)These data reinforce those observed bythe Dunn post hoc test based on the frequency of micronu-cleus indicating the clastogenic action of the E6 oncoproteinMoreover the presence of anaphase bridge was alreadydescribed in cells transfected with recombinant adenoviruscontaining the E6 and E7 ORFs of HPV [29 30] Howeverthere are no studies describing the action of BPVE6 oncopro-tein in the induction of anaphase bridges or studies that showthis same action of E6 oncoprotein of HPV per se Anaphasebridges are indicators of genomic instability [82 83] which isconsidered the first step in carcinogenesis [46 47] Moreoverthe presence of these bridges is an important hallmark ofDNA double strand breaks (DSBs) [84]
Statistical analysis based on the cytokinesis-block prolif-eration index (CBPI) showed that BPV-1 E6 oncoprotein hascytotoxic levels similar to cyclophosphamide (Table 6) Thisresult suggests that the BPV E6 oncoprotein can deregulatethe cell cycle contributing to the cell proliferation andimmortalization To date this mitogenic action is attributedto the E7 oncoprotein [85ndash87] E7 oncoprotein induces
the phosphorylation of pRb resulting in the E2F transcrip-tion factor release [85] E2F when translocated to the nucleusacts as activator and binds to the kinase-dependent cyclinspromoters [85] However the results showed polynucleatedcells in the group treatedwithBPVE6oncoprotein (Figure 5)suggesting that the BPV E6 oncoprotein confers mitogenicstimulation resulting in mitotic stress Moreover studiespointed out that the BPV E6 oncoprotein interacts with theCBPp300 deacetylase promoting p53 downregulation [88]This downregulation increases the expression levels of FoxM1transcription factor (Foxhead box M1) [89] promoting B1cyclin D1 cyclin and cdc25 upregulation [89] The upregu-lation of these genes is associated with increased levels of cellproliferation which is necessary in order to make DNA poly-merases available to virus replication However if this mech-anism guarantees the BPV replication it can contribute to cellimmortalization and cancer progression
The FoxM1 factor also participates in the Wnt120573-cateninsignaling pathway and binds directly to the 120573-catenin [89]This interaction promotes the nuclear 120573-catenin transloca-tion [90] 120573-catenin translocation to nucleus induces thecyclin expression [64] So these data suggest that the BPVE6 oncoprotein not only promotes mitogenic stress but alsocontributes to the epithelial-mesenchymal transition (EMT)Studies based on theHPVE6 oncoprotein have indicated thatthis oncoprotein contributes significantly to the EMT [91]This occurs not only because the E6 oncoprotein induces thetranslocation of 120573-catenin but also due to the proteasomaldegradation of regulatory proteins of apical-basal polarity[92ndash94] Studies also showed that the E6 oncoprotein is ableto bind to Crumbs (Dlg and Patj) proteins resulting in loss ofcell polarity [93 94] Based on these results the E6 oncopro-tein emerges as a possible therapeutic target with biotechno-logical value in cancer treatment
Mitogenic action of E6 oncoprotein can be moreexpressed in cells infected by papillomaviruses once thesecells also expressed E5 and E7 oncoproteins [74] The E7oncoprotein is able to form a complex E7-p600 which pro-motes E6 upregulation [85] Furthermore the E6 oncoproteincan bind to the E6AP ubiquitin ligase forming the E6-E6APcomplex increasing the hTERT levels of expression con-tributing to cell immortalization [95] Moreover E6-E6APinduces NFX1 expression promoting MHC-II downregula-tion [96]Thismechanism reduces the antigenic presentationmediated by CD4+ T lymphocytes contributing to immuneevasion This action guarantees the PV infection for longperiods with or without clinical symptoms [24] Althoughthe BPV infection can be asymptomatic the BPV presence inperipheral blood is associated with DNA damage [1 14 24]
CBMNA analysis pointed out the presence of cells withintracellular cytokinesis (Figure 5) This result suggests theneotic action of BPV E6 oncoprotein Neosis is characterizedby the presence of (1) DNA damage (2) loss of checkpointcontrol (3) repair system failures and (4) endoreduplication[97] In this scenario the E6 oncoprotein attempts all theseneosis criteria Moreover the oncoprotein not only increasesthe cytogenetic damage leading to increasing of micronu-cleus frequency but also induces clastogenesis as shown bythe comet assay and anaphase bridges So statistical analysis
BioMed Research International 11
performed based on the comet scores reinforced the CBMNAresults Comet assay allows detecting both simple and dou-ble DNA breaks being more sensitive than the CBMNAClastogenesis is considered the most serious type of DNAdamage [98] DNA breaks can be repaired by homologous ornonhomologous recombination However studies point outthat the E6 oncoprotein can link to the Holliday junctionduring the homologous repair avoiding the junction reso-lution [99] Moreover E6 oncoprotein promotes the TP53gene deacetylation resulting in p53 downregulation [51]Thisepigenetic effect results in cell cycle deregulation affectingthe checkpoints
Studies have shown that the HPV-induced DNA breaksare required for the virus integration into the host genome[100] Although there are no studies showing the BPV inte-gration to date the results observed in this work in additionto those already published [24] indicate the necessity ofmorestudies to evaluate the virus-host interaction
Studies also showed that the E6 oncoprotein inducesclathrin-transporter adapter protein (AP-1) increasing thelevel of this protein in the plasma membrane [76 77] Theincreasing quantity of AP-1 in membrane can facilitate theBPV virions infection This occurs because the infectionprocess is clathrin dependent In this scenario the E6 onco-protein can contribute to the virus infection [101]
In summary the CBMNA and CA results showed thatthe BPV E6 oncoprotein participates not only in cancerpromotion but also in initiation inducing DNA breaksThese DNA breaks represent mutagenic effects consideredthe first step in the oncogenesis process which are associatedwith genomic instability [47 48 102 103] Thus the E6recombinant oncoprotein has been suggested as a possiblevaccine candidate [104ndash112] due to its immunogenicity [44]However the mutagenic tests such as CBMNA and CA arerequired in drug validation process [113] In this scenariothis study pointed out that the BPV-1 E6 recombinant onco-protein in tested concentration showedmutagenic potentialThis is the first study that reports the mutagenic potential ofa possible therapeutic vaccine candidate On the one handthese results allowed better understanding the mechanism ofcancer promotion associatedwith the BPVE6oncoprotein aswell as revealing that this oncoprotein can induce carcinogen-esis per se on the other hand this data pointed out thatmaybeBPV E6 recombinant oncoprotein requires protein modifica-tions to be used as vaccine
Conflict of Interests
The authors declare that there is no conflict of interests
Acknowledgments
Theauthors thank Fundacao deAmparo a Pesquisa do Estadode Sao Paulo (FAPESP Process 201420617-5) and FundacaoButantan for the financial support
References
[1] R C Stocco dos Santos C J Lindsey O P Ferraz et al ldquoBovinepapillomavirus transmission and chromosomal aberrations an
experimental modelrdquo Journal of General Virology vol 79 no 9pp 2127ndash2135 1998
[2] A Antonsson and B G Hansson ldquoHealthy skin ofmany animalspecies harbors papillomaviruses which are closely related totheir human counterpartsrdquo Journal of Virology vol 76 no 24pp 12537ndash12542 2002
[3] P Rous and J W Beard ldquoA virus-induced mammalian growthwith the characters of a tumor (the Shope rabbit papilloma) IThe growth on implantationwithin favorable hostsrdquoThe Journalof Experimental Medicine vol 60 no 6 pp 701ndash722 1934
[4] R Shope and E Hurst ldquoInfectious papillomatosis of rabbitsrdquoThe Journal of Experimental Medicine vol 58 no 1 pp 607ndash624 1933
[5] H Delius M A Van Ranst A B Jenson H Zur Hausen and JP Sundberg ldquoCanine oral papillomavirus genomic sequence aunique 15-kb intervening sequence between the E2 andL2 openreading framesrdquo Virology vol 204 no 1 pp 447ndash452 1994
[6] W A Demonbreun and E Goodpasture ldquoInfectious oral papil-lomatosis of dogsrdquo The American Journal of Pathology vol 8no 1 pp 43ndash56 1932
[7] P K Nicholls B A Klaunberg R A Moore et al ldquoNaturallyoccurring nonregressing canine oral papillomavirus infectionhost immunity virus characterization and experimental infec-tionrdquo Virology vol 265 no 2 pp 365ndash374 1999
[8] A Alberti S Pirino F Pintore et al ldquoOvis aries Papillomavirus3 a prototype of a novel genus in the family Papillomaviridaeassociated with ovine squamous cell carcinomardquo Virology vol407 no 2 pp 352ndash359 2010
[9] H zur Hausen E-M de Villiers and L Gissmann ldquoPapil-lomavirus infections and human genital cancerrdquo GynecologicOncology vol 12 no 2 pp S124ndashS128 1981
[10] C Foresta A Bertoldo A Garolla et al ldquoHuman papillo-mavirus proteins are found in peripheral blood and semenCd20+ and Cd56+ cells during Hpv-16 semen infectionrdquo BMCInfectious Diseases vol 13 article 593 2013
[11] S Ljubojevic andM Skerlev ldquoHPV-associated diseasesrdquo Clinicsin Dermatology vol 32 no 2 pp 227ndash234 2014
[12] A P R Cassel R B Barcellos C M D da Silva S E de MatosAlmeida and M L R Rossetti ldquoAssociation between humanpapillomavirus (HPV) DNA and micronuclei in normal cervi-cal cytologyrdquo Genetics and Molecular Biology vol 37 no 2 pp360ndash363 2014
[13] W Jarrett M Campo B OrsquoNeil H Laird and L Coggins ldquoAnovel bovine papillomavirus (BPV-6) causing true epithelialpapillomas of the mammary gland skin a member of a pro-posed newBPV subgrouprdquoVirology vol 136 no 2 pp 255ndash2641984
[14] T CMelo N Diniz S R C Campos et al ldquoCytogenetic studiesin peripheral blood of bovines afflicted by papillomatosisrdquoVeterinary and Comparative Oncology vol 9 no 4 pp 269ndash2742011
[15] S R C Campos T C Melo S Assaf et al ldquoChromosome aber-rations in cells infected with bovine papillomavirus comparingcutaneous papilloma esophagus papilloma and urinary blad-der lesion cellsrdquo ISRN Oncology vol 2013 Article ID 910849 8pages 2013
[16] H zur Hausen ldquoThe search for infectious causes of humancancers where and whyrdquoVirology vol 392 no 1 pp 1ndash10 2009
[17] R P Araldi R F Carvalho T C Melo et al ldquoBovine papillo-mavirus in beef cattle first description of BPV-12 and putativetype BAPV8 in BrazilrdquoGenetics andMolecular Research vol 13no 3 pp 5644ndash5653 2014
12 BioMed Research International
[18] T Rampias C Sasaki and A Psyrri ldquoMolecular mechanisms ofHPV induced carcinogenesis in head and neckrdquoOral Oncologyvol 50 no 5 pp 356ndash363 2014
[19] S Roperto V Russo L Leonardi et al ldquoBovine papillomavirustype 13 expression in the urothelial bladder tumours of cattlerdquoTransboundary and Emerging Diseases 2015
[20] S RCCampos C TrindadeO P Ferraz et al ldquoCan establishedcultured papilloma cells harbor bovine papillomavirusrdquoGenet-ics and Molecular Research vol 7 no 4 pp 1119ndash1126 2008
[21] M S Campo ldquoAnimalmodels of papilloma virus pathogenesisrdquoVirus Research vol 89 no 2 pp 249ndash261 2002
[22] G Borzacchiello ldquoBovine papillomavirus infections in ani-malsrdquo in Communicating Current Research and EducationalTopics and Trends in AppliedMicrobiology pp 673ndash679 Forma-tex Badajoz Spain 2007
[23] F Bocaneti G Altamura A Corteggio E Velescu F RopertoandG Borzacchiello ldquoBovine papillomavirus new insights intoan old diseaserdquo Transboundary and Emerging Diseases 2014
[24] R P Araldi T C Melo N Diniz et al ldquoBovine papillomavirusclastogenic effect analyzed in comet assayrdquo BioMed ResearchInternational vol 2013 Article ID 630683 7 pages 2013
[25] J Munday N Thomson M Dunowska C Knight R Laurieand S Hills ldquoGenomic characterisation of the feline sarcoid-associated papillomavirus and proposed classification as Bostaurus papillomavirus type 14rdquoVeterinaryMicrobiology vol 177no 3-4 pp 289ndash295 2015
[26] G Borzacchiello V Russo C Spoleto et al ldquoBovine papillo-mavirus type-2 DNA and expression of E5 and E7 oncoproteinsin vascular tumours of the urinary bladder in cattlerdquo CancerLetters vol 250 no 1 pp 82ndash91 2007
[27] P Maiolino A Ozkul A Sepici-Dincel et al ldquoBovine papillo-mavirus type 2 infection and microscopic patterns of urothelialtumors of the urinary bladder in water buffaloesrdquo BioMedResearch International vol 2013 Article ID 937918 6 pages2013
[28] S Roperto G Borzacchiello R Brun et al ldquoMultiple glomustumors of the urinary bladder in a cow associated with bovinepapillomavirus type 2 (BPV-2) infectionrdquo Veterinary Pathologyvol 45 no 1 pp 39ndash42 2008
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[30] S Duensing L Y Lee A Duensing et al ldquoThe human papil-lomavirus type 16 E6 and E7 oncoproteins cooperate to inducemitotic defects and genomic instability by uncoupling centro-some duplication from the cell division cyclerdquo Proceedings of theNational Academy of Sciences of theUnited States of America vol97 no 18 pp 10002ndash10007 2000
[31] C A Moody and L A Laimins ldquoHuman papillomavirus onco-proteins pathways to transformationrdquo Nature Reviews Cancervol 10 no 8 pp 550ndash560 2010
[32] C Mansur and E Androphy ldquoCellular transformation bypapillomavirus oncoproteinsrdquo Biochimica et Biophysica Actavol 1155 no 3 pp 323ndash345 1993
[33] J J Chen Y Hong and E J Androphy ldquoMutational analysis oftranscriptional activation by the bovine papillomavirus type 1E6rdquo Virology vol 236 no 1 pp 30ndash36 1997
[34] X Tong andPMHowley ldquoThebovine papillomavirus E6 onco-protein interacts with paxillin and disrupts the actin cytoskele-tonrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 94 no 9 pp 4412ndash4417 1997
[35] X TongW Boll T Kirchhausen andPMHowley ldquoInteractionof the bovine papillomavirus E6 protein with the clathrinadaptor complex AP-1rdquo Journal of Virology vol 72 no 1 pp476ndash482 1998
[36] Y Nomine M Masson S Charbonnier et al ldquoStructural andfunctional analysis of E6 oncoprotein insights in the molecularpathways of human papillomavirus-mediated pathogenesisrdquoMolecular Cell vol 21 no 5 pp 665ndash678 2006
[37] T Ristriani MMasson Y Nomine et al ldquoHPV oncoprotein E6is a structure-dependent DNA-binding protein that recognizesfour-way junctionsrdquo Journal of Molecular Biology vol 296 no5 pp 1189ndash1203 2000
[38] Y Liu and J D Baleja ldquoStructure and function of the papil-lomavirus E6 protein and its interacting proteinsrdquo Frontiers inBioscience vol 13 no 1 pp 121ndash134 2008
[39] F Lipari G A McGibbon E Wardrop and M G CordingleyldquoPurification and biophysical characterization of a minimalfunctional domain and of an N-terminal Zn2+-binding frag-ment from the human papillomavirus type 16 E6 proteinrdquoBiochemistry vol 40 no 5 pp 1196ndash1204 2001
[40] Y Nomine T Ristriani C Laurent J-F Lefevre E Weissand G Trave ldquoA strategy for optimizing the monodispersity offusion proteins application to purification of recombinantHPVE6 oncoproteinrdquo Protein Engineering vol 14 no 4 pp 297ndash3052001
[41] C G Ullman P I Haris D A Galloway V C Emery and SJ Perkins ldquoPredicted 120572-helix120573-sheet secondary structures forthe zinc-binding motifs of human papillomavirus E7 and E6proteins by consensus prediction averaging and spectroscopicstudies of E7rdquo Biochemical Journal vol 319 no 1 pp 229ndash2391996
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[43] K Zanier S Charbonnier A O M O Sidi et al ldquoStructuralbasis for hijacking of cellular LxxLL motifs by papillomavirusE6 oncoproteinsrdquo Science vol 339 no 6120 pp 694ndash698 2013
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[49] K Zanier S Charbonnier M Baltzinger Y Nomine DAltschuh and G Trave ldquoKinetic analysis of the interactionsof human papillomavirus E6 oncoproteins with the ubiquitinligase E6AP using surface plasmon resonancerdquo Journal ofMolecular Biology vol 349 no 2 pp 401ndash412 2005
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[70] O Danos E Georges G Orth and M Yaniv ldquoFine structureof the cottontail rabbit papillomavirus mRNAs expressed in thetransplantable VX2 carcinomardquo Journal of Virology vol 53 no3 pp 735ndash741 1985
[71] M Nasseri and F O Wettstein ldquoDifferences exist between viraltranscripts in cottontail rabbit papillomavirus-induced benignand malignant tumors as well as non-virus-producing andvirus-producing tumorsrdquo Journal of Virology vol 51 no 3 pp706ndash712 1984
[72] X Tong R Salgia J-L Li J D Griffin and P M Howley ldquoThebovine papillomavirus E6 protein binds to the LDmotif repeatsof paxillin and blocks its interaction with vinculin and the focaladhesion kinaserdquo Journal of Biological Chemistry vol 272 no52 pp 33373ndash33376 1997
[73] Y Maeda T Shibahara Y Wada et al ldquoAn outbreak of teatpapillomatosis in cattle caused by bovine papilloma virus (BPV)type 6 and unclassified BPVsrdquo Veterinary Microbiology vol 121no 3-4 pp 242ndash248 2007
[74] R P Araldi T C Melo A Neves et al ldquoHyperproliferativeaction of bovine papillomavirus genetic and histopathologicalaspectsrdquo Genetics and Molecular Research vol 14 no 4 pp12942ndash12954 2015
[75] NDiniz T CMelo J F Santos et al ldquoSimultaneous presence ofbovine papillomavirus in blood and in short-term lymphocytecultures from dairy cattle in Pernambuco Brazilrdquo Genetics andMolecular Research vol 8 no 4 pp 1474ndash1480 2009
[76] O M Brito M F Guimaraes and C C Lanna ldquoCiclofosfamidae funcao ovarianardquo Revista Brasileira de Reumatologia vol 48no 1 pp 39ndash45 2008
[77] D Anderson T-W Yu and D B McGregor ldquoComet assayresponses as indicators of carcinogen exposurerdquo Mutagenesisvol 13 no 6 pp 539ndash555 1998
[78] M L Adam C Pini S Tulio J C L L Cantalice R A Torresand M T Dos Santos Correia ldquoAssessment of the associationbetween micronuclei and the degree of uterine lesions and viralload in women with human papillomavirusrdquo Cancer Genomicsamp Proteomics vol 12 no 2 pp 67ndash72 2015
[79] E Cortes-Gutierrez M Davila-Rodrıguez J Vargas-VillarrealF Hernandez-Garza and R Cerda-Flores ldquoAssociationbetween human papilloma virus-type infections with micro-nuclei frequenciesrdquo Prague Medical Report vol 111 no 1 pp35ndash41 2010
[80] R N Chakrabarti and K Dutta ldquoMicronuclei test in routinesmears fromuterine cervixrdquo European Journal of GynaecologicalOncology vol 9 no 5 pp 370ndash372 1988
[81] E M M Cerqueira C L Santoro N F Donozo et al ldquoGeneticdamage in exfoliated cells of the uterine cervix Association
14 BioMed Research International
and interaction between cigarette smoking and progression tomalignant transformationrdquo Acta Cytologica vol 42 no 3 pp639ndash649 1998
[82] S M Germann V Schramke R T Pedersen et al ldquoTopBP1Dpb11 bindsDNA anaphase bridges to prevent genome instabil-ityrdquoThe Journal of Cell Biology vol 204 no 1 pp 45ndash59 2014
[83] D R Hoffelder L Luo N A Burke S CWatkins S M Gollinand W S Saunders ldquoResolution of anaphase bridges in cancercellsrdquo Chromosoma vol 112 no 8 pp 389ndash397 2004
[84] C Acilan D M Potter and W S Saunders ldquoDNA repairpathways involved in anaphase bridge formationrdquo Genes Chro-mosomes and Cancer vol 46 no 6 pp 522ndash531 2007
[85] J DeMasi M C Chao A S Kumar and P M Howley ldquoBovinepapillomavirus E7 oncoprotein inhibits anoikisrdquo Journal ofVirology vol 81 no 17 pp 9419ndash9425 2007
[86] J DeMasi K-W Huh Y Nakatani K Munger and P MHowley ldquoBovine papillomavirus E7 transformation functioncorrelates with cellular p600 protein bindingrdquo Proceedings of theNational Academy of Sciences of theUnited States of America vol102 no 32 pp 11486ndash11491 2005
[87] E A White and P M Howley ldquoProteomic approaches to thestudy of papillomavirusndashhost interactionsrdquo Virology vol 435no 1 pp 57ndash69 2013
[88] FMantovani and L Banks ldquoThe human papillomavirus E6 pro-tein and its contribution to malignant progressionrdquo Oncogenevol 20 no 54 pp 7874ndash7887 2001
[89] P Chen and H Lee ldquoFOXM1 induced by E6 oncoprotein pro-motes tumor invasion and chemoresistance in HPV-infectedlung cancerrdquo Cancer Cell amp Microenvironment vol 2 no 1 pp6ndash9 2015
[90] N Zhang P Wei A Gong et al ldquoFoxM1 promotes 120573-cateninnuclear localization and controls Wnt target-gene expressionand glioma tumorigenesisrdquo Cancer Cell vol 127 no 4 pp 358ndash366 2012
[91] Z Liu Y Liu Y Hong L Rapp E J Androphy and J JChen ldquoBovine papillomavirus type 1 E6-induced sensitizationto apoptosis is distinct from its transforming activityrdquo Virologyvol 295 no 2 pp 230ndash237 2002
[92] M Thomas P Massimi C Navarro J-P Borg and L BanksldquoThe hScribDlg apico-basal control complex is differentiallytargeted by HPV-16 and HPV-18 E6 proteinsrdquo Oncogene vol24 no 41 pp 6222ndash6230 2005
[93] C H Storrs and S J Silverstein ldquoPATJ a tight junction-associated PDZ protein is a novel degradation target of high-risk human papillomavirus E6 and the alternatively splicedisoform 18 E6rdquo Journal of Virology vol 81 no 8 pp 4080ndash40902007
[94] R Accardi R Rubino M Scalise et al ldquoE6 and E7 from humanpapillomavirus type 16 cooperate to target the PDZ proteinNaH exchange regulatory factor 1rdquo Journal of Virology vol 85no 16 pp 8208ndash8216 2011
[95] S Nakagawa and J M Huibregtse ldquoHuman scribble (Vartul)is targeted for ubiquitin-mediated degradation by the high-risk papillomavirus E6 proteins and the E6APubiquitin-proteinligaserdquoMolecular and Cellular Biology vol 20 no 21 pp 8244ndash8253 2000
[96] L Gewin H Myers T Kiyono and D A Galloway ldquoIdentifi-cation of a novel telomerase repressor that interacts with thehuman papillomavirus type-16 E6E6-AP complexrdquo Genes ampDevelopment vol 18 no 18 pp 2269ndash2282 2004
[97] M Sundaram D L GuernseyMM Rajaraman and R Rajara-man ldquoNeosis a novel type of cell division in cancerrdquo CancerBiology ampTherapy vol 3 no 2 pp 207ndash218 2004
[98] R D Snyder and J W Green ldquoA review of the genotoxicity ofmarketed pharmaceuticalsrdquo Mutation Research vol 488 no 2pp 151ndash169 2001
[99] T Ristriani Y Nomine C Laurent E Weiss and G TraveldquoProtein mutagenesis with monodispersity-based quality prob-ing selective inactivation of p53 degradation and DNA-bindingproperties of HPV E6 oncoproteinrdquo Protein Expression andPurification vol 26 no 3 pp 357ndash367 2002
[100] M Kadaja A Sumerina T Verst M Ojarand E Ustav and MUstav ldquoGenomic instability of the host cell induced by thehuman papillomavirus replicationmachineryrdquoTheEMBO Jour-nal vol 26 no 8 pp 2180ndash2191 2007
[101] PMDay D R Lowy and J T Schiller ldquoPapillomaviruses infectcells via a clathrin-dependent pathwayrdquo Virology vol 307 no 1pp 1ndash11 2003
[102] T D Tlsty A Briot A Gualberto et al ldquoGenomic instabilityand cancerrdquoMutation Research vol 337 no 1 pp 1ndash7 1995
[103] J E Eyfjord and S K Bodvarsdottir ldquoGenomic instabilityand cancer networks involved in response to DNA damagerdquoMutation Research vol 592 no 1-2 pp 18ndash28 2005
[104] Y Yao W Huang X Yang et al ldquoHPV-16 E6 and E7 protein Tcell epitopes prediction analysis based on distributions of HLA-A loci across populations an in silico approachrdquo Vaccine vol31 no 18 pp 2289ndash2294 2013
[105] C-F Hung B Ma A Monie S-W Tsen and T-C Wu ldquoTher-apeutic human papillomavirus vaccines current clinical trialsand future directionsrdquo Expert Opinion on Biological Therapyvol 8 no 4 pp 421ndash439 2008
[106] M J P Welters G G Kenter S J Piersma et al ldquoInductionof tumor-specific CD4+ and CD8+ T-cell immunity in cervicalcancer patients by a human papillomavirus type 16 E6 and E7long peptides vaccinerdquo Clinical Cancer Research vol 14 no 1pp 178ndash187 2008
[107] R Han N M Cladel C A Reed X Peng and N D Chris-tensen ldquoProtection of rabbits from viral challenge by gene gun-based intracutaneous vaccination with a combination of cot-tontail rabbit papillomavirus E1 E2 E6 and E7 genesrdquo Journalof Virology vol 73 no 8 pp 7039ndash7043 1999
[108] Z He A P Wlazlo D W Kowalczyk et al ldquoViral recombinantvaccines to the E6 and E7 antigens of HPV-16rdquo Virology vol270 no 1 pp 146ndash161 2000
[109] A Kaufmann P Stern E Rankin et al ldquoSafety and immuno-genicity of TA-HPV a recombinant vaccinia virus expressingmodified human papillomavirus (HPV)-16 and HPV-18 E6 andE7 genes in women with progressive cervical cancerrdquo ClinicalCancer Research vol 8 no 12 pp 3676ndash3685 2002
[110] L K Borysiewicz A Fiander M Nimako et al ldquoA recombinantvaccinia virus encoding human papillomavirus types 16 and 18E6 and E7 proteins as immunotherapy for cervical cancerrdquoTheLancet vol 347 no 9014 pp 1523ndash1527 1996
[111] A De Jong T OrsquoNeill A Y Khan et al ldquoEnhancement ofhuman papillomavirus (HPV) type 16 E6 and E7-specific T-cellimmunity in healthy volunteers through vaccination with TA-CIN an HPV16 L2E7E6 fusion protein vaccinerdquo Vaccine vol20 no 29-30 pp 3456ndash3464 2002
[112] S Chen C Liao Y Lai et al ldquoDe-oncogenicHPVE6E7 vaccinegets enhanced antigenicity and promotes tumoricidal synergywith cisplatinrdquo Acta Biochimica et Biophysica Sinica vol 46 no1 pp 6ndash14 2014
BioMed Research International 15
[113] J Bonhoeffer E B Imoukhuede G Aldrovandi et al ldquoTemplateprotocol for clinical trials investigating vaccines-focus on safetyelementsrdquo Vaccine vol 31 no 47 pp 5602ndash5620 2013
[114] S Jackson and A Storey ldquoE6 proteins from diverse cutaneonsHPV types inhibit apoptosis in response to UV damagerdquoOncogene vol 19 no 4 pp 592ndash598 2000
[115] M Thomas and L Banks ldquoPDZRN3LNX3 is a novel targetof human papillomavirus type 16 (HPV-16) and HPV-18 E6rdquoJournal of Virology vol 89 no 2 pp 1439ndash1444 2015
[116] P R Daniels C M Sanders P Coulson and N J MaitlandldquoMolecular analysis of the interaction between HPV type 16 E6and human E6-associated proteinrdquo FEBS Letters vol 416 no 1pp 6ndash10 1997
[117] W H Kao S L Beaudenon A L Talis J M Huibregtse andP M Howley ldquoHuman papillomavirus type 16 E6 induces self-ubiquitination of the E6AP ubiquitin-protein ligaserdquo Journal ofVirology vol 74 no 14 pp 6408ndash6417 2000
[118] T Iftner M Elbel B Schopp et al ldquoInterference of papillo-mavirus E6 protein with single-strand break repair by interac-tion with XRCC1rdquo The EMBO Journal vol 21 no 17 pp 4741ndash4748 2002
[119] Y Imai Y Tsunokawa T Sugimura and M Terada ldquoPurifi-cation and DNA-binding properties of human papillomavirustype 16 E6 protein expressed in Escherichia colirdquo Biochemicaland Biophysical Research Communications vol 164 no 3 pp1402ndash1410 1989
[120] M Lusky L Berg H Weiher and M Botchan ldquoBovinepapilloma virus contains an activator of gene expression at thedistal end of the early transcription unitrdquoMolecular andCellularBiology vol 3 no 6 pp 1108ndash1122 1983
[121] S Dalal Q Gao E J Androphy and V Band ldquoMutationalanalysis of human papillomavirus type 16 E6 demonstrates thatp53 degradation is necessary for immortalization of mammaryepithelial cellsrdquo Journal of Virology vol 70 no 2 pp 683ndash6881996
[122] J M Huibregtse M Scheffner and P M Howley ldquoA cellularprotein mediates association of p53 with the E6 oncoprotein ofhuman papillomavirus types 16 or 18rdquo The EMBO Journal vol10 no 13 pp 4129ndash4135 1991
[123] M Scheffner B A Werness J M Huibregtse A J Levine andP M Howley ldquoThe E6 oncoprotein encoded by human papillo-mavirus types 16 and 18 promotes the degradation of p53rdquo Cellvol 63 no 6 pp 1129ndash1136 1990
[124] M Elbel S Carl S Spaderna and T Iftner ldquoA comparativeanalysis of the interactions of the E6 proteins from cutaneousand genital papillomaviruses with p53 and E6AP in correlationto their transforming potentialrdquoVirology vol 239 no 1 pp 132ndash149 1997
[125] M C Thomas and C-M Chiang ldquoE6 oncoprotein repressesp53-dependent gene activation via inhibition of protein acety-lation independently of inducing p53 degradationrdquo MolecularCell vol 17 no 2 pp 251ndash264 2005
[126] H Zimmermann R Degenkolbe H-U Bernard and M JOrsquoConnor ldquoThe human papillomavirus type 16 E6 oncoproteincan down-regulate p53 activity by targeting the transcriptionalcoactivator CBPp300rdquo Journal of Virology vol 73 no 8 pp6209ndash6219 1999
[127] D A Thompson G Belinsky T H-T Chang D L Jones RSchlegel and K Munger ldquoThe human papillomavirus-16 E6oncoprotein decreases the vigilance of mitotic checkpointsrdquoOncogene vol 15 no 25 pp 3025ndash3035 1997
[128] J Bohl K Das B Dasgupta and S B Vande Pol ldquoCompetitivebinding to a charged leucine motif represses transformation bya papillomavirus E6 oncoproteinrdquo Virology vol 271 no 1 pp163ndash170 2000
[129] S Song H C Pitot and P F Lambert ldquoThe human papillo-mavirus type 16 E6 gene alone is sufficient to induce carcinomasin transgenic animalsrdquo Journal of Virology vol 73 no 7 pp5887ndash5893 1999
[130] S Yasumoto A L Burkhardt J Doniger and J A DiPaololdquoHumanpapillomavirus type 16DNA-inducedmalignant trans-formation of NIH 3T3 cellsrdquo Journal of Virology vol 57 no 2pp 572ndash577 1986
[131] S B Vande Pol M C Brown and C E Turner ldquoAssociation ofBovine Papillomavirus Type 1 E6 oncoprotein with the focaladhesion protein paxillin through a conserved protein interac-tion motifrdquo Oncogene vol 16 no 1 pp 43ndash52 1998
[132] R Wade N Brimer and S Vande Pol ldquoTransformation bybovine papillomavirus type 1 E6 requires paxillinrdquo Journal ofVirology vol 82 no 12 pp 5962ndash5966 2008
[133] A J Klingelhutz S A Foster and J KMcDougall ldquoTelomeraseactivation by the E6 gene product of humanpapillomavirus type16rdquo Nature vol 380 no 6569 pp 79ndash82 1996
[134] S T Oh S Kyo and L A Laimins ldquoTelomerase activation byhuman papillomavirus type 16 E6 protein Induction of humantelomerase reverse transcriptase expression through Myc andGC-rich Sp1 binding sitesrdquo Journal of Virology vol 75 no 12pp 5559ndash5566 2001
[135] L V Ronco A Y KarpovaMVidal and PMHowley ldquoHumanpapillomavirus 16 E6 oncoprotein binds to interferon regulatoryfactor-3 and inhibits its transcriptional activityrdquo Genes andDevelopment vol 12 no 13 pp 2061ndash2072 1998
Figure 3 (a) Photomicroscopy of binucleated lymphocyte without micronucleus (LB) observed in negative control Image of binucleatedlymphocyte with micronucleus (LMN) observed in positive control (b) and group treated with E6 recombinant oncoprotein (c) Image ofbinucleated lymphocyte with anaphase bridge and micronucleus (PA and MN) observed in group treated with E6 (d) Images obtained withtotal magnification of 1000x
60
50
40
30
20
10
0
Num
ber o
f mic
ronu
clei
GroupCminus C+ E6
Figure 4 Comparative boxplot based on maximum minimumand median values of micronucleus observed per group
a genotoxicity potential being able to induce DNAbreaks in epithelial cells verified by the presence of MNs(Figure 8)
Figure 5 Evidence of neosis Photomicroscopy of lymphocyteshowing endoreduplication (black arrow) suggesting neosis Cellsanalyzed in total magnification of 1000x
4 Discussion
Transforming potential of E6 oncoprotein has been discussedsince the 1980s based on studies of cottontail rabbit papil-lomaviruses (CRPV) [70 71] Although there are lines ofevidence that the E6 oncoprotein can induce transformation
8 BioMed Research International
(a) (b)
(c) (d)
Figure 6 Images of cometsrsquo nucleoids (a) Image of class 0 (without DNA damage) observed in negative control group Images of class 2(maximum DNA damage) observed in positive control (b) and experimental (c) groups (d) Image of hedgehog comet characterized by thehead absence observed in experimental group Images captured in total magnification of 400x
200
150
100
50
0
Com
et sc
ore
GroupsCminus C+ E6
Figure 7 Boxplot of comet score
[72] the mechanisms of initiation and promotion of cancerassociated with the BPV E6 oncoprotein are unknown
Molecular diagnosis of five peripheral blood samplesfrom five calves showed the absence of amplicons for thefive different primers used (Figure 2) However both specific(BPV-1 BPV-2 and BPV-4) and degenerate (Delta-Epsilonand Xi) primers sets amplified the controls genomes of BPV-1 BPV-2 and BPV-4 None of the negative controls revealedthe amplicon presence These results indicate that the fivesampleswere uninfected by BPVMoreover the use of specific
and degenerate primers increases the capacity of BPV iden-tification This occurs because specific primers are moresensitive than degenerates [66] This sensitivity is due tothe absence of degeneration in the 51015840 region which reducesthe primer ability to recognize and to link with DNA targetsequence [73]The absence of BPV infection in the peripheralblood samples is required for the CBMNA and CA allowinginvestigating the aneugenic andor clastogenic action of BPV-1 E6 recombinant oncoprotein This is because the presenceof BPV sequences is associated with the presence of oncopro-teins transcripts [74]Thus studies have shown that the pres-ence of BPVDNA sequences in peripheral blood is associatedwith cytogenetic damage including clastogenesis [1 14 75]Complementary studies show the association of the BPVpresence with cytogenetic damage in both benign lesions(papillomas) [74] and carcinomas [15]
Chi-square test based on the number of micronucleatedcells pointed out significant statistical values in both positivecontrol and group treated with BPV-1 E6 recombinant onco-protein (Table 3) Negative control showed fewer number ofmicronucleated cells This result was expected because thebiological material was transported and processed after threehours of its collection This procedure reduced the influenceof exogenous environmental factors which could induceDNA damage Furthermore the absence of BPV infection
BioMed Research International 9
(a) (b)
8
6
4
2
0
Num
ber o
f mic
ronu
cleat
ed ce
lls
Neg
ativ
e con
trol
Posit
ive c
ontro
l
E6on
copr
otei
n
(c)
Figure 8 Photomicroscope showing CRIB cell not treated with any drug (negative control) indicating absence of micronucleus (a) andtreated with BPV-1 E6 recombinant oncoprotein showing the presence of MN indicated by arrow (b) (c) Number of micronucleated cellsobserved in each group
in the peripheral blood reduces the influence of endogenousenvironmental factors that could interfere in the analysisPositive control showed a high number of micronucle-ated lymphocytes This result was also expected once thisgroup was treated with 50 120583gmL of cyclophosphamide achemotherapeutic drug with cytotoxic and teratogenic effect[76 77] The cells treated with 1120583gmL of BPV-1 E6 recom-binant oncoprotein also showed significant statistical valuesKruskal-Wallis test followed by the Dunn post hoc testwas performed to compare the frequencies of micronucleusamong three groups This test indicated statistical differencebetween negative control and the cells treated with BPV-1 E6 recombinant oncoprotein However the test did notreveal differences between the cells treated with cyclophos-phamide and BPV-1 E6 recombinant oncoprotein (Table 4Figure 4) Similar results were also observed in epithelial cells(Figure 8) These results suggest that the BPV-1 E6 oncopro-tein has an aneugenic andor clastogenic potential similarto or higher than the observed with the cyclophosphamideThis is because the cells treated with the oncoprotein showed
a frequency of micronucleus formation (MN1199030) 14003
higher than those observed in positive controlThe elevated mutagenic potential of the BPV-1 E6 onco-
protein can justify the cytogenetic damage already describedin the literature [1 14 15 24] Similar results were observedin HPV-infected cells [12 78ndash81] The micronucleus induce-ment was already described in cytological samples collectedduring Papanicolaursquos test from healthy cervix infected byHPV [12] For this reason the micronucleus assay has beenproposed as a complementary method for Papanicolaursquos testbeing a suggestive biomarker of lesion degree [78] Howeverthe association between the BPV and the micronucleusinducement was not yet reported
The high frequency of micronucleated lymphocytes insamples infected by HPV has been attributed to the synergicaction of E5 E6 and E7 oncoproteins [29 30 78 79]However this is the first study that pointed out themutagenicaction of BPV-1 E6 protein per se
Student-Newman-Keuls post hoc test based on the fre-quency of anaphase bridges showed significant statistical
10 BioMed Research International
Table 7 Comet assay results showing the number of nucleoidsobserved per class the number of hedgehog comets and cometscore
Sample Class 0 Class 1 Class 2 Hedgehog ScoreNegative control
Groups Rank differences Calculated 119885 Critical 119885 119901
Cminus and C+ 68 24042 2394 lt05Cminus and E6 82 28991 2394 lt05C+ and E6 14 04950 2394 nsns nonsignificant differences
difference between the negative control and the group treatedwith the BPV-1 E6 recombinant oncoprotein (Table 5) How-ever the test did not reveal differences between the cellstreated with cyclophosphamide and BPV-1 E6 recombinantoncoprotein (Table 5)These data reinforce those observed bythe Dunn post hoc test based on the frequency of micronu-cleus indicating the clastogenic action of the E6 oncoproteinMoreover the presence of anaphase bridge was alreadydescribed in cells transfected with recombinant adenoviruscontaining the E6 and E7 ORFs of HPV [29 30] Howeverthere are no studies describing the action of BPVE6 oncopro-tein in the induction of anaphase bridges or studies that showthis same action of E6 oncoprotein of HPV per se Anaphasebridges are indicators of genomic instability [82 83] which isconsidered the first step in carcinogenesis [46 47] Moreoverthe presence of these bridges is an important hallmark ofDNA double strand breaks (DSBs) [84]
Statistical analysis based on the cytokinesis-block prolif-eration index (CBPI) showed that BPV-1 E6 oncoprotein hascytotoxic levels similar to cyclophosphamide (Table 6) Thisresult suggests that the BPV E6 oncoprotein can deregulatethe cell cycle contributing to the cell proliferation andimmortalization To date this mitogenic action is attributedto the E7 oncoprotein [85ndash87] E7 oncoprotein induces
the phosphorylation of pRb resulting in the E2F transcrip-tion factor release [85] E2F when translocated to the nucleusacts as activator and binds to the kinase-dependent cyclinspromoters [85] However the results showed polynucleatedcells in the group treatedwithBPVE6oncoprotein (Figure 5)suggesting that the BPV E6 oncoprotein confers mitogenicstimulation resulting in mitotic stress Moreover studiespointed out that the BPV E6 oncoprotein interacts with theCBPp300 deacetylase promoting p53 downregulation [88]This downregulation increases the expression levels of FoxM1transcription factor (Foxhead box M1) [89] promoting B1cyclin D1 cyclin and cdc25 upregulation [89] The upregu-lation of these genes is associated with increased levels of cellproliferation which is necessary in order to make DNA poly-merases available to virus replication However if this mech-anism guarantees the BPV replication it can contribute to cellimmortalization and cancer progression
The FoxM1 factor also participates in the Wnt120573-cateninsignaling pathway and binds directly to the 120573-catenin [89]This interaction promotes the nuclear 120573-catenin transloca-tion [90] 120573-catenin translocation to nucleus induces thecyclin expression [64] So these data suggest that the BPVE6 oncoprotein not only promotes mitogenic stress but alsocontributes to the epithelial-mesenchymal transition (EMT)Studies based on theHPVE6 oncoprotein have indicated thatthis oncoprotein contributes significantly to the EMT [91]This occurs not only because the E6 oncoprotein induces thetranslocation of 120573-catenin but also due to the proteasomaldegradation of regulatory proteins of apical-basal polarity[92ndash94] Studies also showed that the E6 oncoprotein is ableto bind to Crumbs (Dlg and Patj) proteins resulting in loss ofcell polarity [93 94] Based on these results the E6 oncopro-tein emerges as a possible therapeutic target with biotechno-logical value in cancer treatment
Mitogenic action of E6 oncoprotein can be moreexpressed in cells infected by papillomaviruses once thesecells also expressed E5 and E7 oncoproteins [74] The E7oncoprotein is able to form a complex E7-p600 which pro-motes E6 upregulation [85] Furthermore the E6 oncoproteincan bind to the E6AP ubiquitin ligase forming the E6-E6APcomplex increasing the hTERT levels of expression con-tributing to cell immortalization [95] Moreover E6-E6APinduces NFX1 expression promoting MHC-II downregula-tion [96]Thismechanism reduces the antigenic presentationmediated by CD4+ T lymphocytes contributing to immuneevasion This action guarantees the PV infection for longperiods with or without clinical symptoms [24] Althoughthe BPV infection can be asymptomatic the BPV presence inperipheral blood is associated with DNA damage [1 14 24]
CBMNA analysis pointed out the presence of cells withintracellular cytokinesis (Figure 5) This result suggests theneotic action of BPV E6 oncoprotein Neosis is characterizedby the presence of (1) DNA damage (2) loss of checkpointcontrol (3) repair system failures and (4) endoreduplication[97] In this scenario the E6 oncoprotein attempts all theseneosis criteria Moreover the oncoprotein not only increasesthe cytogenetic damage leading to increasing of micronu-cleus frequency but also induces clastogenesis as shown bythe comet assay and anaphase bridges So statistical analysis
BioMed Research International 11
performed based on the comet scores reinforced the CBMNAresults Comet assay allows detecting both simple and dou-ble DNA breaks being more sensitive than the CBMNAClastogenesis is considered the most serious type of DNAdamage [98] DNA breaks can be repaired by homologous ornonhomologous recombination However studies point outthat the E6 oncoprotein can link to the Holliday junctionduring the homologous repair avoiding the junction reso-lution [99] Moreover E6 oncoprotein promotes the TP53gene deacetylation resulting in p53 downregulation [51]Thisepigenetic effect results in cell cycle deregulation affectingthe checkpoints
Studies have shown that the HPV-induced DNA breaksare required for the virus integration into the host genome[100] Although there are no studies showing the BPV inte-gration to date the results observed in this work in additionto those already published [24] indicate the necessity ofmorestudies to evaluate the virus-host interaction
Studies also showed that the E6 oncoprotein inducesclathrin-transporter adapter protein (AP-1) increasing thelevel of this protein in the plasma membrane [76 77] Theincreasing quantity of AP-1 in membrane can facilitate theBPV virions infection This occurs because the infectionprocess is clathrin dependent In this scenario the E6 onco-protein can contribute to the virus infection [101]
In summary the CBMNA and CA results showed thatthe BPV E6 oncoprotein participates not only in cancerpromotion but also in initiation inducing DNA breaksThese DNA breaks represent mutagenic effects consideredthe first step in the oncogenesis process which are associatedwith genomic instability [47 48 102 103] Thus the E6recombinant oncoprotein has been suggested as a possiblevaccine candidate [104ndash112] due to its immunogenicity [44]However the mutagenic tests such as CBMNA and CA arerequired in drug validation process [113] In this scenariothis study pointed out that the BPV-1 E6 recombinant onco-protein in tested concentration showedmutagenic potentialThis is the first study that reports the mutagenic potential ofa possible therapeutic vaccine candidate On the one handthese results allowed better understanding the mechanism ofcancer promotion associatedwith the BPVE6oncoprotein aswell as revealing that this oncoprotein can induce carcinogen-esis per se on the other hand this data pointed out thatmaybeBPV E6 recombinant oncoprotein requires protein modifica-tions to be used as vaccine
Conflict of Interests
The authors declare that there is no conflict of interests
Acknowledgments
Theauthors thank Fundacao deAmparo a Pesquisa do Estadode Sao Paulo (FAPESP Process 201420617-5) and FundacaoButantan for the financial support
References
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experimental modelrdquo Journal of General Virology vol 79 no 9pp 2127ndash2135 1998
[2] A Antonsson and B G Hansson ldquoHealthy skin ofmany animalspecies harbors papillomaviruses which are closely related totheir human counterpartsrdquo Journal of Virology vol 76 no 24pp 12537ndash12542 2002
[3] P Rous and J W Beard ldquoA virus-induced mammalian growthwith the characters of a tumor (the Shope rabbit papilloma) IThe growth on implantationwithin favorable hostsrdquoThe Journalof Experimental Medicine vol 60 no 6 pp 701ndash722 1934
[4] R Shope and E Hurst ldquoInfectious papillomatosis of rabbitsrdquoThe Journal of Experimental Medicine vol 58 no 1 pp 607ndash624 1933
[5] H Delius M A Van Ranst A B Jenson H Zur Hausen and JP Sundberg ldquoCanine oral papillomavirus genomic sequence aunique 15-kb intervening sequence between the E2 andL2 openreading framesrdquo Virology vol 204 no 1 pp 447ndash452 1994
[6] W A Demonbreun and E Goodpasture ldquoInfectious oral papil-lomatosis of dogsrdquo The American Journal of Pathology vol 8no 1 pp 43ndash56 1932
[7] P K Nicholls B A Klaunberg R A Moore et al ldquoNaturallyoccurring nonregressing canine oral papillomavirus infectionhost immunity virus characterization and experimental infec-tionrdquo Virology vol 265 no 2 pp 365ndash374 1999
[8] A Alberti S Pirino F Pintore et al ldquoOvis aries Papillomavirus3 a prototype of a novel genus in the family Papillomaviridaeassociated with ovine squamous cell carcinomardquo Virology vol407 no 2 pp 352ndash359 2010
[9] H zur Hausen E-M de Villiers and L Gissmann ldquoPapil-lomavirus infections and human genital cancerrdquo GynecologicOncology vol 12 no 2 pp S124ndashS128 1981
[10] C Foresta A Bertoldo A Garolla et al ldquoHuman papillo-mavirus proteins are found in peripheral blood and semenCd20+ and Cd56+ cells during Hpv-16 semen infectionrdquo BMCInfectious Diseases vol 13 article 593 2013
[11] S Ljubojevic andM Skerlev ldquoHPV-associated diseasesrdquo Clinicsin Dermatology vol 32 no 2 pp 227ndash234 2014
[12] A P R Cassel R B Barcellos C M D da Silva S E de MatosAlmeida and M L R Rossetti ldquoAssociation between humanpapillomavirus (HPV) DNA and micronuclei in normal cervi-cal cytologyrdquo Genetics and Molecular Biology vol 37 no 2 pp360ndash363 2014
[13] W Jarrett M Campo B OrsquoNeil H Laird and L Coggins ldquoAnovel bovine papillomavirus (BPV-6) causing true epithelialpapillomas of the mammary gland skin a member of a pro-posed newBPV subgrouprdquoVirology vol 136 no 2 pp 255ndash2641984
[14] T CMelo N Diniz S R C Campos et al ldquoCytogenetic studiesin peripheral blood of bovines afflicted by papillomatosisrdquoVeterinary and Comparative Oncology vol 9 no 4 pp 269ndash2742011
[15] S R C Campos T C Melo S Assaf et al ldquoChromosome aber-rations in cells infected with bovine papillomavirus comparingcutaneous papilloma esophagus papilloma and urinary blad-der lesion cellsrdquo ISRN Oncology vol 2013 Article ID 910849 8pages 2013
[16] H zur Hausen ldquoThe search for infectious causes of humancancers where and whyrdquoVirology vol 392 no 1 pp 1ndash10 2009
[17] R P Araldi R F Carvalho T C Melo et al ldquoBovine papillo-mavirus in beef cattle first description of BPV-12 and putativetype BAPV8 in BrazilrdquoGenetics andMolecular Research vol 13no 3 pp 5644ndash5653 2014
12 BioMed Research International
[18] T Rampias C Sasaki and A Psyrri ldquoMolecular mechanisms ofHPV induced carcinogenesis in head and neckrdquoOral Oncologyvol 50 no 5 pp 356ndash363 2014
[19] S Roperto V Russo L Leonardi et al ldquoBovine papillomavirustype 13 expression in the urothelial bladder tumours of cattlerdquoTransboundary and Emerging Diseases 2015
[20] S RCCampos C TrindadeO P Ferraz et al ldquoCan establishedcultured papilloma cells harbor bovine papillomavirusrdquoGenet-ics and Molecular Research vol 7 no 4 pp 1119ndash1126 2008
[21] M S Campo ldquoAnimalmodels of papilloma virus pathogenesisrdquoVirus Research vol 89 no 2 pp 249ndash261 2002
[22] G Borzacchiello ldquoBovine papillomavirus infections in ani-malsrdquo in Communicating Current Research and EducationalTopics and Trends in AppliedMicrobiology pp 673ndash679 Forma-tex Badajoz Spain 2007
[23] F Bocaneti G Altamura A Corteggio E Velescu F RopertoandG Borzacchiello ldquoBovine papillomavirus new insights intoan old diseaserdquo Transboundary and Emerging Diseases 2014
[24] R P Araldi T C Melo N Diniz et al ldquoBovine papillomavirusclastogenic effect analyzed in comet assayrdquo BioMed ResearchInternational vol 2013 Article ID 630683 7 pages 2013
[25] J Munday N Thomson M Dunowska C Knight R Laurieand S Hills ldquoGenomic characterisation of the feline sarcoid-associated papillomavirus and proposed classification as Bostaurus papillomavirus type 14rdquoVeterinaryMicrobiology vol 177no 3-4 pp 289ndash295 2015
[26] G Borzacchiello V Russo C Spoleto et al ldquoBovine papillo-mavirus type-2 DNA and expression of E5 and E7 oncoproteinsin vascular tumours of the urinary bladder in cattlerdquo CancerLetters vol 250 no 1 pp 82ndash91 2007
[27] P Maiolino A Ozkul A Sepici-Dincel et al ldquoBovine papillo-mavirus type 2 infection and microscopic patterns of urothelialtumors of the urinary bladder in water buffaloesrdquo BioMedResearch International vol 2013 Article ID 937918 6 pages2013
[28] S Roperto G Borzacchiello R Brun et al ldquoMultiple glomustumors of the urinary bladder in a cow associated with bovinepapillomavirus type 2 (BPV-2) infectionrdquo Veterinary Pathologyvol 45 no 1 pp 39ndash42 2008
[29] S Duensing and K Munger ldquoThe human papillomavirus type16 E6 and E7 oncoproteins independently induce numerical andstructural chromosome instabilityrdquoCancer Research vol 62 no23 pp 7075ndash7082 2002
[30] S Duensing L Y Lee A Duensing et al ldquoThe human papil-lomavirus type 16 E6 and E7 oncoproteins cooperate to inducemitotic defects and genomic instability by uncoupling centro-some duplication from the cell division cyclerdquo Proceedings of theNational Academy of Sciences of theUnited States of America vol97 no 18 pp 10002ndash10007 2000
[31] C A Moody and L A Laimins ldquoHuman papillomavirus onco-proteins pathways to transformationrdquo Nature Reviews Cancervol 10 no 8 pp 550ndash560 2010
[32] C Mansur and E Androphy ldquoCellular transformation bypapillomavirus oncoproteinsrdquo Biochimica et Biophysica Actavol 1155 no 3 pp 323ndash345 1993
[33] J J Chen Y Hong and E J Androphy ldquoMutational analysis oftranscriptional activation by the bovine papillomavirus type 1E6rdquo Virology vol 236 no 1 pp 30ndash36 1997
[34] X Tong andPMHowley ldquoThebovine papillomavirus E6 onco-protein interacts with paxillin and disrupts the actin cytoskele-tonrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 94 no 9 pp 4412ndash4417 1997
[35] X TongW Boll T Kirchhausen andPMHowley ldquoInteractionof the bovine papillomavirus E6 protein with the clathrinadaptor complex AP-1rdquo Journal of Virology vol 72 no 1 pp476ndash482 1998
[36] Y Nomine M Masson S Charbonnier et al ldquoStructural andfunctional analysis of E6 oncoprotein insights in the molecularpathways of human papillomavirus-mediated pathogenesisrdquoMolecular Cell vol 21 no 5 pp 665ndash678 2006
[37] T Ristriani MMasson Y Nomine et al ldquoHPV oncoprotein E6is a structure-dependent DNA-binding protein that recognizesfour-way junctionsrdquo Journal of Molecular Biology vol 296 no5 pp 1189ndash1203 2000
[38] Y Liu and J D Baleja ldquoStructure and function of the papil-lomavirus E6 protein and its interacting proteinsrdquo Frontiers inBioscience vol 13 no 1 pp 121ndash134 2008
[39] F Lipari G A McGibbon E Wardrop and M G CordingleyldquoPurification and biophysical characterization of a minimalfunctional domain and of an N-terminal Zn2+-binding frag-ment from the human papillomavirus type 16 E6 proteinrdquoBiochemistry vol 40 no 5 pp 1196ndash1204 2001
[40] Y Nomine T Ristriani C Laurent J-F Lefevre E Weissand G Trave ldquoA strategy for optimizing the monodispersity offusion proteins application to purification of recombinantHPVE6 oncoproteinrdquo Protein Engineering vol 14 no 4 pp 297ndash3052001
[41] C G Ullman P I Haris D A Galloway V C Emery and SJ Perkins ldquoPredicted 120572-helix120573-sheet secondary structures forthe zinc-binding motifs of human papillomavirus E7 and E6proteins by consensus prediction averaging and spectroscopicstudies of E7rdquo Biochemical Journal vol 319 no 1 pp 229ndash2391996
[42] A Sidi K Babah N Brimer et al ldquoStrategies for bacterialexpression of protein-peptide complex application to solubi-lization of papillomavirus E6rdquo Protein Expression and Purifica-tion vol 29 no 6 pp 997ndash1003 2011
[43] K Zanier S Charbonnier A O M O Sidi et al ldquoStructuralbasis for hijacking of cellular LxxLL motifs by papillomavirusE6 oncoproteinsrdquo Science vol 339 no 6120 pp 694ndash698 2013
[44] J Mazzuchelli-de-Souza R F Carvalho R M Ruiz et alldquoExpression and in silico analysis of the recombinant bovinepapillomavirus e6 protein as a model for viral oncoproteinsstudiesrdquo BioMed Research International vol 2013 Article ID421398 9 pages 2013
[45] L Banks P Spence E Androphy et al ldquoIdentification of humanpapillomavirus type 18 E6 polypeptide in cells derived fromhuman cervical carcinomasrdquo Journal of General Virology vol68 no 5 pp 1351ndash1359 1987
[46] D Hanahan and R AWeinberg ldquoHallmarks of cancer the nextgenerationrdquo Cell vol 144 no 5 pp 646ndash674 2011
[47] G S Charames and B Bapat ldquoGenomic instability and cancerrdquoCurrent Molecular Medicine vol 3 no 7 pp 589ndash596 2003
[48] S Negrini V G Gorgoulis and T D Halazonetis ldquoGenomicinstabilitymdashan evolving hallmark of cancerrdquo Nature ReviewsMolecular Cell Biology vol 11 no 3 pp 220ndash228 2010
[49] K Zanier S Charbonnier M Baltzinger Y Nomine DAltschuh and G Trave ldquoKinetic analysis of the interactionsof human papillomavirus E6 oncoproteins with the ubiquitinligase E6AP using surface plasmon resonancerdquo Journal ofMolecular Biology vol 349 no 2 pp 401ndash412 2005
BioMed Research International 13
[50] M Scheffner J M Huibregtse R D Vierstra and PM HowleyldquoThe HPV-16 E6 and E6-AP complex functions as a ubiquitin-protein ligase in the ubiquitination of p53rdquo Cell vol 75 no 3pp 495ndash505 1993
[51] H Zimmermann C-H Koh R Degenkolbe et al ldquoInteractionwith CBPp300 enables the bovine papillomavirus type 1 E6oncoprotein to downregulate CBPp300-mediated transactiva-tion by p53rdquo Journal of General Virology vol 81 no 11 pp 2617ndash2623 2000
[52] M Fenech ldquoThe in vitro micronucleus techniquerdquo MutationResearchmdashFundamental and Molecular Mechanisms of Mutage-nesis vol 455 no 1-2 pp 81ndash95 2000
[53] R P Araldi T C de Melo T B Mendes et al ldquoUsing the cometand micronucleus assays for genotoxicity studies a reviewrdquoBiomedicine amp Pharmacotherapy vol 72 pp 74ndash82 2015
[54] P Thomas and M Fenech ldquoCytokinesis-block micronucleuscytome assay in lymphocytesrdquo Methods in Molecular Biologyvol 682 pp 217ndash234 2011
[55] M Kirsch-Volders T Sofuni M Aardema et al ldquoReport fromthe in vitro micronucleus assay working grouprdquo MutationResearch Genetic Toxicology and Environmental Mutagenesisvol 540 no 2 pp 153ndash163 2003
[56] S Samanta and P Dey ldquoMicronucleus and its applicationsrdquoDiagnostic Cytopathology vol 40 no 1 pp 84ndash90 2012
[57] M Hayashi J T MacGregor D G Gatehouse et al ldquoIn vivoerythrocyte micronucleus assay III Validation and regulatoryacceptance of automated scoring and the use of rat periph-eral blood reticulocytes with discussion of non-hematopoietictarget cells and a single dose-level limit testrdquo MutationResearchGenetic Toxicology and Environmental Mutagenesisvol 627 no 1 pp 10ndash30 2007
[58] A Azqueta and A R Collins ldquoThe essential comet assay acomprehensive guide to measuring DNA damage and repairrdquoArchives of Toxicology vol 87 no 6 pp 949ndash968 2013
[59] N P Singh M T McCoy R R Tice and E L Schneider ldquoAsimple technique for quantitation of low levels of DNA damagein individual cellsrdquo Experimental Cell Research vol 175 no 1pp 184ndash191 1988
[60] J H Calinisan S R Chan A King and P J Chan ldquoHumanpapillomavirus and blastocyst apoptosisrdquo Journal of AssistedReproduction and Genetics vol 19 no 3 pp 132ndash136 2002
[61] R Oliveira Studies on the biopathological actions of Pteridiumaquilinum [PhD thesis] nstituto de Ciencias Biomedicas AbelSalazar 2012
[62] D M Potter and M S Baird ldquoCarcinogenic effects of ptaquilo-side in bracken fern and related compoundsrdquo British Journal ofCancer vol 83 no 7 pp 914ndash920 2000
[63] R B Lucena D R Rissi G D Kommers et al ldquoA retrospectivestudy of 586 tumours in Brazilian cattlerdquo Journal of ComparativePathology vol 145 no 1 pp 20ndash24 2011
[64] A Yaguiu M L Z Dagli E H Birgel Jr et al ldquoSimultaneouspresence of bovine papillomavirus and bovine leukemia virusin different bovine tissues in situ hybridization and cytogeneticanalysisrdquoGenetics andMolecular Research vol 7 no 2 pp 487ndash497 2008
[65] T C Melo R F Carvalho J Mazzucchelli-de-Souza et alldquoPhylogenetic classification and clinical aspects of a new puta-tive Deltapapillomavirus associated with skin lesions in cattlerdquoGenetics and Molecular Research vol 13 no 2 pp 2458ndash24692014
[66] R P Araldi D N S Giovanni T CMelo et al ldquoBovine papillo-mavirus isolation by ultracentrifugationrdquo Journal of VirologicalMethods vol 208 pp 119ndash124 2014
[67] L M Chandrachud B W OrsquoNeil W F H Jarrett G J Grind-lay G M McGarvie and M S Campo ldquoHumoral immuneresponse to the E7 protein of bovine papillomavirus type 4 andidentification of B-cell epitopesrdquoVirology vol 200 no 1 pp 98ndash104 1994
[68] M Ayres M J Ayres D L Ayres and A A Santos BioEstatmdashAplicacoes Estatısticas nas Areas de Ciencias Bio-Medicas Insti-tuto Mamiraua Belem Brazil 2007
[69] K Das J Bohl and S B Vande Pol ldquoIdentification of a secondtransforming function in bovine papillomavirus type 1 E6 andthe role of E6 interactions with paxillin E6BP and E6APrdquoJournal of Virology vol 74 no 2 pp 812ndash816 2000
[70] O Danos E Georges G Orth and M Yaniv ldquoFine structureof the cottontail rabbit papillomavirus mRNAs expressed in thetransplantable VX2 carcinomardquo Journal of Virology vol 53 no3 pp 735ndash741 1985
[71] M Nasseri and F O Wettstein ldquoDifferences exist between viraltranscripts in cottontail rabbit papillomavirus-induced benignand malignant tumors as well as non-virus-producing andvirus-producing tumorsrdquo Journal of Virology vol 51 no 3 pp706ndash712 1984
[72] X Tong R Salgia J-L Li J D Griffin and P M Howley ldquoThebovine papillomavirus E6 protein binds to the LDmotif repeatsof paxillin and blocks its interaction with vinculin and the focaladhesion kinaserdquo Journal of Biological Chemistry vol 272 no52 pp 33373ndash33376 1997
[73] Y Maeda T Shibahara Y Wada et al ldquoAn outbreak of teatpapillomatosis in cattle caused by bovine papilloma virus (BPV)type 6 and unclassified BPVsrdquo Veterinary Microbiology vol 121no 3-4 pp 242ndash248 2007
[74] R P Araldi T C Melo A Neves et al ldquoHyperproliferativeaction of bovine papillomavirus genetic and histopathologicalaspectsrdquo Genetics and Molecular Research vol 14 no 4 pp12942ndash12954 2015
[75] NDiniz T CMelo J F Santos et al ldquoSimultaneous presence ofbovine papillomavirus in blood and in short-term lymphocytecultures from dairy cattle in Pernambuco Brazilrdquo Genetics andMolecular Research vol 8 no 4 pp 1474ndash1480 2009
[76] O M Brito M F Guimaraes and C C Lanna ldquoCiclofosfamidae funcao ovarianardquo Revista Brasileira de Reumatologia vol 48no 1 pp 39ndash45 2008
[77] D Anderson T-W Yu and D B McGregor ldquoComet assayresponses as indicators of carcinogen exposurerdquo Mutagenesisvol 13 no 6 pp 539ndash555 1998
[78] M L Adam C Pini S Tulio J C L L Cantalice R A Torresand M T Dos Santos Correia ldquoAssessment of the associationbetween micronuclei and the degree of uterine lesions and viralload in women with human papillomavirusrdquo Cancer Genomicsamp Proteomics vol 12 no 2 pp 67ndash72 2015
[79] E Cortes-Gutierrez M Davila-Rodrıguez J Vargas-VillarrealF Hernandez-Garza and R Cerda-Flores ldquoAssociationbetween human papilloma virus-type infections with micro-nuclei frequenciesrdquo Prague Medical Report vol 111 no 1 pp35ndash41 2010
[80] R N Chakrabarti and K Dutta ldquoMicronuclei test in routinesmears fromuterine cervixrdquo European Journal of GynaecologicalOncology vol 9 no 5 pp 370ndash372 1988
[81] E M M Cerqueira C L Santoro N F Donozo et al ldquoGeneticdamage in exfoliated cells of the uterine cervix Association
14 BioMed Research International
and interaction between cigarette smoking and progression tomalignant transformationrdquo Acta Cytologica vol 42 no 3 pp639ndash649 1998
[82] S M Germann V Schramke R T Pedersen et al ldquoTopBP1Dpb11 bindsDNA anaphase bridges to prevent genome instabil-ityrdquoThe Journal of Cell Biology vol 204 no 1 pp 45ndash59 2014
[83] D R Hoffelder L Luo N A Burke S CWatkins S M Gollinand W S Saunders ldquoResolution of anaphase bridges in cancercellsrdquo Chromosoma vol 112 no 8 pp 389ndash397 2004
[84] C Acilan D M Potter and W S Saunders ldquoDNA repairpathways involved in anaphase bridge formationrdquo Genes Chro-mosomes and Cancer vol 46 no 6 pp 522ndash531 2007
[85] J DeMasi M C Chao A S Kumar and P M Howley ldquoBovinepapillomavirus E7 oncoprotein inhibits anoikisrdquo Journal ofVirology vol 81 no 17 pp 9419ndash9425 2007
[86] J DeMasi K-W Huh Y Nakatani K Munger and P MHowley ldquoBovine papillomavirus E7 transformation functioncorrelates with cellular p600 protein bindingrdquo Proceedings of theNational Academy of Sciences of theUnited States of America vol102 no 32 pp 11486ndash11491 2005
[87] E A White and P M Howley ldquoProteomic approaches to thestudy of papillomavirusndashhost interactionsrdquo Virology vol 435no 1 pp 57ndash69 2013
[88] FMantovani and L Banks ldquoThe human papillomavirus E6 pro-tein and its contribution to malignant progressionrdquo Oncogenevol 20 no 54 pp 7874ndash7887 2001
[89] P Chen and H Lee ldquoFOXM1 induced by E6 oncoprotein pro-motes tumor invasion and chemoresistance in HPV-infectedlung cancerrdquo Cancer Cell amp Microenvironment vol 2 no 1 pp6ndash9 2015
[90] N Zhang P Wei A Gong et al ldquoFoxM1 promotes 120573-cateninnuclear localization and controls Wnt target-gene expressionand glioma tumorigenesisrdquo Cancer Cell vol 127 no 4 pp 358ndash366 2012
[91] Z Liu Y Liu Y Hong L Rapp E J Androphy and J JChen ldquoBovine papillomavirus type 1 E6-induced sensitizationto apoptosis is distinct from its transforming activityrdquo Virologyvol 295 no 2 pp 230ndash237 2002
[92] M Thomas P Massimi C Navarro J-P Borg and L BanksldquoThe hScribDlg apico-basal control complex is differentiallytargeted by HPV-16 and HPV-18 E6 proteinsrdquo Oncogene vol24 no 41 pp 6222ndash6230 2005
[93] C H Storrs and S J Silverstein ldquoPATJ a tight junction-associated PDZ protein is a novel degradation target of high-risk human papillomavirus E6 and the alternatively splicedisoform 18 E6rdquo Journal of Virology vol 81 no 8 pp 4080ndash40902007
[94] R Accardi R Rubino M Scalise et al ldquoE6 and E7 from humanpapillomavirus type 16 cooperate to target the PDZ proteinNaH exchange regulatory factor 1rdquo Journal of Virology vol 85no 16 pp 8208ndash8216 2011
[95] S Nakagawa and J M Huibregtse ldquoHuman scribble (Vartul)is targeted for ubiquitin-mediated degradation by the high-risk papillomavirus E6 proteins and the E6APubiquitin-proteinligaserdquoMolecular and Cellular Biology vol 20 no 21 pp 8244ndash8253 2000
[96] L Gewin H Myers T Kiyono and D A Galloway ldquoIdentifi-cation of a novel telomerase repressor that interacts with thehuman papillomavirus type-16 E6E6-AP complexrdquo Genes ampDevelopment vol 18 no 18 pp 2269ndash2282 2004
[97] M Sundaram D L GuernseyMM Rajaraman and R Rajara-man ldquoNeosis a novel type of cell division in cancerrdquo CancerBiology ampTherapy vol 3 no 2 pp 207ndash218 2004
[98] R D Snyder and J W Green ldquoA review of the genotoxicity ofmarketed pharmaceuticalsrdquo Mutation Research vol 488 no 2pp 151ndash169 2001
[99] T Ristriani Y Nomine C Laurent E Weiss and G TraveldquoProtein mutagenesis with monodispersity-based quality prob-ing selective inactivation of p53 degradation and DNA-bindingproperties of HPV E6 oncoproteinrdquo Protein Expression andPurification vol 26 no 3 pp 357ndash367 2002
[100] M Kadaja A Sumerina T Verst M Ojarand E Ustav and MUstav ldquoGenomic instability of the host cell induced by thehuman papillomavirus replicationmachineryrdquoTheEMBO Jour-nal vol 26 no 8 pp 2180ndash2191 2007
[101] PMDay D R Lowy and J T Schiller ldquoPapillomaviruses infectcells via a clathrin-dependent pathwayrdquo Virology vol 307 no 1pp 1ndash11 2003
[102] T D Tlsty A Briot A Gualberto et al ldquoGenomic instabilityand cancerrdquoMutation Research vol 337 no 1 pp 1ndash7 1995
[103] J E Eyfjord and S K Bodvarsdottir ldquoGenomic instabilityand cancer networks involved in response to DNA damagerdquoMutation Research vol 592 no 1-2 pp 18ndash28 2005
[104] Y Yao W Huang X Yang et al ldquoHPV-16 E6 and E7 protein Tcell epitopes prediction analysis based on distributions of HLA-A loci across populations an in silico approachrdquo Vaccine vol31 no 18 pp 2289ndash2294 2013
[105] C-F Hung B Ma A Monie S-W Tsen and T-C Wu ldquoTher-apeutic human papillomavirus vaccines current clinical trialsand future directionsrdquo Expert Opinion on Biological Therapyvol 8 no 4 pp 421ndash439 2008
[106] M J P Welters G G Kenter S J Piersma et al ldquoInductionof tumor-specific CD4+ and CD8+ T-cell immunity in cervicalcancer patients by a human papillomavirus type 16 E6 and E7long peptides vaccinerdquo Clinical Cancer Research vol 14 no 1pp 178ndash187 2008
[107] R Han N M Cladel C A Reed X Peng and N D Chris-tensen ldquoProtection of rabbits from viral challenge by gene gun-based intracutaneous vaccination with a combination of cot-tontail rabbit papillomavirus E1 E2 E6 and E7 genesrdquo Journalof Virology vol 73 no 8 pp 7039ndash7043 1999
[108] Z He A P Wlazlo D W Kowalczyk et al ldquoViral recombinantvaccines to the E6 and E7 antigens of HPV-16rdquo Virology vol270 no 1 pp 146ndash161 2000
[109] A Kaufmann P Stern E Rankin et al ldquoSafety and immuno-genicity of TA-HPV a recombinant vaccinia virus expressingmodified human papillomavirus (HPV)-16 and HPV-18 E6 andE7 genes in women with progressive cervical cancerrdquo ClinicalCancer Research vol 8 no 12 pp 3676ndash3685 2002
[110] L K Borysiewicz A Fiander M Nimako et al ldquoA recombinantvaccinia virus encoding human papillomavirus types 16 and 18E6 and E7 proteins as immunotherapy for cervical cancerrdquoTheLancet vol 347 no 9014 pp 1523ndash1527 1996
[111] A De Jong T OrsquoNeill A Y Khan et al ldquoEnhancement ofhuman papillomavirus (HPV) type 16 E6 and E7-specific T-cellimmunity in healthy volunteers through vaccination with TA-CIN an HPV16 L2E7E6 fusion protein vaccinerdquo Vaccine vol20 no 29-30 pp 3456ndash3464 2002
[112] S Chen C Liao Y Lai et al ldquoDe-oncogenicHPVE6E7 vaccinegets enhanced antigenicity and promotes tumoricidal synergywith cisplatinrdquo Acta Biochimica et Biophysica Sinica vol 46 no1 pp 6ndash14 2014
BioMed Research International 15
[113] J Bonhoeffer E B Imoukhuede G Aldrovandi et al ldquoTemplateprotocol for clinical trials investigating vaccines-focus on safetyelementsrdquo Vaccine vol 31 no 47 pp 5602ndash5620 2013
[114] S Jackson and A Storey ldquoE6 proteins from diverse cutaneonsHPV types inhibit apoptosis in response to UV damagerdquoOncogene vol 19 no 4 pp 592ndash598 2000
[115] M Thomas and L Banks ldquoPDZRN3LNX3 is a novel targetof human papillomavirus type 16 (HPV-16) and HPV-18 E6rdquoJournal of Virology vol 89 no 2 pp 1439ndash1444 2015
[116] P R Daniels C M Sanders P Coulson and N J MaitlandldquoMolecular analysis of the interaction between HPV type 16 E6and human E6-associated proteinrdquo FEBS Letters vol 416 no 1pp 6ndash10 1997
[117] W H Kao S L Beaudenon A L Talis J M Huibregtse andP M Howley ldquoHuman papillomavirus type 16 E6 induces self-ubiquitination of the E6AP ubiquitin-protein ligaserdquo Journal ofVirology vol 74 no 14 pp 6408ndash6417 2000
[118] T Iftner M Elbel B Schopp et al ldquoInterference of papillo-mavirus E6 protein with single-strand break repair by interac-tion with XRCC1rdquo The EMBO Journal vol 21 no 17 pp 4741ndash4748 2002
[119] Y Imai Y Tsunokawa T Sugimura and M Terada ldquoPurifi-cation and DNA-binding properties of human papillomavirustype 16 E6 protein expressed in Escherichia colirdquo Biochemicaland Biophysical Research Communications vol 164 no 3 pp1402ndash1410 1989
[120] M Lusky L Berg H Weiher and M Botchan ldquoBovinepapilloma virus contains an activator of gene expression at thedistal end of the early transcription unitrdquoMolecular andCellularBiology vol 3 no 6 pp 1108ndash1122 1983
[121] S Dalal Q Gao E J Androphy and V Band ldquoMutationalanalysis of human papillomavirus type 16 E6 demonstrates thatp53 degradation is necessary for immortalization of mammaryepithelial cellsrdquo Journal of Virology vol 70 no 2 pp 683ndash6881996
[122] J M Huibregtse M Scheffner and P M Howley ldquoA cellularprotein mediates association of p53 with the E6 oncoprotein ofhuman papillomavirus types 16 or 18rdquo The EMBO Journal vol10 no 13 pp 4129ndash4135 1991
[123] M Scheffner B A Werness J M Huibregtse A J Levine andP M Howley ldquoThe E6 oncoprotein encoded by human papillo-mavirus types 16 and 18 promotes the degradation of p53rdquo Cellvol 63 no 6 pp 1129ndash1136 1990
[124] M Elbel S Carl S Spaderna and T Iftner ldquoA comparativeanalysis of the interactions of the E6 proteins from cutaneousand genital papillomaviruses with p53 and E6AP in correlationto their transforming potentialrdquoVirology vol 239 no 1 pp 132ndash149 1997
[125] M C Thomas and C-M Chiang ldquoE6 oncoprotein repressesp53-dependent gene activation via inhibition of protein acety-lation independently of inducing p53 degradationrdquo MolecularCell vol 17 no 2 pp 251ndash264 2005
[126] H Zimmermann R Degenkolbe H-U Bernard and M JOrsquoConnor ldquoThe human papillomavirus type 16 E6 oncoproteincan down-regulate p53 activity by targeting the transcriptionalcoactivator CBPp300rdquo Journal of Virology vol 73 no 8 pp6209ndash6219 1999
[127] D A Thompson G Belinsky T H-T Chang D L Jones RSchlegel and K Munger ldquoThe human papillomavirus-16 E6oncoprotein decreases the vigilance of mitotic checkpointsrdquoOncogene vol 15 no 25 pp 3025ndash3035 1997
[128] J Bohl K Das B Dasgupta and S B Vande Pol ldquoCompetitivebinding to a charged leucine motif represses transformation bya papillomavirus E6 oncoproteinrdquo Virology vol 271 no 1 pp163ndash170 2000
[129] S Song H C Pitot and P F Lambert ldquoThe human papillo-mavirus type 16 E6 gene alone is sufficient to induce carcinomasin transgenic animalsrdquo Journal of Virology vol 73 no 7 pp5887ndash5893 1999
[130] S Yasumoto A L Burkhardt J Doniger and J A DiPaololdquoHumanpapillomavirus type 16DNA-inducedmalignant trans-formation of NIH 3T3 cellsrdquo Journal of Virology vol 57 no 2pp 572ndash577 1986
[131] S B Vande Pol M C Brown and C E Turner ldquoAssociation ofBovine Papillomavirus Type 1 E6 oncoprotein with the focaladhesion protein paxillin through a conserved protein interac-tion motifrdquo Oncogene vol 16 no 1 pp 43ndash52 1998
[132] R Wade N Brimer and S Vande Pol ldquoTransformation bybovine papillomavirus type 1 E6 requires paxillinrdquo Journal ofVirology vol 82 no 12 pp 5962ndash5966 2008
[133] A J Klingelhutz S A Foster and J KMcDougall ldquoTelomeraseactivation by the E6 gene product of humanpapillomavirus type16rdquo Nature vol 380 no 6569 pp 79ndash82 1996
[134] S T Oh S Kyo and L A Laimins ldquoTelomerase activation byhuman papillomavirus type 16 E6 protein Induction of humantelomerase reverse transcriptase expression through Myc andGC-rich Sp1 binding sitesrdquo Journal of Virology vol 75 no 12pp 5559ndash5566 2001
[135] L V Ronco A Y KarpovaMVidal and PMHowley ldquoHumanpapillomavirus 16 E6 oncoprotein binds to interferon regulatoryfactor-3 and inhibits its transcriptional activityrdquo Genes andDevelopment vol 12 no 13 pp 2061ndash2072 1998
Figure 6 Images of cometsrsquo nucleoids (a) Image of class 0 (without DNA damage) observed in negative control group Images of class 2(maximum DNA damage) observed in positive control (b) and experimental (c) groups (d) Image of hedgehog comet characterized by thehead absence observed in experimental group Images captured in total magnification of 400x
200
150
100
50
0
Com
et sc
ore
GroupsCminus C+ E6
Figure 7 Boxplot of comet score
[72] the mechanisms of initiation and promotion of cancerassociated with the BPV E6 oncoprotein are unknown
Molecular diagnosis of five peripheral blood samplesfrom five calves showed the absence of amplicons for thefive different primers used (Figure 2) However both specific(BPV-1 BPV-2 and BPV-4) and degenerate (Delta-Epsilonand Xi) primers sets amplified the controls genomes of BPV-1 BPV-2 and BPV-4 None of the negative controls revealedthe amplicon presence These results indicate that the fivesampleswere uninfected by BPVMoreover the use of specific
and degenerate primers increases the capacity of BPV iden-tification This occurs because specific primers are moresensitive than degenerates [66] This sensitivity is due tothe absence of degeneration in the 51015840 region which reducesthe primer ability to recognize and to link with DNA targetsequence [73]The absence of BPV infection in the peripheralblood samples is required for the CBMNA and CA allowinginvestigating the aneugenic andor clastogenic action of BPV-1 E6 recombinant oncoprotein This is because the presenceof BPV sequences is associated with the presence of oncopro-teins transcripts [74]Thus studies have shown that the pres-ence of BPVDNA sequences in peripheral blood is associatedwith cytogenetic damage including clastogenesis [1 14 75]Complementary studies show the association of the BPVpresence with cytogenetic damage in both benign lesions(papillomas) [74] and carcinomas [15]
Chi-square test based on the number of micronucleatedcells pointed out significant statistical values in both positivecontrol and group treated with BPV-1 E6 recombinant onco-protein (Table 3) Negative control showed fewer number ofmicronucleated cells This result was expected because thebiological material was transported and processed after threehours of its collection This procedure reduced the influenceof exogenous environmental factors which could induceDNA damage Furthermore the absence of BPV infection
BioMed Research International 9
(a) (b)
8
6
4
2
0
Num
ber o
f mic
ronu
cleat
ed ce
lls
Neg
ativ
e con
trol
Posit
ive c
ontro
l
E6on
copr
otei
n
(c)
Figure 8 Photomicroscope showing CRIB cell not treated with any drug (negative control) indicating absence of micronucleus (a) andtreated with BPV-1 E6 recombinant oncoprotein showing the presence of MN indicated by arrow (b) (c) Number of micronucleated cellsobserved in each group
in the peripheral blood reduces the influence of endogenousenvironmental factors that could interfere in the analysisPositive control showed a high number of micronucle-ated lymphocytes This result was also expected once thisgroup was treated with 50 120583gmL of cyclophosphamide achemotherapeutic drug with cytotoxic and teratogenic effect[76 77] The cells treated with 1120583gmL of BPV-1 E6 recom-binant oncoprotein also showed significant statistical valuesKruskal-Wallis test followed by the Dunn post hoc testwas performed to compare the frequencies of micronucleusamong three groups This test indicated statistical differencebetween negative control and the cells treated with BPV-1 E6 recombinant oncoprotein However the test did notreveal differences between the cells treated with cyclophos-phamide and BPV-1 E6 recombinant oncoprotein (Table 4Figure 4) Similar results were also observed in epithelial cells(Figure 8) These results suggest that the BPV-1 E6 oncopro-tein has an aneugenic andor clastogenic potential similarto or higher than the observed with the cyclophosphamideThis is because the cells treated with the oncoprotein showed
a frequency of micronucleus formation (MN1199030) 14003
higher than those observed in positive controlThe elevated mutagenic potential of the BPV-1 E6 onco-
protein can justify the cytogenetic damage already describedin the literature [1 14 15 24] Similar results were observedin HPV-infected cells [12 78ndash81] The micronucleus induce-ment was already described in cytological samples collectedduring Papanicolaursquos test from healthy cervix infected byHPV [12] For this reason the micronucleus assay has beenproposed as a complementary method for Papanicolaursquos testbeing a suggestive biomarker of lesion degree [78] Howeverthe association between the BPV and the micronucleusinducement was not yet reported
The high frequency of micronucleated lymphocytes insamples infected by HPV has been attributed to the synergicaction of E5 E6 and E7 oncoproteins [29 30 78 79]However this is the first study that pointed out themutagenicaction of BPV-1 E6 protein per se
Student-Newman-Keuls post hoc test based on the fre-quency of anaphase bridges showed significant statistical
10 BioMed Research International
Table 7 Comet assay results showing the number of nucleoidsobserved per class the number of hedgehog comets and cometscore
Sample Class 0 Class 1 Class 2 Hedgehog ScoreNegative control
Groups Rank differences Calculated 119885 Critical 119885 119901
Cminus and C+ 68 24042 2394 lt05Cminus and E6 82 28991 2394 lt05C+ and E6 14 04950 2394 nsns nonsignificant differences
difference between the negative control and the group treatedwith the BPV-1 E6 recombinant oncoprotein (Table 5) How-ever the test did not reveal differences between the cellstreated with cyclophosphamide and BPV-1 E6 recombinantoncoprotein (Table 5)These data reinforce those observed bythe Dunn post hoc test based on the frequency of micronu-cleus indicating the clastogenic action of the E6 oncoproteinMoreover the presence of anaphase bridge was alreadydescribed in cells transfected with recombinant adenoviruscontaining the E6 and E7 ORFs of HPV [29 30] Howeverthere are no studies describing the action of BPVE6 oncopro-tein in the induction of anaphase bridges or studies that showthis same action of E6 oncoprotein of HPV per se Anaphasebridges are indicators of genomic instability [82 83] which isconsidered the first step in carcinogenesis [46 47] Moreoverthe presence of these bridges is an important hallmark ofDNA double strand breaks (DSBs) [84]
Statistical analysis based on the cytokinesis-block prolif-eration index (CBPI) showed that BPV-1 E6 oncoprotein hascytotoxic levels similar to cyclophosphamide (Table 6) Thisresult suggests that the BPV E6 oncoprotein can deregulatethe cell cycle contributing to the cell proliferation andimmortalization To date this mitogenic action is attributedto the E7 oncoprotein [85ndash87] E7 oncoprotein induces
the phosphorylation of pRb resulting in the E2F transcrip-tion factor release [85] E2F when translocated to the nucleusacts as activator and binds to the kinase-dependent cyclinspromoters [85] However the results showed polynucleatedcells in the group treatedwithBPVE6oncoprotein (Figure 5)suggesting that the BPV E6 oncoprotein confers mitogenicstimulation resulting in mitotic stress Moreover studiespointed out that the BPV E6 oncoprotein interacts with theCBPp300 deacetylase promoting p53 downregulation [88]This downregulation increases the expression levels of FoxM1transcription factor (Foxhead box M1) [89] promoting B1cyclin D1 cyclin and cdc25 upregulation [89] The upregu-lation of these genes is associated with increased levels of cellproliferation which is necessary in order to make DNA poly-merases available to virus replication However if this mech-anism guarantees the BPV replication it can contribute to cellimmortalization and cancer progression
The FoxM1 factor also participates in the Wnt120573-cateninsignaling pathway and binds directly to the 120573-catenin [89]This interaction promotes the nuclear 120573-catenin transloca-tion [90] 120573-catenin translocation to nucleus induces thecyclin expression [64] So these data suggest that the BPVE6 oncoprotein not only promotes mitogenic stress but alsocontributes to the epithelial-mesenchymal transition (EMT)Studies based on theHPVE6 oncoprotein have indicated thatthis oncoprotein contributes significantly to the EMT [91]This occurs not only because the E6 oncoprotein induces thetranslocation of 120573-catenin but also due to the proteasomaldegradation of regulatory proteins of apical-basal polarity[92ndash94] Studies also showed that the E6 oncoprotein is ableto bind to Crumbs (Dlg and Patj) proteins resulting in loss ofcell polarity [93 94] Based on these results the E6 oncopro-tein emerges as a possible therapeutic target with biotechno-logical value in cancer treatment
Mitogenic action of E6 oncoprotein can be moreexpressed in cells infected by papillomaviruses once thesecells also expressed E5 and E7 oncoproteins [74] The E7oncoprotein is able to form a complex E7-p600 which pro-motes E6 upregulation [85] Furthermore the E6 oncoproteincan bind to the E6AP ubiquitin ligase forming the E6-E6APcomplex increasing the hTERT levels of expression con-tributing to cell immortalization [95] Moreover E6-E6APinduces NFX1 expression promoting MHC-II downregula-tion [96]Thismechanism reduces the antigenic presentationmediated by CD4+ T lymphocytes contributing to immuneevasion This action guarantees the PV infection for longperiods with or without clinical symptoms [24] Althoughthe BPV infection can be asymptomatic the BPV presence inperipheral blood is associated with DNA damage [1 14 24]
CBMNA analysis pointed out the presence of cells withintracellular cytokinesis (Figure 5) This result suggests theneotic action of BPV E6 oncoprotein Neosis is characterizedby the presence of (1) DNA damage (2) loss of checkpointcontrol (3) repair system failures and (4) endoreduplication[97] In this scenario the E6 oncoprotein attempts all theseneosis criteria Moreover the oncoprotein not only increasesthe cytogenetic damage leading to increasing of micronu-cleus frequency but also induces clastogenesis as shown bythe comet assay and anaphase bridges So statistical analysis
BioMed Research International 11
performed based on the comet scores reinforced the CBMNAresults Comet assay allows detecting both simple and dou-ble DNA breaks being more sensitive than the CBMNAClastogenesis is considered the most serious type of DNAdamage [98] DNA breaks can be repaired by homologous ornonhomologous recombination However studies point outthat the E6 oncoprotein can link to the Holliday junctionduring the homologous repair avoiding the junction reso-lution [99] Moreover E6 oncoprotein promotes the TP53gene deacetylation resulting in p53 downregulation [51]Thisepigenetic effect results in cell cycle deregulation affectingthe checkpoints
Studies have shown that the HPV-induced DNA breaksare required for the virus integration into the host genome[100] Although there are no studies showing the BPV inte-gration to date the results observed in this work in additionto those already published [24] indicate the necessity ofmorestudies to evaluate the virus-host interaction
Studies also showed that the E6 oncoprotein inducesclathrin-transporter adapter protein (AP-1) increasing thelevel of this protein in the plasma membrane [76 77] Theincreasing quantity of AP-1 in membrane can facilitate theBPV virions infection This occurs because the infectionprocess is clathrin dependent In this scenario the E6 onco-protein can contribute to the virus infection [101]
In summary the CBMNA and CA results showed thatthe BPV E6 oncoprotein participates not only in cancerpromotion but also in initiation inducing DNA breaksThese DNA breaks represent mutagenic effects consideredthe first step in the oncogenesis process which are associatedwith genomic instability [47 48 102 103] Thus the E6recombinant oncoprotein has been suggested as a possiblevaccine candidate [104ndash112] due to its immunogenicity [44]However the mutagenic tests such as CBMNA and CA arerequired in drug validation process [113] In this scenariothis study pointed out that the BPV-1 E6 recombinant onco-protein in tested concentration showedmutagenic potentialThis is the first study that reports the mutagenic potential ofa possible therapeutic vaccine candidate On the one handthese results allowed better understanding the mechanism ofcancer promotion associatedwith the BPVE6oncoprotein aswell as revealing that this oncoprotein can induce carcinogen-esis per se on the other hand this data pointed out thatmaybeBPV E6 recombinant oncoprotein requires protein modifica-tions to be used as vaccine
Conflict of Interests
The authors declare that there is no conflict of interests
Acknowledgments
Theauthors thank Fundacao deAmparo a Pesquisa do Estadode Sao Paulo (FAPESP Process 201420617-5) and FundacaoButantan for the financial support
References
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experimental modelrdquo Journal of General Virology vol 79 no 9pp 2127ndash2135 1998
[2] A Antonsson and B G Hansson ldquoHealthy skin ofmany animalspecies harbors papillomaviruses which are closely related totheir human counterpartsrdquo Journal of Virology vol 76 no 24pp 12537ndash12542 2002
[3] P Rous and J W Beard ldquoA virus-induced mammalian growthwith the characters of a tumor (the Shope rabbit papilloma) IThe growth on implantationwithin favorable hostsrdquoThe Journalof Experimental Medicine vol 60 no 6 pp 701ndash722 1934
[4] R Shope and E Hurst ldquoInfectious papillomatosis of rabbitsrdquoThe Journal of Experimental Medicine vol 58 no 1 pp 607ndash624 1933
[5] H Delius M A Van Ranst A B Jenson H Zur Hausen and JP Sundberg ldquoCanine oral papillomavirus genomic sequence aunique 15-kb intervening sequence between the E2 andL2 openreading framesrdquo Virology vol 204 no 1 pp 447ndash452 1994
[6] W A Demonbreun and E Goodpasture ldquoInfectious oral papil-lomatosis of dogsrdquo The American Journal of Pathology vol 8no 1 pp 43ndash56 1932
[7] P K Nicholls B A Klaunberg R A Moore et al ldquoNaturallyoccurring nonregressing canine oral papillomavirus infectionhost immunity virus characterization and experimental infec-tionrdquo Virology vol 265 no 2 pp 365ndash374 1999
[8] A Alberti S Pirino F Pintore et al ldquoOvis aries Papillomavirus3 a prototype of a novel genus in the family Papillomaviridaeassociated with ovine squamous cell carcinomardquo Virology vol407 no 2 pp 352ndash359 2010
[9] H zur Hausen E-M de Villiers and L Gissmann ldquoPapil-lomavirus infections and human genital cancerrdquo GynecologicOncology vol 12 no 2 pp S124ndashS128 1981
[10] C Foresta A Bertoldo A Garolla et al ldquoHuman papillo-mavirus proteins are found in peripheral blood and semenCd20+ and Cd56+ cells during Hpv-16 semen infectionrdquo BMCInfectious Diseases vol 13 article 593 2013
[11] S Ljubojevic andM Skerlev ldquoHPV-associated diseasesrdquo Clinicsin Dermatology vol 32 no 2 pp 227ndash234 2014
[12] A P R Cassel R B Barcellos C M D da Silva S E de MatosAlmeida and M L R Rossetti ldquoAssociation between humanpapillomavirus (HPV) DNA and micronuclei in normal cervi-cal cytologyrdquo Genetics and Molecular Biology vol 37 no 2 pp360ndash363 2014
[13] W Jarrett M Campo B OrsquoNeil H Laird and L Coggins ldquoAnovel bovine papillomavirus (BPV-6) causing true epithelialpapillomas of the mammary gland skin a member of a pro-posed newBPV subgrouprdquoVirology vol 136 no 2 pp 255ndash2641984
[14] T CMelo N Diniz S R C Campos et al ldquoCytogenetic studiesin peripheral blood of bovines afflicted by papillomatosisrdquoVeterinary and Comparative Oncology vol 9 no 4 pp 269ndash2742011
[15] S R C Campos T C Melo S Assaf et al ldquoChromosome aber-rations in cells infected with bovine papillomavirus comparingcutaneous papilloma esophagus papilloma and urinary blad-der lesion cellsrdquo ISRN Oncology vol 2013 Article ID 910849 8pages 2013
[16] H zur Hausen ldquoThe search for infectious causes of humancancers where and whyrdquoVirology vol 392 no 1 pp 1ndash10 2009
[17] R P Araldi R F Carvalho T C Melo et al ldquoBovine papillo-mavirus in beef cattle first description of BPV-12 and putativetype BAPV8 in BrazilrdquoGenetics andMolecular Research vol 13no 3 pp 5644ndash5653 2014
12 BioMed Research International
[18] T Rampias C Sasaki and A Psyrri ldquoMolecular mechanisms ofHPV induced carcinogenesis in head and neckrdquoOral Oncologyvol 50 no 5 pp 356ndash363 2014
[19] S Roperto V Russo L Leonardi et al ldquoBovine papillomavirustype 13 expression in the urothelial bladder tumours of cattlerdquoTransboundary and Emerging Diseases 2015
[20] S RCCampos C TrindadeO P Ferraz et al ldquoCan establishedcultured papilloma cells harbor bovine papillomavirusrdquoGenet-ics and Molecular Research vol 7 no 4 pp 1119ndash1126 2008
[21] M S Campo ldquoAnimalmodels of papilloma virus pathogenesisrdquoVirus Research vol 89 no 2 pp 249ndash261 2002
[22] G Borzacchiello ldquoBovine papillomavirus infections in ani-malsrdquo in Communicating Current Research and EducationalTopics and Trends in AppliedMicrobiology pp 673ndash679 Forma-tex Badajoz Spain 2007
[23] F Bocaneti G Altamura A Corteggio E Velescu F RopertoandG Borzacchiello ldquoBovine papillomavirus new insights intoan old diseaserdquo Transboundary and Emerging Diseases 2014
[24] R P Araldi T C Melo N Diniz et al ldquoBovine papillomavirusclastogenic effect analyzed in comet assayrdquo BioMed ResearchInternational vol 2013 Article ID 630683 7 pages 2013
[25] J Munday N Thomson M Dunowska C Knight R Laurieand S Hills ldquoGenomic characterisation of the feline sarcoid-associated papillomavirus and proposed classification as Bostaurus papillomavirus type 14rdquoVeterinaryMicrobiology vol 177no 3-4 pp 289ndash295 2015
[26] G Borzacchiello V Russo C Spoleto et al ldquoBovine papillo-mavirus type-2 DNA and expression of E5 and E7 oncoproteinsin vascular tumours of the urinary bladder in cattlerdquo CancerLetters vol 250 no 1 pp 82ndash91 2007
[27] P Maiolino A Ozkul A Sepici-Dincel et al ldquoBovine papillo-mavirus type 2 infection and microscopic patterns of urothelialtumors of the urinary bladder in water buffaloesrdquo BioMedResearch International vol 2013 Article ID 937918 6 pages2013
[28] S Roperto G Borzacchiello R Brun et al ldquoMultiple glomustumors of the urinary bladder in a cow associated with bovinepapillomavirus type 2 (BPV-2) infectionrdquo Veterinary Pathologyvol 45 no 1 pp 39ndash42 2008
[29] S Duensing and K Munger ldquoThe human papillomavirus type16 E6 and E7 oncoproteins independently induce numerical andstructural chromosome instabilityrdquoCancer Research vol 62 no23 pp 7075ndash7082 2002
[30] S Duensing L Y Lee A Duensing et al ldquoThe human papil-lomavirus type 16 E6 and E7 oncoproteins cooperate to inducemitotic defects and genomic instability by uncoupling centro-some duplication from the cell division cyclerdquo Proceedings of theNational Academy of Sciences of theUnited States of America vol97 no 18 pp 10002ndash10007 2000
[31] C A Moody and L A Laimins ldquoHuman papillomavirus onco-proteins pathways to transformationrdquo Nature Reviews Cancervol 10 no 8 pp 550ndash560 2010
[32] C Mansur and E Androphy ldquoCellular transformation bypapillomavirus oncoproteinsrdquo Biochimica et Biophysica Actavol 1155 no 3 pp 323ndash345 1993
[33] J J Chen Y Hong and E J Androphy ldquoMutational analysis oftranscriptional activation by the bovine papillomavirus type 1E6rdquo Virology vol 236 no 1 pp 30ndash36 1997
[34] X Tong andPMHowley ldquoThebovine papillomavirus E6 onco-protein interacts with paxillin and disrupts the actin cytoskele-tonrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 94 no 9 pp 4412ndash4417 1997
[35] X TongW Boll T Kirchhausen andPMHowley ldquoInteractionof the bovine papillomavirus E6 protein with the clathrinadaptor complex AP-1rdquo Journal of Virology vol 72 no 1 pp476ndash482 1998
[36] Y Nomine M Masson S Charbonnier et al ldquoStructural andfunctional analysis of E6 oncoprotein insights in the molecularpathways of human papillomavirus-mediated pathogenesisrdquoMolecular Cell vol 21 no 5 pp 665ndash678 2006
[37] T Ristriani MMasson Y Nomine et al ldquoHPV oncoprotein E6is a structure-dependent DNA-binding protein that recognizesfour-way junctionsrdquo Journal of Molecular Biology vol 296 no5 pp 1189ndash1203 2000
[38] Y Liu and J D Baleja ldquoStructure and function of the papil-lomavirus E6 protein and its interacting proteinsrdquo Frontiers inBioscience vol 13 no 1 pp 121ndash134 2008
[39] F Lipari G A McGibbon E Wardrop and M G CordingleyldquoPurification and biophysical characterization of a minimalfunctional domain and of an N-terminal Zn2+-binding frag-ment from the human papillomavirus type 16 E6 proteinrdquoBiochemistry vol 40 no 5 pp 1196ndash1204 2001
[40] Y Nomine T Ristriani C Laurent J-F Lefevre E Weissand G Trave ldquoA strategy for optimizing the monodispersity offusion proteins application to purification of recombinantHPVE6 oncoproteinrdquo Protein Engineering vol 14 no 4 pp 297ndash3052001
[41] C G Ullman P I Haris D A Galloway V C Emery and SJ Perkins ldquoPredicted 120572-helix120573-sheet secondary structures forthe zinc-binding motifs of human papillomavirus E7 and E6proteins by consensus prediction averaging and spectroscopicstudies of E7rdquo Biochemical Journal vol 319 no 1 pp 229ndash2391996
[42] A Sidi K Babah N Brimer et al ldquoStrategies for bacterialexpression of protein-peptide complex application to solubi-lization of papillomavirus E6rdquo Protein Expression and Purifica-tion vol 29 no 6 pp 997ndash1003 2011
[43] K Zanier S Charbonnier A O M O Sidi et al ldquoStructuralbasis for hijacking of cellular LxxLL motifs by papillomavirusE6 oncoproteinsrdquo Science vol 339 no 6120 pp 694ndash698 2013
[44] J Mazzuchelli-de-Souza R F Carvalho R M Ruiz et alldquoExpression and in silico analysis of the recombinant bovinepapillomavirus e6 protein as a model for viral oncoproteinsstudiesrdquo BioMed Research International vol 2013 Article ID421398 9 pages 2013
[45] L Banks P Spence E Androphy et al ldquoIdentification of humanpapillomavirus type 18 E6 polypeptide in cells derived fromhuman cervical carcinomasrdquo Journal of General Virology vol68 no 5 pp 1351ndash1359 1987
[46] D Hanahan and R AWeinberg ldquoHallmarks of cancer the nextgenerationrdquo Cell vol 144 no 5 pp 646ndash674 2011
[47] G S Charames and B Bapat ldquoGenomic instability and cancerrdquoCurrent Molecular Medicine vol 3 no 7 pp 589ndash596 2003
[48] S Negrini V G Gorgoulis and T D Halazonetis ldquoGenomicinstabilitymdashan evolving hallmark of cancerrdquo Nature ReviewsMolecular Cell Biology vol 11 no 3 pp 220ndash228 2010
[49] K Zanier S Charbonnier M Baltzinger Y Nomine DAltschuh and G Trave ldquoKinetic analysis of the interactionsof human papillomavirus E6 oncoproteins with the ubiquitinligase E6AP using surface plasmon resonancerdquo Journal ofMolecular Biology vol 349 no 2 pp 401ndash412 2005
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[51] H Zimmermann C-H Koh R Degenkolbe et al ldquoInteractionwith CBPp300 enables the bovine papillomavirus type 1 E6oncoprotein to downregulate CBPp300-mediated transactiva-tion by p53rdquo Journal of General Virology vol 81 no 11 pp 2617ndash2623 2000
[52] M Fenech ldquoThe in vitro micronucleus techniquerdquo MutationResearchmdashFundamental and Molecular Mechanisms of Mutage-nesis vol 455 no 1-2 pp 81ndash95 2000
[53] R P Araldi T C de Melo T B Mendes et al ldquoUsing the cometand micronucleus assays for genotoxicity studies a reviewrdquoBiomedicine amp Pharmacotherapy vol 72 pp 74ndash82 2015
[54] P Thomas and M Fenech ldquoCytokinesis-block micronucleuscytome assay in lymphocytesrdquo Methods in Molecular Biologyvol 682 pp 217ndash234 2011
[55] M Kirsch-Volders T Sofuni M Aardema et al ldquoReport fromthe in vitro micronucleus assay working grouprdquo MutationResearch Genetic Toxicology and Environmental Mutagenesisvol 540 no 2 pp 153ndash163 2003
[56] S Samanta and P Dey ldquoMicronucleus and its applicationsrdquoDiagnostic Cytopathology vol 40 no 1 pp 84ndash90 2012
[57] M Hayashi J T MacGregor D G Gatehouse et al ldquoIn vivoerythrocyte micronucleus assay III Validation and regulatoryacceptance of automated scoring and the use of rat periph-eral blood reticulocytes with discussion of non-hematopoietictarget cells and a single dose-level limit testrdquo MutationResearchGenetic Toxicology and Environmental Mutagenesisvol 627 no 1 pp 10ndash30 2007
[58] A Azqueta and A R Collins ldquoThe essential comet assay acomprehensive guide to measuring DNA damage and repairrdquoArchives of Toxicology vol 87 no 6 pp 949ndash968 2013
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[64] A Yaguiu M L Z Dagli E H Birgel Jr et al ldquoSimultaneouspresence of bovine papillomavirus and bovine leukemia virusin different bovine tissues in situ hybridization and cytogeneticanalysisrdquoGenetics andMolecular Research vol 7 no 2 pp 487ndash497 2008
[65] T C Melo R F Carvalho J Mazzucchelli-de-Souza et alldquoPhylogenetic classification and clinical aspects of a new puta-tive Deltapapillomavirus associated with skin lesions in cattlerdquoGenetics and Molecular Research vol 13 no 2 pp 2458ndash24692014
[66] R P Araldi D N S Giovanni T CMelo et al ldquoBovine papillo-mavirus isolation by ultracentrifugationrdquo Journal of VirologicalMethods vol 208 pp 119ndash124 2014
[67] L M Chandrachud B W OrsquoNeil W F H Jarrett G J Grind-lay G M McGarvie and M S Campo ldquoHumoral immuneresponse to the E7 protein of bovine papillomavirus type 4 andidentification of B-cell epitopesrdquoVirology vol 200 no 1 pp 98ndash104 1994
[68] M Ayres M J Ayres D L Ayres and A A Santos BioEstatmdashAplicacoes Estatısticas nas Areas de Ciencias Bio-Medicas Insti-tuto Mamiraua Belem Brazil 2007
[69] K Das J Bohl and S B Vande Pol ldquoIdentification of a secondtransforming function in bovine papillomavirus type 1 E6 andthe role of E6 interactions with paxillin E6BP and E6APrdquoJournal of Virology vol 74 no 2 pp 812ndash816 2000
[70] O Danos E Georges G Orth and M Yaniv ldquoFine structureof the cottontail rabbit papillomavirus mRNAs expressed in thetransplantable VX2 carcinomardquo Journal of Virology vol 53 no3 pp 735ndash741 1985
[71] M Nasseri and F O Wettstein ldquoDifferences exist between viraltranscripts in cottontail rabbit papillomavirus-induced benignand malignant tumors as well as non-virus-producing andvirus-producing tumorsrdquo Journal of Virology vol 51 no 3 pp706ndash712 1984
[72] X Tong R Salgia J-L Li J D Griffin and P M Howley ldquoThebovine papillomavirus E6 protein binds to the LDmotif repeatsof paxillin and blocks its interaction with vinculin and the focaladhesion kinaserdquo Journal of Biological Chemistry vol 272 no52 pp 33373ndash33376 1997
[73] Y Maeda T Shibahara Y Wada et al ldquoAn outbreak of teatpapillomatosis in cattle caused by bovine papilloma virus (BPV)type 6 and unclassified BPVsrdquo Veterinary Microbiology vol 121no 3-4 pp 242ndash248 2007
[74] R P Araldi T C Melo A Neves et al ldquoHyperproliferativeaction of bovine papillomavirus genetic and histopathologicalaspectsrdquo Genetics and Molecular Research vol 14 no 4 pp12942ndash12954 2015
[75] NDiniz T CMelo J F Santos et al ldquoSimultaneous presence ofbovine papillomavirus in blood and in short-term lymphocytecultures from dairy cattle in Pernambuco Brazilrdquo Genetics andMolecular Research vol 8 no 4 pp 1474ndash1480 2009
[76] O M Brito M F Guimaraes and C C Lanna ldquoCiclofosfamidae funcao ovarianardquo Revista Brasileira de Reumatologia vol 48no 1 pp 39ndash45 2008
[77] D Anderson T-W Yu and D B McGregor ldquoComet assayresponses as indicators of carcinogen exposurerdquo Mutagenesisvol 13 no 6 pp 539ndash555 1998
[78] M L Adam C Pini S Tulio J C L L Cantalice R A Torresand M T Dos Santos Correia ldquoAssessment of the associationbetween micronuclei and the degree of uterine lesions and viralload in women with human papillomavirusrdquo Cancer Genomicsamp Proteomics vol 12 no 2 pp 67ndash72 2015
[79] E Cortes-Gutierrez M Davila-Rodrıguez J Vargas-VillarrealF Hernandez-Garza and R Cerda-Flores ldquoAssociationbetween human papilloma virus-type infections with micro-nuclei frequenciesrdquo Prague Medical Report vol 111 no 1 pp35ndash41 2010
[80] R N Chakrabarti and K Dutta ldquoMicronuclei test in routinesmears fromuterine cervixrdquo European Journal of GynaecologicalOncology vol 9 no 5 pp 370ndash372 1988
[81] E M M Cerqueira C L Santoro N F Donozo et al ldquoGeneticdamage in exfoliated cells of the uterine cervix Association
14 BioMed Research International
and interaction between cigarette smoking and progression tomalignant transformationrdquo Acta Cytologica vol 42 no 3 pp639ndash649 1998
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[83] D R Hoffelder L Luo N A Burke S CWatkins S M Gollinand W S Saunders ldquoResolution of anaphase bridges in cancercellsrdquo Chromosoma vol 112 no 8 pp 389ndash397 2004
[84] C Acilan D M Potter and W S Saunders ldquoDNA repairpathways involved in anaphase bridge formationrdquo Genes Chro-mosomes and Cancer vol 46 no 6 pp 522ndash531 2007
[85] J DeMasi M C Chao A S Kumar and P M Howley ldquoBovinepapillomavirus E7 oncoprotein inhibits anoikisrdquo Journal ofVirology vol 81 no 17 pp 9419ndash9425 2007
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[87] E A White and P M Howley ldquoProteomic approaches to thestudy of papillomavirusndashhost interactionsrdquo Virology vol 435no 1 pp 57ndash69 2013
[88] FMantovani and L Banks ldquoThe human papillomavirus E6 pro-tein and its contribution to malignant progressionrdquo Oncogenevol 20 no 54 pp 7874ndash7887 2001
[89] P Chen and H Lee ldquoFOXM1 induced by E6 oncoprotein pro-motes tumor invasion and chemoresistance in HPV-infectedlung cancerrdquo Cancer Cell amp Microenvironment vol 2 no 1 pp6ndash9 2015
[90] N Zhang P Wei A Gong et al ldquoFoxM1 promotes 120573-cateninnuclear localization and controls Wnt target-gene expressionand glioma tumorigenesisrdquo Cancer Cell vol 127 no 4 pp 358ndash366 2012
[91] Z Liu Y Liu Y Hong L Rapp E J Androphy and J JChen ldquoBovine papillomavirus type 1 E6-induced sensitizationto apoptosis is distinct from its transforming activityrdquo Virologyvol 295 no 2 pp 230ndash237 2002
[92] M Thomas P Massimi C Navarro J-P Borg and L BanksldquoThe hScribDlg apico-basal control complex is differentiallytargeted by HPV-16 and HPV-18 E6 proteinsrdquo Oncogene vol24 no 41 pp 6222ndash6230 2005
[93] C H Storrs and S J Silverstein ldquoPATJ a tight junction-associated PDZ protein is a novel degradation target of high-risk human papillomavirus E6 and the alternatively splicedisoform 18 E6rdquo Journal of Virology vol 81 no 8 pp 4080ndash40902007
[94] R Accardi R Rubino M Scalise et al ldquoE6 and E7 from humanpapillomavirus type 16 cooperate to target the PDZ proteinNaH exchange regulatory factor 1rdquo Journal of Virology vol 85no 16 pp 8208ndash8216 2011
[95] S Nakagawa and J M Huibregtse ldquoHuman scribble (Vartul)is targeted for ubiquitin-mediated degradation by the high-risk papillomavirus E6 proteins and the E6APubiquitin-proteinligaserdquoMolecular and Cellular Biology vol 20 no 21 pp 8244ndash8253 2000
[96] L Gewin H Myers T Kiyono and D A Galloway ldquoIdentifi-cation of a novel telomerase repressor that interacts with thehuman papillomavirus type-16 E6E6-AP complexrdquo Genes ampDevelopment vol 18 no 18 pp 2269ndash2282 2004
[97] M Sundaram D L GuernseyMM Rajaraman and R Rajara-man ldquoNeosis a novel type of cell division in cancerrdquo CancerBiology ampTherapy vol 3 no 2 pp 207ndash218 2004
[98] R D Snyder and J W Green ldquoA review of the genotoxicity ofmarketed pharmaceuticalsrdquo Mutation Research vol 488 no 2pp 151ndash169 2001
[99] T Ristriani Y Nomine C Laurent E Weiss and G TraveldquoProtein mutagenesis with monodispersity-based quality prob-ing selective inactivation of p53 degradation and DNA-bindingproperties of HPV E6 oncoproteinrdquo Protein Expression andPurification vol 26 no 3 pp 357ndash367 2002
[100] M Kadaja A Sumerina T Verst M Ojarand E Ustav and MUstav ldquoGenomic instability of the host cell induced by thehuman papillomavirus replicationmachineryrdquoTheEMBO Jour-nal vol 26 no 8 pp 2180ndash2191 2007
[101] PMDay D R Lowy and J T Schiller ldquoPapillomaviruses infectcells via a clathrin-dependent pathwayrdquo Virology vol 307 no 1pp 1ndash11 2003
[102] T D Tlsty A Briot A Gualberto et al ldquoGenomic instabilityand cancerrdquoMutation Research vol 337 no 1 pp 1ndash7 1995
[103] J E Eyfjord and S K Bodvarsdottir ldquoGenomic instabilityand cancer networks involved in response to DNA damagerdquoMutation Research vol 592 no 1-2 pp 18ndash28 2005
[104] Y Yao W Huang X Yang et al ldquoHPV-16 E6 and E7 protein Tcell epitopes prediction analysis based on distributions of HLA-A loci across populations an in silico approachrdquo Vaccine vol31 no 18 pp 2289ndash2294 2013
[105] C-F Hung B Ma A Monie S-W Tsen and T-C Wu ldquoTher-apeutic human papillomavirus vaccines current clinical trialsand future directionsrdquo Expert Opinion on Biological Therapyvol 8 no 4 pp 421ndash439 2008
[106] M J P Welters G G Kenter S J Piersma et al ldquoInductionof tumor-specific CD4+ and CD8+ T-cell immunity in cervicalcancer patients by a human papillomavirus type 16 E6 and E7long peptides vaccinerdquo Clinical Cancer Research vol 14 no 1pp 178ndash187 2008
[107] R Han N M Cladel C A Reed X Peng and N D Chris-tensen ldquoProtection of rabbits from viral challenge by gene gun-based intracutaneous vaccination with a combination of cot-tontail rabbit papillomavirus E1 E2 E6 and E7 genesrdquo Journalof Virology vol 73 no 8 pp 7039ndash7043 1999
[108] Z He A P Wlazlo D W Kowalczyk et al ldquoViral recombinantvaccines to the E6 and E7 antigens of HPV-16rdquo Virology vol270 no 1 pp 146ndash161 2000
[109] A Kaufmann P Stern E Rankin et al ldquoSafety and immuno-genicity of TA-HPV a recombinant vaccinia virus expressingmodified human papillomavirus (HPV)-16 and HPV-18 E6 andE7 genes in women with progressive cervical cancerrdquo ClinicalCancer Research vol 8 no 12 pp 3676ndash3685 2002
[110] L K Borysiewicz A Fiander M Nimako et al ldquoA recombinantvaccinia virus encoding human papillomavirus types 16 and 18E6 and E7 proteins as immunotherapy for cervical cancerrdquoTheLancet vol 347 no 9014 pp 1523ndash1527 1996
[111] A De Jong T OrsquoNeill A Y Khan et al ldquoEnhancement ofhuman papillomavirus (HPV) type 16 E6 and E7-specific T-cellimmunity in healthy volunteers through vaccination with TA-CIN an HPV16 L2E7E6 fusion protein vaccinerdquo Vaccine vol20 no 29-30 pp 3456ndash3464 2002
[112] S Chen C Liao Y Lai et al ldquoDe-oncogenicHPVE6E7 vaccinegets enhanced antigenicity and promotes tumoricidal synergywith cisplatinrdquo Acta Biochimica et Biophysica Sinica vol 46 no1 pp 6ndash14 2014
BioMed Research International 15
[113] J Bonhoeffer E B Imoukhuede G Aldrovandi et al ldquoTemplateprotocol for clinical trials investigating vaccines-focus on safetyelementsrdquo Vaccine vol 31 no 47 pp 5602ndash5620 2013
[114] S Jackson and A Storey ldquoE6 proteins from diverse cutaneonsHPV types inhibit apoptosis in response to UV damagerdquoOncogene vol 19 no 4 pp 592ndash598 2000
[115] M Thomas and L Banks ldquoPDZRN3LNX3 is a novel targetof human papillomavirus type 16 (HPV-16) and HPV-18 E6rdquoJournal of Virology vol 89 no 2 pp 1439ndash1444 2015
[116] P R Daniels C M Sanders P Coulson and N J MaitlandldquoMolecular analysis of the interaction between HPV type 16 E6and human E6-associated proteinrdquo FEBS Letters vol 416 no 1pp 6ndash10 1997
[117] W H Kao S L Beaudenon A L Talis J M Huibregtse andP M Howley ldquoHuman papillomavirus type 16 E6 induces self-ubiquitination of the E6AP ubiquitin-protein ligaserdquo Journal ofVirology vol 74 no 14 pp 6408ndash6417 2000
[118] T Iftner M Elbel B Schopp et al ldquoInterference of papillo-mavirus E6 protein with single-strand break repair by interac-tion with XRCC1rdquo The EMBO Journal vol 21 no 17 pp 4741ndash4748 2002
[119] Y Imai Y Tsunokawa T Sugimura and M Terada ldquoPurifi-cation and DNA-binding properties of human papillomavirustype 16 E6 protein expressed in Escherichia colirdquo Biochemicaland Biophysical Research Communications vol 164 no 3 pp1402ndash1410 1989
[120] M Lusky L Berg H Weiher and M Botchan ldquoBovinepapilloma virus contains an activator of gene expression at thedistal end of the early transcription unitrdquoMolecular andCellularBiology vol 3 no 6 pp 1108ndash1122 1983
[121] S Dalal Q Gao E J Androphy and V Band ldquoMutationalanalysis of human papillomavirus type 16 E6 demonstrates thatp53 degradation is necessary for immortalization of mammaryepithelial cellsrdquo Journal of Virology vol 70 no 2 pp 683ndash6881996
[122] J M Huibregtse M Scheffner and P M Howley ldquoA cellularprotein mediates association of p53 with the E6 oncoprotein ofhuman papillomavirus types 16 or 18rdquo The EMBO Journal vol10 no 13 pp 4129ndash4135 1991
[123] M Scheffner B A Werness J M Huibregtse A J Levine andP M Howley ldquoThe E6 oncoprotein encoded by human papillo-mavirus types 16 and 18 promotes the degradation of p53rdquo Cellvol 63 no 6 pp 1129ndash1136 1990
[124] M Elbel S Carl S Spaderna and T Iftner ldquoA comparativeanalysis of the interactions of the E6 proteins from cutaneousand genital papillomaviruses with p53 and E6AP in correlationto their transforming potentialrdquoVirology vol 239 no 1 pp 132ndash149 1997
[125] M C Thomas and C-M Chiang ldquoE6 oncoprotein repressesp53-dependent gene activation via inhibition of protein acety-lation independently of inducing p53 degradationrdquo MolecularCell vol 17 no 2 pp 251ndash264 2005
[126] H Zimmermann R Degenkolbe H-U Bernard and M JOrsquoConnor ldquoThe human papillomavirus type 16 E6 oncoproteincan down-regulate p53 activity by targeting the transcriptionalcoactivator CBPp300rdquo Journal of Virology vol 73 no 8 pp6209ndash6219 1999
[127] D A Thompson G Belinsky T H-T Chang D L Jones RSchlegel and K Munger ldquoThe human papillomavirus-16 E6oncoprotein decreases the vigilance of mitotic checkpointsrdquoOncogene vol 15 no 25 pp 3025ndash3035 1997
[128] J Bohl K Das B Dasgupta and S B Vande Pol ldquoCompetitivebinding to a charged leucine motif represses transformation bya papillomavirus E6 oncoproteinrdquo Virology vol 271 no 1 pp163ndash170 2000
[129] S Song H C Pitot and P F Lambert ldquoThe human papillo-mavirus type 16 E6 gene alone is sufficient to induce carcinomasin transgenic animalsrdquo Journal of Virology vol 73 no 7 pp5887ndash5893 1999
[130] S Yasumoto A L Burkhardt J Doniger and J A DiPaololdquoHumanpapillomavirus type 16DNA-inducedmalignant trans-formation of NIH 3T3 cellsrdquo Journal of Virology vol 57 no 2pp 572ndash577 1986
[131] S B Vande Pol M C Brown and C E Turner ldquoAssociation ofBovine Papillomavirus Type 1 E6 oncoprotein with the focaladhesion protein paxillin through a conserved protein interac-tion motifrdquo Oncogene vol 16 no 1 pp 43ndash52 1998
[132] R Wade N Brimer and S Vande Pol ldquoTransformation bybovine papillomavirus type 1 E6 requires paxillinrdquo Journal ofVirology vol 82 no 12 pp 5962ndash5966 2008
[133] A J Klingelhutz S A Foster and J KMcDougall ldquoTelomeraseactivation by the E6 gene product of humanpapillomavirus type16rdquo Nature vol 380 no 6569 pp 79ndash82 1996
[134] S T Oh S Kyo and L A Laimins ldquoTelomerase activation byhuman papillomavirus type 16 E6 protein Induction of humantelomerase reverse transcriptase expression through Myc andGC-rich Sp1 binding sitesrdquo Journal of Virology vol 75 no 12pp 5559ndash5566 2001
[135] L V Ronco A Y KarpovaMVidal and PMHowley ldquoHumanpapillomavirus 16 E6 oncoprotein binds to interferon regulatoryfactor-3 and inhibits its transcriptional activityrdquo Genes andDevelopment vol 12 no 13 pp 2061ndash2072 1998
Figure 8 Photomicroscope showing CRIB cell not treated with any drug (negative control) indicating absence of micronucleus (a) andtreated with BPV-1 E6 recombinant oncoprotein showing the presence of MN indicated by arrow (b) (c) Number of micronucleated cellsobserved in each group
in the peripheral blood reduces the influence of endogenousenvironmental factors that could interfere in the analysisPositive control showed a high number of micronucle-ated lymphocytes This result was also expected once thisgroup was treated with 50 120583gmL of cyclophosphamide achemotherapeutic drug with cytotoxic and teratogenic effect[76 77] The cells treated with 1120583gmL of BPV-1 E6 recom-binant oncoprotein also showed significant statistical valuesKruskal-Wallis test followed by the Dunn post hoc testwas performed to compare the frequencies of micronucleusamong three groups This test indicated statistical differencebetween negative control and the cells treated with BPV-1 E6 recombinant oncoprotein However the test did notreveal differences between the cells treated with cyclophos-phamide and BPV-1 E6 recombinant oncoprotein (Table 4Figure 4) Similar results were also observed in epithelial cells(Figure 8) These results suggest that the BPV-1 E6 oncopro-tein has an aneugenic andor clastogenic potential similarto or higher than the observed with the cyclophosphamideThis is because the cells treated with the oncoprotein showed
a frequency of micronucleus formation (MN1199030) 14003
higher than those observed in positive controlThe elevated mutagenic potential of the BPV-1 E6 onco-
protein can justify the cytogenetic damage already describedin the literature [1 14 15 24] Similar results were observedin HPV-infected cells [12 78ndash81] The micronucleus induce-ment was already described in cytological samples collectedduring Papanicolaursquos test from healthy cervix infected byHPV [12] For this reason the micronucleus assay has beenproposed as a complementary method for Papanicolaursquos testbeing a suggestive biomarker of lesion degree [78] Howeverthe association between the BPV and the micronucleusinducement was not yet reported
The high frequency of micronucleated lymphocytes insamples infected by HPV has been attributed to the synergicaction of E5 E6 and E7 oncoproteins [29 30 78 79]However this is the first study that pointed out themutagenicaction of BPV-1 E6 protein per se
Student-Newman-Keuls post hoc test based on the fre-quency of anaphase bridges showed significant statistical
10 BioMed Research International
Table 7 Comet assay results showing the number of nucleoidsobserved per class the number of hedgehog comets and cometscore
Sample Class 0 Class 1 Class 2 Hedgehog ScoreNegative control
Groups Rank differences Calculated 119885 Critical 119885 119901
Cminus and C+ 68 24042 2394 lt05Cminus and E6 82 28991 2394 lt05C+ and E6 14 04950 2394 nsns nonsignificant differences
difference between the negative control and the group treatedwith the BPV-1 E6 recombinant oncoprotein (Table 5) How-ever the test did not reveal differences between the cellstreated with cyclophosphamide and BPV-1 E6 recombinantoncoprotein (Table 5)These data reinforce those observed bythe Dunn post hoc test based on the frequency of micronu-cleus indicating the clastogenic action of the E6 oncoproteinMoreover the presence of anaphase bridge was alreadydescribed in cells transfected with recombinant adenoviruscontaining the E6 and E7 ORFs of HPV [29 30] Howeverthere are no studies describing the action of BPVE6 oncopro-tein in the induction of anaphase bridges or studies that showthis same action of E6 oncoprotein of HPV per se Anaphasebridges are indicators of genomic instability [82 83] which isconsidered the first step in carcinogenesis [46 47] Moreoverthe presence of these bridges is an important hallmark ofDNA double strand breaks (DSBs) [84]
Statistical analysis based on the cytokinesis-block prolif-eration index (CBPI) showed that BPV-1 E6 oncoprotein hascytotoxic levels similar to cyclophosphamide (Table 6) Thisresult suggests that the BPV E6 oncoprotein can deregulatethe cell cycle contributing to the cell proliferation andimmortalization To date this mitogenic action is attributedto the E7 oncoprotein [85ndash87] E7 oncoprotein induces
the phosphorylation of pRb resulting in the E2F transcrip-tion factor release [85] E2F when translocated to the nucleusacts as activator and binds to the kinase-dependent cyclinspromoters [85] However the results showed polynucleatedcells in the group treatedwithBPVE6oncoprotein (Figure 5)suggesting that the BPV E6 oncoprotein confers mitogenicstimulation resulting in mitotic stress Moreover studiespointed out that the BPV E6 oncoprotein interacts with theCBPp300 deacetylase promoting p53 downregulation [88]This downregulation increases the expression levels of FoxM1transcription factor (Foxhead box M1) [89] promoting B1cyclin D1 cyclin and cdc25 upregulation [89] The upregu-lation of these genes is associated with increased levels of cellproliferation which is necessary in order to make DNA poly-merases available to virus replication However if this mech-anism guarantees the BPV replication it can contribute to cellimmortalization and cancer progression
The FoxM1 factor also participates in the Wnt120573-cateninsignaling pathway and binds directly to the 120573-catenin [89]This interaction promotes the nuclear 120573-catenin transloca-tion [90] 120573-catenin translocation to nucleus induces thecyclin expression [64] So these data suggest that the BPVE6 oncoprotein not only promotes mitogenic stress but alsocontributes to the epithelial-mesenchymal transition (EMT)Studies based on theHPVE6 oncoprotein have indicated thatthis oncoprotein contributes significantly to the EMT [91]This occurs not only because the E6 oncoprotein induces thetranslocation of 120573-catenin but also due to the proteasomaldegradation of regulatory proteins of apical-basal polarity[92ndash94] Studies also showed that the E6 oncoprotein is ableto bind to Crumbs (Dlg and Patj) proteins resulting in loss ofcell polarity [93 94] Based on these results the E6 oncopro-tein emerges as a possible therapeutic target with biotechno-logical value in cancer treatment
Mitogenic action of E6 oncoprotein can be moreexpressed in cells infected by papillomaviruses once thesecells also expressed E5 and E7 oncoproteins [74] The E7oncoprotein is able to form a complex E7-p600 which pro-motes E6 upregulation [85] Furthermore the E6 oncoproteincan bind to the E6AP ubiquitin ligase forming the E6-E6APcomplex increasing the hTERT levels of expression con-tributing to cell immortalization [95] Moreover E6-E6APinduces NFX1 expression promoting MHC-II downregula-tion [96]Thismechanism reduces the antigenic presentationmediated by CD4+ T lymphocytes contributing to immuneevasion This action guarantees the PV infection for longperiods with or without clinical symptoms [24] Althoughthe BPV infection can be asymptomatic the BPV presence inperipheral blood is associated with DNA damage [1 14 24]
CBMNA analysis pointed out the presence of cells withintracellular cytokinesis (Figure 5) This result suggests theneotic action of BPV E6 oncoprotein Neosis is characterizedby the presence of (1) DNA damage (2) loss of checkpointcontrol (3) repair system failures and (4) endoreduplication[97] In this scenario the E6 oncoprotein attempts all theseneosis criteria Moreover the oncoprotein not only increasesthe cytogenetic damage leading to increasing of micronu-cleus frequency but also induces clastogenesis as shown bythe comet assay and anaphase bridges So statistical analysis
BioMed Research International 11
performed based on the comet scores reinforced the CBMNAresults Comet assay allows detecting both simple and dou-ble DNA breaks being more sensitive than the CBMNAClastogenesis is considered the most serious type of DNAdamage [98] DNA breaks can be repaired by homologous ornonhomologous recombination However studies point outthat the E6 oncoprotein can link to the Holliday junctionduring the homologous repair avoiding the junction reso-lution [99] Moreover E6 oncoprotein promotes the TP53gene deacetylation resulting in p53 downregulation [51]Thisepigenetic effect results in cell cycle deregulation affectingthe checkpoints
Studies have shown that the HPV-induced DNA breaksare required for the virus integration into the host genome[100] Although there are no studies showing the BPV inte-gration to date the results observed in this work in additionto those already published [24] indicate the necessity ofmorestudies to evaluate the virus-host interaction
Studies also showed that the E6 oncoprotein inducesclathrin-transporter adapter protein (AP-1) increasing thelevel of this protein in the plasma membrane [76 77] Theincreasing quantity of AP-1 in membrane can facilitate theBPV virions infection This occurs because the infectionprocess is clathrin dependent In this scenario the E6 onco-protein can contribute to the virus infection [101]
In summary the CBMNA and CA results showed thatthe BPV E6 oncoprotein participates not only in cancerpromotion but also in initiation inducing DNA breaksThese DNA breaks represent mutagenic effects consideredthe first step in the oncogenesis process which are associatedwith genomic instability [47 48 102 103] Thus the E6recombinant oncoprotein has been suggested as a possiblevaccine candidate [104ndash112] due to its immunogenicity [44]However the mutagenic tests such as CBMNA and CA arerequired in drug validation process [113] In this scenariothis study pointed out that the BPV-1 E6 recombinant onco-protein in tested concentration showedmutagenic potentialThis is the first study that reports the mutagenic potential ofa possible therapeutic vaccine candidate On the one handthese results allowed better understanding the mechanism ofcancer promotion associatedwith the BPVE6oncoprotein aswell as revealing that this oncoprotein can induce carcinogen-esis per se on the other hand this data pointed out thatmaybeBPV E6 recombinant oncoprotein requires protein modifica-tions to be used as vaccine
Conflict of Interests
The authors declare that there is no conflict of interests
Acknowledgments
Theauthors thank Fundacao deAmparo a Pesquisa do Estadode Sao Paulo (FAPESP Process 201420617-5) and FundacaoButantan for the financial support
References
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experimental modelrdquo Journal of General Virology vol 79 no 9pp 2127ndash2135 1998
[2] A Antonsson and B G Hansson ldquoHealthy skin ofmany animalspecies harbors papillomaviruses which are closely related totheir human counterpartsrdquo Journal of Virology vol 76 no 24pp 12537ndash12542 2002
[3] P Rous and J W Beard ldquoA virus-induced mammalian growthwith the characters of a tumor (the Shope rabbit papilloma) IThe growth on implantationwithin favorable hostsrdquoThe Journalof Experimental Medicine vol 60 no 6 pp 701ndash722 1934
[4] R Shope and E Hurst ldquoInfectious papillomatosis of rabbitsrdquoThe Journal of Experimental Medicine vol 58 no 1 pp 607ndash624 1933
[5] H Delius M A Van Ranst A B Jenson H Zur Hausen and JP Sundberg ldquoCanine oral papillomavirus genomic sequence aunique 15-kb intervening sequence between the E2 andL2 openreading framesrdquo Virology vol 204 no 1 pp 447ndash452 1994
[6] W A Demonbreun and E Goodpasture ldquoInfectious oral papil-lomatosis of dogsrdquo The American Journal of Pathology vol 8no 1 pp 43ndash56 1932
[7] P K Nicholls B A Klaunberg R A Moore et al ldquoNaturallyoccurring nonregressing canine oral papillomavirus infectionhost immunity virus characterization and experimental infec-tionrdquo Virology vol 265 no 2 pp 365ndash374 1999
[8] A Alberti S Pirino F Pintore et al ldquoOvis aries Papillomavirus3 a prototype of a novel genus in the family Papillomaviridaeassociated with ovine squamous cell carcinomardquo Virology vol407 no 2 pp 352ndash359 2010
[9] H zur Hausen E-M de Villiers and L Gissmann ldquoPapil-lomavirus infections and human genital cancerrdquo GynecologicOncology vol 12 no 2 pp S124ndashS128 1981
[10] C Foresta A Bertoldo A Garolla et al ldquoHuman papillo-mavirus proteins are found in peripheral blood and semenCd20+ and Cd56+ cells during Hpv-16 semen infectionrdquo BMCInfectious Diseases vol 13 article 593 2013
[11] S Ljubojevic andM Skerlev ldquoHPV-associated diseasesrdquo Clinicsin Dermatology vol 32 no 2 pp 227ndash234 2014
[12] A P R Cassel R B Barcellos C M D da Silva S E de MatosAlmeida and M L R Rossetti ldquoAssociation between humanpapillomavirus (HPV) DNA and micronuclei in normal cervi-cal cytologyrdquo Genetics and Molecular Biology vol 37 no 2 pp360ndash363 2014
[13] W Jarrett M Campo B OrsquoNeil H Laird and L Coggins ldquoAnovel bovine papillomavirus (BPV-6) causing true epithelialpapillomas of the mammary gland skin a member of a pro-posed newBPV subgrouprdquoVirology vol 136 no 2 pp 255ndash2641984
[14] T CMelo N Diniz S R C Campos et al ldquoCytogenetic studiesin peripheral blood of bovines afflicted by papillomatosisrdquoVeterinary and Comparative Oncology vol 9 no 4 pp 269ndash2742011
[15] S R C Campos T C Melo S Assaf et al ldquoChromosome aber-rations in cells infected with bovine papillomavirus comparingcutaneous papilloma esophagus papilloma and urinary blad-der lesion cellsrdquo ISRN Oncology vol 2013 Article ID 910849 8pages 2013
[16] H zur Hausen ldquoThe search for infectious causes of humancancers where and whyrdquoVirology vol 392 no 1 pp 1ndash10 2009
[17] R P Araldi R F Carvalho T C Melo et al ldquoBovine papillo-mavirus in beef cattle first description of BPV-12 and putativetype BAPV8 in BrazilrdquoGenetics andMolecular Research vol 13no 3 pp 5644ndash5653 2014
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[18] T Rampias C Sasaki and A Psyrri ldquoMolecular mechanisms ofHPV induced carcinogenesis in head and neckrdquoOral Oncologyvol 50 no 5 pp 356ndash363 2014
[19] S Roperto V Russo L Leonardi et al ldquoBovine papillomavirustype 13 expression in the urothelial bladder tumours of cattlerdquoTransboundary and Emerging Diseases 2015
[20] S RCCampos C TrindadeO P Ferraz et al ldquoCan establishedcultured papilloma cells harbor bovine papillomavirusrdquoGenet-ics and Molecular Research vol 7 no 4 pp 1119ndash1126 2008
[21] M S Campo ldquoAnimalmodels of papilloma virus pathogenesisrdquoVirus Research vol 89 no 2 pp 249ndash261 2002
[22] G Borzacchiello ldquoBovine papillomavirus infections in ani-malsrdquo in Communicating Current Research and EducationalTopics and Trends in AppliedMicrobiology pp 673ndash679 Forma-tex Badajoz Spain 2007
[23] F Bocaneti G Altamura A Corteggio E Velescu F RopertoandG Borzacchiello ldquoBovine papillomavirus new insights intoan old diseaserdquo Transboundary and Emerging Diseases 2014
[24] R P Araldi T C Melo N Diniz et al ldquoBovine papillomavirusclastogenic effect analyzed in comet assayrdquo BioMed ResearchInternational vol 2013 Article ID 630683 7 pages 2013
[25] J Munday N Thomson M Dunowska C Knight R Laurieand S Hills ldquoGenomic characterisation of the feline sarcoid-associated papillomavirus and proposed classification as Bostaurus papillomavirus type 14rdquoVeterinaryMicrobiology vol 177no 3-4 pp 289ndash295 2015
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[30] S Duensing L Y Lee A Duensing et al ldquoThe human papil-lomavirus type 16 E6 and E7 oncoproteins cooperate to inducemitotic defects and genomic instability by uncoupling centro-some duplication from the cell division cyclerdquo Proceedings of theNational Academy of Sciences of theUnited States of America vol97 no 18 pp 10002ndash10007 2000
[31] C A Moody and L A Laimins ldquoHuman papillomavirus onco-proteins pathways to transformationrdquo Nature Reviews Cancervol 10 no 8 pp 550ndash560 2010
[32] C Mansur and E Androphy ldquoCellular transformation bypapillomavirus oncoproteinsrdquo Biochimica et Biophysica Actavol 1155 no 3 pp 323ndash345 1993
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[34] X Tong andPMHowley ldquoThebovine papillomavirus E6 onco-protein interacts with paxillin and disrupts the actin cytoskele-tonrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 94 no 9 pp 4412ndash4417 1997
[35] X TongW Boll T Kirchhausen andPMHowley ldquoInteractionof the bovine papillomavirus E6 protein with the clathrinadaptor complex AP-1rdquo Journal of Virology vol 72 no 1 pp476ndash482 1998
[36] Y Nomine M Masson S Charbonnier et al ldquoStructural andfunctional analysis of E6 oncoprotein insights in the molecularpathways of human papillomavirus-mediated pathogenesisrdquoMolecular Cell vol 21 no 5 pp 665ndash678 2006
[37] T Ristriani MMasson Y Nomine et al ldquoHPV oncoprotein E6is a structure-dependent DNA-binding protein that recognizesfour-way junctionsrdquo Journal of Molecular Biology vol 296 no5 pp 1189ndash1203 2000
[38] Y Liu and J D Baleja ldquoStructure and function of the papil-lomavirus E6 protein and its interacting proteinsrdquo Frontiers inBioscience vol 13 no 1 pp 121ndash134 2008
[39] F Lipari G A McGibbon E Wardrop and M G CordingleyldquoPurification and biophysical characterization of a minimalfunctional domain and of an N-terminal Zn2+-binding frag-ment from the human papillomavirus type 16 E6 proteinrdquoBiochemistry vol 40 no 5 pp 1196ndash1204 2001
[40] Y Nomine T Ristriani C Laurent J-F Lefevre E Weissand G Trave ldquoA strategy for optimizing the monodispersity offusion proteins application to purification of recombinantHPVE6 oncoproteinrdquo Protein Engineering vol 14 no 4 pp 297ndash3052001
[41] C G Ullman P I Haris D A Galloway V C Emery and SJ Perkins ldquoPredicted 120572-helix120573-sheet secondary structures forthe zinc-binding motifs of human papillomavirus E7 and E6proteins by consensus prediction averaging and spectroscopicstudies of E7rdquo Biochemical Journal vol 319 no 1 pp 229ndash2391996
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[43] K Zanier S Charbonnier A O M O Sidi et al ldquoStructuralbasis for hijacking of cellular LxxLL motifs by papillomavirusE6 oncoproteinsrdquo Science vol 339 no 6120 pp 694ndash698 2013
[44] J Mazzuchelli-de-Souza R F Carvalho R M Ruiz et alldquoExpression and in silico analysis of the recombinant bovinepapillomavirus e6 protein as a model for viral oncoproteinsstudiesrdquo BioMed Research International vol 2013 Article ID421398 9 pages 2013
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[48] S Negrini V G Gorgoulis and T D Halazonetis ldquoGenomicinstabilitymdashan evolving hallmark of cancerrdquo Nature ReviewsMolecular Cell Biology vol 11 no 3 pp 220ndash228 2010
[49] K Zanier S Charbonnier M Baltzinger Y Nomine DAltschuh and G Trave ldquoKinetic analysis of the interactionsof human papillomavirus E6 oncoproteins with the ubiquitinligase E6AP using surface plasmon resonancerdquo Journal ofMolecular Biology vol 349 no 2 pp 401ndash412 2005
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[50] M Scheffner J M Huibregtse R D Vierstra and PM HowleyldquoThe HPV-16 E6 and E6-AP complex functions as a ubiquitin-protein ligase in the ubiquitination of p53rdquo Cell vol 75 no 3pp 495ndash505 1993
[51] H Zimmermann C-H Koh R Degenkolbe et al ldquoInteractionwith CBPp300 enables the bovine papillomavirus type 1 E6oncoprotein to downregulate CBPp300-mediated transactiva-tion by p53rdquo Journal of General Virology vol 81 no 11 pp 2617ndash2623 2000
[52] M Fenech ldquoThe in vitro micronucleus techniquerdquo MutationResearchmdashFundamental and Molecular Mechanisms of Mutage-nesis vol 455 no 1-2 pp 81ndash95 2000
[53] R P Araldi T C de Melo T B Mendes et al ldquoUsing the cometand micronucleus assays for genotoxicity studies a reviewrdquoBiomedicine amp Pharmacotherapy vol 72 pp 74ndash82 2015
[54] P Thomas and M Fenech ldquoCytokinesis-block micronucleuscytome assay in lymphocytesrdquo Methods in Molecular Biologyvol 682 pp 217ndash234 2011
[55] M Kirsch-Volders T Sofuni M Aardema et al ldquoReport fromthe in vitro micronucleus assay working grouprdquo MutationResearch Genetic Toxicology and Environmental Mutagenesisvol 540 no 2 pp 153ndash163 2003
[56] S Samanta and P Dey ldquoMicronucleus and its applicationsrdquoDiagnostic Cytopathology vol 40 no 1 pp 84ndash90 2012
[57] M Hayashi J T MacGregor D G Gatehouse et al ldquoIn vivoerythrocyte micronucleus assay III Validation and regulatoryacceptance of automated scoring and the use of rat periph-eral blood reticulocytes with discussion of non-hematopoietictarget cells and a single dose-level limit testrdquo MutationResearchGenetic Toxicology and Environmental Mutagenesisvol 627 no 1 pp 10ndash30 2007
[58] A Azqueta and A R Collins ldquoThe essential comet assay acomprehensive guide to measuring DNA damage and repairrdquoArchives of Toxicology vol 87 no 6 pp 949ndash968 2013
[59] N P Singh M T McCoy R R Tice and E L Schneider ldquoAsimple technique for quantitation of low levels of DNA damagein individual cellsrdquo Experimental Cell Research vol 175 no 1pp 184ndash191 1988
[60] J H Calinisan S R Chan A King and P J Chan ldquoHumanpapillomavirus and blastocyst apoptosisrdquo Journal of AssistedReproduction and Genetics vol 19 no 3 pp 132ndash136 2002
[61] R Oliveira Studies on the biopathological actions of Pteridiumaquilinum [PhD thesis] nstituto de Ciencias Biomedicas AbelSalazar 2012
[62] D M Potter and M S Baird ldquoCarcinogenic effects of ptaquilo-side in bracken fern and related compoundsrdquo British Journal ofCancer vol 83 no 7 pp 914ndash920 2000
[63] R B Lucena D R Rissi G D Kommers et al ldquoA retrospectivestudy of 586 tumours in Brazilian cattlerdquo Journal of ComparativePathology vol 145 no 1 pp 20ndash24 2011
[64] A Yaguiu M L Z Dagli E H Birgel Jr et al ldquoSimultaneouspresence of bovine papillomavirus and bovine leukemia virusin different bovine tissues in situ hybridization and cytogeneticanalysisrdquoGenetics andMolecular Research vol 7 no 2 pp 487ndash497 2008
[65] T C Melo R F Carvalho J Mazzucchelli-de-Souza et alldquoPhylogenetic classification and clinical aspects of a new puta-tive Deltapapillomavirus associated with skin lesions in cattlerdquoGenetics and Molecular Research vol 13 no 2 pp 2458ndash24692014
[66] R P Araldi D N S Giovanni T CMelo et al ldquoBovine papillo-mavirus isolation by ultracentrifugationrdquo Journal of VirologicalMethods vol 208 pp 119ndash124 2014
[67] L M Chandrachud B W OrsquoNeil W F H Jarrett G J Grind-lay G M McGarvie and M S Campo ldquoHumoral immuneresponse to the E7 protein of bovine papillomavirus type 4 andidentification of B-cell epitopesrdquoVirology vol 200 no 1 pp 98ndash104 1994
[68] M Ayres M J Ayres D L Ayres and A A Santos BioEstatmdashAplicacoes Estatısticas nas Areas de Ciencias Bio-Medicas Insti-tuto Mamiraua Belem Brazil 2007
[69] K Das J Bohl and S B Vande Pol ldquoIdentification of a secondtransforming function in bovine papillomavirus type 1 E6 andthe role of E6 interactions with paxillin E6BP and E6APrdquoJournal of Virology vol 74 no 2 pp 812ndash816 2000
[70] O Danos E Georges G Orth and M Yaniv ldquoFine structureof the cottontail rabbit papillomavirus mRNAs expressed in thetransplantable VX2 carcinomardquo Journal of Virology vol 53 no3 pp 735ndash741 1985
[71] M Nasseri and F O Wettstein ldquoDifferences exist between viraltranscripts in cottontail rabbit papillomavirus-induced benignand malignant tumors as well as non-virus-producing andvirus-producing tumorsrdquo Journal of Virology vol 51 no 3 pp706ndash712 1984
[72] X Tong R Salgia J-L Li J D Griffin and P M Howley ldquoThebovine papillomavirus E6 protein binds to the LDmotif repeatsof paxillin and blocks its interaction with vinculin and the focaladhesion kinaserdquo Journal of Biological Chemistry vol 272 no52 pp 33373ndash33376 1997
[73] Y Maeda T Shibahara Y Wada et al ldquoAn outbreak of teatpapillomatosis in cattle caused by bovine papilloma virus (BPV)type 6 and unclassified BPVsrdquo Veterinary Microbiology vol 121no 3-4 pp 242ndash248 2007
[74] R P Araldi T C Melo A Neves et al ldquoHyperproliferativeaction of bovine papillomavirus genetic and histopathologicalaspectsrdquo Genetics and Molecular Research vol 14 no 4 pp12942ndash12954 2015
[75] NDiniz T CMelo J F Santos et al ldquoSimultaneous presence ofbovine papillomavirus in blood and in short-term lymphocytecultures from dairy cattle in Pernambuco Brazilrdquo Genetics andMolecular Research vol 8 no 4 pp 1474ndash1480 2009
[76] O M Brito M F Guimaraes and C C Lanna ldquoCiclofosfamidae funcao ovarianardquo Revista Brasileira de Reumatologia vol 48no 1 pp 39ndash45 2008
[77] D Anderson T-W Yu and D B McGregor ldquoComet assayresponses as indicators of carcinogen exposurerdquo Mutagenesisvol 13 no 6 pp 539ndash555 1998
[78] M L Adam C Pini S Tulio J C L L Cantalice R A Torresand M T Dos Santos Correia ldquoAssessment of the associationbetween micronuclei and the degree of uterine lesions and viralload in women with human papillomavirusrdquo Cancer Genomicsamp Proteomics vol 12 no 2 pp 67ndash72 2015
[79] E Cortes-Gutierrez M Davila-Rodrıguez J Vargas-VillarrealF Hernandez-Garza and R Cerda-Flores ldquoAssociationbetween human papilloma virus-type infections with micro-nuclei frequenciesrdquo Prague Medical Report vol 111 no 1 pp35ndash41 2010
[80] R N Chakrabarti and K Dutta ldquoMicronuclei test in routinesmears fromuterine cervixrdquo European Journal of GynaecologicalOncology vol 9 no 5 pp 370ndash372 1988
[81] E M M Cerqueira C L Santoro N F Donozo et al ldquoGeneticdamage in exfoliated cells of the uterine cervix Association
14 BioMed Research International
and interaction between cigarette smoking and progression tomalignant transformationrdquo Acta Cytologica vol 42 no 3 pp639ndash649 1998
[82] S M Germann V Schramke R T Pedersen et al ldquoTopBP1Dpb11 bindsDNA anaphase bridges to prevent genome instabil-ityrdquoThe Journal of Cell Biology vol 204 no 1 pp 45ndash59 2014
[83] D R Hoffelder L Luo N A Burke S CWatkins S M Gollinand W S Saunders ldquoResolution of anaphase bridges in cancercellsrdquo Chromosoma vol 112 no 8 pp 389ndash397 2004
[84] C Acilan D M Potter and W S Saunders ldquoDNA repairpathways involved in anaphase bridge formationrdquo Genes Chro-mosomes and Cancer vol 46 no 6 pp 522ndash531 2007
[85] J DeMasi M C Chao A S Kumar and P M Howley ldquoBovinepapillomavirus E7 oncoprotein inhibits anoikisrdquo Journal ofVirology vol 81 no 17 pp 9419ndash9425 2007
[86] J DeMasi K-W Huh Y Nakatani K Munger and P MHowley ldquoBovine papillomavirus E7 transformation functioncorrelates with cellular p600 protein bindingrdquo Proceedings of theNational Academy of Sciences of theUnited States of America vol102 no 32 pp 11486ndash11491 2005
[87] E A White and P M Howley ldquoProteomic approaches to thestudy of papillomavirusndashhost interactionsrdquo Virology vol 435no 1 pp 57ndash69 2013
[88] FMantovani and L Banks ldquoThe human papillomavirus E6 pro-tein and its contribution to malignant progressionrdquo Oncogenevol 20 no 54 pp 7874ndash7887 2001
[89] P Chen and H Lee ldquoFOXM1 induced by E6 oncoprotein pro-motes tumor invasion and chemoresistance in HPV-infectedlung cancerrdquo Cancer Cell amp Microenvironment vol 2 no 1 pp6ndash9 2015
[90] N Zhang P Wei A Gong et al ldquoFoxM1 promotes 120573-cateninnuclear localization and controls Wnt target-gene expressionand glioma tumorigenesisrdquo Cancer Cell vol 127 no 4 pp 358ndash366 2012
[91] Z Liu Y Liu Y Hong L Rapp E J Androphy and J JChen ldquoBovine papillomavirus type 1 E6-induced sensitizationto apoptosis is distinct from its transforming activityrdquo Virologyvol 295 no 2 pp 230ndash237 2002
[92] M Thomas P Massimi C Navarro J-P Borg and L BanksldquoThe hScribDlg apico-basal control complex is differentiallytargeted by HPV-16 and HPV-18 E6 proteinsrdquo Oncogene vol24 no 41 pp 6222ndash6230 2005
[93] C H Storrs and S J Silverstein ldquoPATJ a tight junction-associated PDZ protein is a novel degradation target of high-risk human papillomavirus E6 and the alternatively splicedisoform 18 E6rdquo Journal of Virology vol 81 no 8 pp 4080ndash40902007
[94] R Accardi R Rubino M Scalise et al ldquoE6 and E7 from humanpapillomavirus type 16 cooperate to target the PDZ proteinNaH exchange regulatory factor 1rdquo Journal of Virology vol 85no 16 pp 8208ndash8216 2011
[95] S Nakagawa and J M Huibregtse ldquoHuman scribble (Vartul)is targeted for ubiquitin-mediated degradation by the high-risk papillomavirus E6 proteins and the E6APubiquitin-proteinligaserdquoMolecular and Cellular Biology vol 20 no 21 pp 8244ndash8253 2000
[96] L Gewin H Myers T Kiyono and D A Galloway ldquoIdentifi-cation of a novel telomerase repressor that interacts with thehuman papillomavirus type-16 E6E6-AP complexrdquo Genes ampDevelopment vol 18 no 18 pp 2269ndash2282 2004
[97] M Sundaram D L GuernseyMM Rajaraman and R Rajara-man ldquoNeosis a novel type of cell division in cancerrdquo CancerBiology ampTherapy vol 3 no 2 pp 207ndash218 2004
[98] R D Snyder and J W Green ldquoA review of the genotoxicity ofmarketed pharmaceuticalsrdquo Mutation Research vol 488 no 2pp 151ndash169 2001
[99] T Ristriani Y Nomine C Laurent E Weiss and G TraveldquoProtein mutagenesis with monodispersity-based quality prob-ing selective inactivation of p53 degradation and DNA-bindingproperties of HPV E6 oncoproteinrdquo Protein Expression andPurification vol 26 no 3 pp 357ndash367 2002
[100] M Kadaja A Sumerina T Verst M Ojarand E Ustav and MUstav ldquoGenomic instability of the host cell induced by thehuman papillomavirus replicationmachineryrdquoTheEMBO Jour-nal vol 26 no 8 pp 2180ndash2191 2007
[101] PMDay D R Lowy and J T Schiller ldquoPapillomaviruses infectcells via a clathrin-dependent pathwayrdquo Virology vol 307 no 1pp 1ndash11 2003
[102] T D Tlsty A Briot A Gualberto et al ldquoGenomic instabilityand cancerrdquoMutation Research vol 337 no 1 pp 1ndash7 1995
[103] J E Eyfjord and S K Bodvarsdottir ldquoGenomic instabilityand cancer networks involved in response to DNA damagerdquoMutation Research vol 592 no 1-2 pp 18ndash28 2005
[104] Y Yao W Huang X Yang et al ldquoHPV-16 E6 and E7 protein Tcell epitopes prediction analysis based on distributions of HLA-A loci across populations an in silico approachrdquo Vaccine vol31 no 18 pp 2289ndash2294 2013
[105] C-F Hung B Ma A Monie S-W Tsen and T-C Wu ldquoTher-apeutic human papillomavirus vaccines current clinical trialsand future directionsrdquo Expert Opinion on Biological Therapyvol 8 no 4 pp 421ndash439 2008
[106] M J P Welters G G Kenter S J Piersma et al ldquoInductionof tumor-specific CD4+ and CD8+ T-cell immunity in cervicalcancer patients by a human papillomavirus type 16 E6 and E7long peptides vaccinerdquo Clinical Cancer Research vol 14 no 1pp 178ndash187 2008
[107] R Han N M Cladel C A Reed X Peng and N D Chris-tensen ldquoProtection of rabbits from viral challenge by gene gun-based intracutaneous vaccination with a combination of cot-tontail rabbit papillomavirus E1 E2 E6 and E7 genesrdquo Journalof Virology vol 73 no 8 pp 7039ndash7043 1999
[108] Z He A P Wlazlo D W Kowalczyk et al ldquoViral recombinantvaccines to the E6 and E7 antigens of HPV-16rdquo Virology vol270 no 1 pp 146ndash161 2000
[109] A Kaufmann P Stern E Rankin et al ldquoSafety and immuno-genicity of TA-HPV a recombinant vaccinia virus expressingmodified human papillomavirus (HPV)-16 and HPV-18 E6 andE7 genes in women with progressive cervical cancerrdquo ClinicalCancer Research vol 8 no 12 pp 3676ndash3685 2002
[110] L K Borysiewicz A Fiander M Nimako et al ldquoA recombinantvaccinia virus encoding human papillomavirus types 16 and 18E6 and E7 proteins as immunotherapy for cervical cancerrdquoTheLancet vol 347 no 9014 pp 1523ndash1527 1996
[111] A De Jong T OrsquoNeill A Y Khan et al ldquoEnhancement ofhuman papillomavirus (HPV) type 16 E6 and E7-specific T-cellimmunity in healthy volunteers through vaccination with TA-CIN an HPV16 L2E7E6 fusion protein vaccinerdquo Vaccine vol20 no 29-30 pp 3456ndash3464 2002
[112] S Chen C Liao Y Lai et al ldquoDe-oncogenicHPVE6E7 vaccinegets enhanced antigenicity and promotes tumoricidal synergywith cisplatinrdquo Acta Biochimica et Biophysica Sinica vol 46 no1 pp 6ndash14 2014
BioMed Research International 15
[113] J Bonhoeffer E B Imoukhuede G Aldrovandi et al ldquoTemplateprotocol for clinical trials investigating vaccines-focus on safetyelementsrdquo Vaccine vol 31 no 47 pp 5602ndash5620 2013
[114] S Jackson and A Storey ldquoE6 proteins from diverse cutaneonsHPV types inhibit apoptosis in response to UV damagerdquoOncogene vol 19 no 4 pp 592ndash598 2000
[115] M Thomas and L Banks ldquoPDZRN3LNX3 is a novel targetof human papillomavirus type 16 (HPV-16) and HPV-18 E6rdquoJournal of Virology vol 89 no 2 pp 1439ndash1444 2015
[116] P R Daniels C M Sanders P Coulson and N J MaitlandldquoMolecular analysis of the interaction between HPV type 16 E6and human E6-associated proteinrdquo FEBS Letters vol 416 no 1pp 6ndash10 1997
[117] W H Kao S L Beaudenon A L Talis J M Huibregtse andP M Howley ldquoHuman papillomavirus type 16 E6 induces self-ubiquitination of the E6AP ubiquitin-protein ligaserdquo Journal ofVirology vol 74 no 14 pp 6408ndash6417 2000
[118] T Iftner M Elbel B Schopp et al ldquoInterference of papillo-mavirus E6 protein with single-strand break repair by interac-tion with XRCC1rdquo The EMBO Journal vol 21 no 17 pp 4741ndash4748 2002
[119] Y Imai Y Tsunokawa T Sugimura and M Terada ldquoPurifi-cation and DNA-binding properties of human papillomavirustype 16 E6 protein expressed in Escherichia colirdquo Biochemicaland Biophysical Research Communications vol 164 no 3 pp1402ndash1410 1989
[120] M Lusky L Berg H Weiher and M Botchan ldquoBovinepapilloma virus contains an activator of gene expression at thedistal end of the early transcription unitrdquoMolecular andCellularBiology vol 3 no 6 pp 1108ndash1122 1983
[121] S Dalal Q Gao E J Androphy and V Band ldquoMutationalanalysis of human papillomavirus type 16 E6 demonstrates thatp53 degradation is necessary for immortalization of mammaryepithelial cellsrdquo Journal of Virology vol 70 no 2 pp 683ndash6881996
[122] J M Huibregtse M Scheffner and P M Howley ldquoA cellularprotein mediates association of p53 with the E6 oncoprotein ofhuman papillomavirus types 16 or 18rdquo The EMBO Journal vol10 no 13 pp 4129ndash4135 1991
[123] M Scheffner B A Werness J M Huibregtse A J Levine andP M Howley ldquoThe E6 oncoprotein encoded by human papillo-mavirus types 16 and 18 promotes the degradation of p53rdquo Cellvol 63 no 6 pp 1129ndash1136 1990
[124] M Elbel S Carl S Spaderna and T Iftner ldquoA comparativeanalysis of the interactions of the E6 proteins from cutaneousand genital papillomaviruses with p53 and E6AP in correlationto their transforming potentialrdquoVirology vol 239 no 1 pp 132ndash149 1997
[125] M C Thomas and C-M Chiang ldquoE6 oncoprotein repressesp53-dependent gene activation via inhibition of protein acety-lation independently of inducing p53 degradationrdquo MolecularCell vol 17 no 2 pp 251ndash264 2005
[126] H Zimmermann R Degenkolbe H-U Bernard and M JOrsquoConnor ldquoThe human papillomavirus type 16 E6 oncoproteincan down-regulate p53 activity by targeting the transcriptionalcoactivator CBPp300rdquo Journal of Virology vol 73 no 8 pp6209ndash6219 1999
[127] D A Thompson G Belinsky T H-T Chang D L Jones RSchlegel and K Munger ldquoThe human papillomavirus-16 E6oncoprotein decreases the vigilance of mitotic checkpointsrdquoOncogene vol 15 no 25 pp 3025ndash3035 1997
[128] J Bohl K Das B Dasgupta and S B Vande Pol ldquoCompetitivebinding to a charged leucine motif represses transformation bya papillomavirus E6 oncoproteinrdquo Virology vol 271 no 1 pp163ndash170 2000
[129] S Song H C Pitot and P F Lambert ldquoThe human papillo-mavirus type 16 E6 gene alone is sufficient to induce carcinomasin transgenic animalsrdquo Journal of Virology vol 73 no 7 pp5887ndash5893 1999
[130] S Yasumoto A L Burkhardt J Doniger and J A DiPaololdquoHumanpapillomavirus type 16DNA-inducedmalignant trans-formation of NIH 3T3 cellsrdquo Journal of Virology vol 57 no 2pp 572ndash577 1986
[131] S B Vande Pol M C Brown and C E Turner ldquoAssociation ofBovine Papillomavirus Type 1 E6 oncoprotein with the focaladhesion protein paxillin through a conserved protein interac-tion motifrdquo Oncogene vol 16 no 1 pp 43ndash52 1998
[132] R Wade N Brimer and S Vande Pol ldquoTransformation bybovine papillomavirus type 1 E6 requires paxillinrdquo Journal ofVirology vol 82 no 12 pp 5962ndash5966 2008
[133] A J Klingelhutz S A Foster and J KMcDougall ldquoTelomeraseactivation by the E6 gene product of humanpapillomavirus type16rdquo Nature vol 380 no 6569 pp 79ndash82 1996
[134] S T Oh S Kyo and L A Laimins ldquoTelomerase activation byhuman papillomavirus type 16 E6 protein Induction of humantelomerase reverse transcriptase expression through Myc andGC-rich Sp1 binding sitesrdquo Journal of Virology vol 75 no 12pp 5559ndash5566 2001
[135] L V Ronco A Y KarpovaMVidal and PMHowley ldquoHumanpapillomavirus 16 E6 oncoprotein binds to interferon regulatoryfactor-3 and inhibits its transcriptional activityrdquo Genes andDevelopment vol 12 no 13 pp 2061ndash2072 1998
Groups Rank differences Calculated 119885 Critical 119885 119901
Cminus and C+ 68 24042 2394 lt05Cminus and E6 82 28991 2394 lt05C+ and E6 14 04950 2394 nsns nonsignificant differences
difference between the negative control and the group treatedwith the BPV-1 E6 recombinant oncoprotein (Table 5) How-ever the test did not reveal differences between the cellstreated with cyclophosphamide and BPV-1 E6 recombinantoncoprotein (Table 5)These data reinforce those observed bythe Dunn post hoc test based on the frequency of micronu-cleus indicating the clastogenic action of the E6 oncoproteinMoreover the presence of anaphase bridge was alreadydescribed in cells transfected with recombinant adenoviruscontaining the E6 and E7 ORFs of HPV [29 30] Howeverthere are no studies describing the action of BPVE6 oncopro-tein in the induction of anaphase bridges or studies that showthis same action of E6 oncoprotein of HPV per se Anaphasebridges are indicators of genomic instability [82 83] which isconsidered the first step in carcinogenesis [46 47] Moreoverthe presence of these bridges is an important hallmark ofDNA double strand breaks (DSBs) [84]
Statistical analysis based on the cytokinesis-block prolif-eration index (CBPI) showed that BPV-1 E6 oncoprotein hascytotoxic levels similar to cyclophosphamide (Table 6) Thisresult suggests that the BPV E6 oncoprotein can deregulatethe cell cycle contributing to the cell proliferation andimmortalization To date this mitogenic action is attributedto the E7 oncoprotein [85ndash87] E7 oncoprotein induces
the phosphorylation of pRb resulting in the E2F transcrip-tion factor release [85] E2F when translocated to the nucleusacts as activator and binds to the kinase-dependent cyclinspromoters [85] However the results showed polynucleatedcells in the group treatedwithBPVE6oncoprotein (Figure 5)suggesting that the BPV E6 oncoprotein confers mitogenicstimulation resulting in mitotic stress Moreover studiespointed out that the BPV E6 oncoprotein interacts with theCBPp300 deacetylase promoting p53 downregulation [88]This downregulation increases the expression levels of FoxM1transcription factor (Foxhead box M1) [89] promoting B1cyclin D1 cyclin and cdc25 upregulation [89] The upregu-lation of these genes is associated with increased levels of cellproliferation which is necessary in order to make DNA poly-merases available to virus replication However if this mech-anism guarantees the BPV replication it can contribute to cellimmortalization and cancer progression
The FoxM1 factor also participates in the Wnt120573-cateninsignaling pathway and binds directly to the 120573-catenin [89]This interaction promotes the nuclear 120573-catenin transloca-tion [90] 120573-catenin translocation to nucleus induces thecyclin expression [64] So these data suggest that the BPVE6 oncoprotein not only promotes mitogenic stress but alsocontributes to the epithelial-mesenchymal transition (EMT)Studies based on theHPVE6 oncoprotein have indicated thatthis oncoprotein contributes significantly to the EMT [91]This occurs not only because the E6 oncoprotein induces thetranslocation of 120573-catenin but also due to the proteasomaldegradation of regulatory proteins of apical-basal polarity[92ndash94] Studies also showed that the E6 oncoprotein is ableto bind to Crumbs (Dlg and Patj) proteins resulting in loss ofcell polarity [93 94] Based on these results the E6 oncopro-tein emerges as a possible therapeutic target with biotechno-logical value in cancer treatment
Mitogenic action of E6 oncoprotein can be moreexpressed in cells infected by papillomaviruses once thesecells also expressed E5 and E7 oncoproteins [74] The E7oncoprotein is able to form a complex E7-p600 which pro-motes E6 upregulation [85] Furthermore the E6 oncoproteincan bind to the E6AP ubiquitin ligase forming the E6-E6APcomplex increasing the hTERT levels of expression con-tributing to cell immortalization [95] Moreover E6-E6APinduces NFX1 expression promoting MHC-II downregula-tion [96]Thismechanism reduces the antigenic presentationmediated by CD4+ T lymphocytes contributing to immuneevasion This action guarantees the PV infection for longperiods with or without clinical symptoms [24] Althoughthe BPV infection can be asymptomatic the BPV presence inperipheral blood is associated with DNA damage [1 14 24]
CBMNA analysis pointed out the presence of cells withintracellular cytokinesis (Figure 5) This result suggests theneotic action of BPV E6 oncoprotein Neosis is characterizedby the presence of (1) DNA damage (2) loss of checkpointcontrol (3) repair system failures and (4) endoreduplication[97] In this scenario the E6 oncoprotein attempts all theseneosis criteria Moreover the oncoprotein not only increasesthe cytogenetic damage leading to increasing of micronu-cleus frequency but also induces clastogenesis as shown bythe comet assay and anaphase bridges So statistical analysis
BioMed Research International 11
performed based on the comet scores reinforced the CBMNAresults Comet assay allows detecting both simple and dou-ble DNA breaks being more sensitive than the CBMNAClastogenesis is considered the most serious type of DNAdamage [98] DNA breaks can be repaired by homologous ornonhomologous recombination However studies point outthat the E6 oncoprotein can link to the Holliday junctionduring the homologous repair avoiding the junction reso-lution [99] Moreover E6 oncoprotein promotes the TP53gene deacetylation resulting in p53 downregulation [51]Thisepigenetic effect results in cell cycle deregulation affectingthe checkpoints
Studies have shown that the HPV-induced DNA breaksare required for the virus integration into the host genome[100] Although there are no studies showing the BPV inte-gration to date the results observed in this work in additionto those already published [24] indicate the necessity ofmorestudies to evaluate the virus-host interaction
Studies also showed that the E6 oncoprotein inducesclathrin-transporter adapter protein (AP-1) increasing thelevel of this protein in the plasma membrane [76 77] Theincreasing quantity of AP-1 in membrane can facilitate theBPV virions infection This occurs because the infectionprocess is clathrin dependent In this scenario the E6 onco-protein can contribute to the virus infection [101]
In summary the CBMNA and CA results showed thatthe BPV E6 oncoprotein participates not only in cancerpromotion but also in initiation inducing DNA breaksThese DNA breaks represent mutagenic effects consideredthe first step in the oncogenesis process which are associatedwith genomic instability [47 48 102 103] Thus the E6recombinant oncoprotein has been suggested as a possiblevaccine candidate [104ndash112] due to its immunogenicity [44]However the mutagenic tests such as CBMNA and CA arerequired in drug validation process [113] In this scenariothis study pointed out that the BPV-1 E6 recombinant onco-protein in tested concentration showedmutagenic potentialThis is the first study that reports the mutagenic potential ofa possible therapeutic vaccine candidate On the one handthese results allowed better understanding the mechanism ofcancer promotion associatedwith the BPVE6oncoprotein aswell as revealing that this oncoprotein can induce carcinogen-esis per se on the other hand this data pointed out thatmaybeBPV E6 recombinant oncoprotein requires protein modifica-tions to be used as vaccine
Conflict of Interests
The authors declare that there is no conflict of interests
Acknowledgments
Theauthors thank Fundacao deAmparo a Pesquisa do Estadode Sao Paulo (FAPESP Process 201420617-5) and FundacaoButantan for the financial support
References
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experimental modelrdquo Journal of General Virology vol 79 no 9pp 2127ndash2135 1998
[2] A Antonsson and B G Hansson ldquoHealthy skin ofmany animalspecies harbors papillomaviruses which are closely related totheir human counterpartsrdquo Journal of Virology vol 76 no 24pp 12537ndash12542 2002
[3] P Rous and J W Beard ldquoA virus-induced mammalian growthwith the characters of a tumor (the Shope rabbit papilloma) IThe growth on implantationwithin favorable hostsrdquoThe Journalof Experimental Medicine vol 60 no 6 pp 701ndash722 1934
[4] R Shope and E Hurst ldquoInfectious papillomatosis of rabbitsrdquoThe Journal of Experimental Medicine vol 58 no 1 pp 607ndash624 1933
[5] H Delius M A Van Ranst A B Jenson H Zur Hausen and JP Sundberg ldquoCanine oral papillomavirus genomic sequence aunique 15-kb intervening sequence between the E2 andL2 openreading framesrdquo Virology vol 204 no 1 pp 447ndash452 1994
[6] W A Demonbreun and E Goodpasture ldquoInfectious oral papil-lomatosis of dogsrdquo The American Journal of Pathology vol 8no 1 pp 43ndash56 1932
[7] P K Nicholls B A Klaunberg R A Moore et al ldquoNaturallyoccurring nonregressing canine oral papillomavirus infectionhost immunity virus characterization and experimental infec-tionrdquo Virology vol 265 no 2 pp 365ndash374 1999
[8] A Alberti S Pirino F Pintore et al ldquoOvis aries Papillomavirus3 a prototype of a novel genus in the family Papillomaviridaeassociated with ovine squamous cell carcinomardquo Virology vol407 no 2 pp 352ndash359 2010
[9] H zur Hausen E-M de Villiers and L Gissmann ldquoPapil-lomavirus infections and human genital cancerrdquo GynecologicOncology vol 12 no 2 pp S124ndashS128 1981
[10] C Foresta A Bertoldo A Garolla et al ldquoHuman papillo-mavirus proteins are found in peripheral blood and semenCd20+ and Cd56+ cells during Hpv-16 semen infectionrdquo BMCInfectious Diseases vol 13 article 593 2013
[11] S Ljubojevic andM Skerlev ldquoHPV-associated diseasesrdquo Clinicsin Dermatology vol 32 no 2 pp 227ndash234 2014
[12] A P R Cassel R B Barcellos C M D da Silva S E de MatosAlmeida and M L R Rossetti ldquoAssociation between humanpapillomavirus (HPV) DNA and micronuclei in normal cervi-cal cytologyrdquo Genetics and Molecular Biology vol 37 no 2 pp360ndash363 2014
[13] W Jarrett M Campo B OrsquoNeil H Laird and L Coggins ldquoAnovel bovine papillomavirus (BPV-6) causing true epithelialpapillomas of the mammary gland skin a member of a pro-posed newBPV subgrouprdquoVirology vol 136 no 2 pp 255ndash2641984
[14] T CMelo N Diniz S R C Campos et al ldquoCytogenetic studiesin peripheral blood of bovines afflicted by papillomatosisrdquoVeterinary and Comparative Oncology vol 9 no 4 pp 269ndash2742011
[15] S R C Campos T C Melo S Assaf et al ldquoChromosome aber-rations in cells infected with bovine papillomavirus comparingcutaneous papilloma esophagus papilloma and urinary blad-der lesion cellsrdquo ISRN Oncology vol 2013 Article ID 910849 8pages 2013
[16] H zur Hausen ldquoThe search for infectious causes of humancancers where and whyrdquoVirology vol 392 no 1 pp 1ndash10 2009
[17] R P Araldi R F Carvalho T C Melo et al ldquoBovine papillo-mavirus in beef cattle first description of BPV-12 and putativetype BAPV8 in BrazilrdquoGenetics andMolecular Research vol 13no 3 pp 5644ndash5653 2014
12 BioMed Research International
[18] T Rampias C Sasaki and A Psyrri ldquoMolecular mechanisms ofHPV induced carcinogenesis in head and neckrdquoOral Oncologyvol 50 no 5 pp 356ndash363 2014
[19] S Roperto V Russo L Leonardi et al ldquoBovine papillomavirustype 13 expression in the urothelial bladder tumours of cattlerdquoTransboundary and Emerging Diseases 2015
[20] S RCCampos C TrindadeO P Ferraz et al ldquoCan establishedcultured papilloma cells harbor bovine papillomavirusrdquoGenet-ics and Molecular Research vol 7 no 4 pp 1119ndash1126 2008
[21] M S Campo ldquoAnimalmodels of papilloma virus pathogenesisrdquoVirus Research vol 89 no 2 pp 249ndash261 2002
[22] G Borzacchiello ldquoBovine papillomavirus infections in ani-malsrdquo in Communicating Current Research and EducationalTopics and Trends in AppliedMicrobiology pp 673ndash679 Forma-tex Badajoz Spain 2007
[23] F Bocaneti G Altamura A Corteggio E Velescu F RopertoandG Borzacchiello ldquoBovine papillomavirus new insights intoan old diseaserdquo Transboundary and Emerging Diseases 2014
[24] R P Araldi T C Melo N Diniz et al ldquoBovine papillomavirusclastogenic effect analyzed in comet assayrdquo BioMed ResearchInternational vol 2013 Article ID 630683 7 pages 2013
[25] J Munday N Thomson M Dunowska C Knight R Laurieand S Hills ldquoGenomic characterisation of the feline sarcoid-associated papillomavirus and proposed classification as Bostaurus papillomavirus type 14rdquoVeterinaryMicrobiology vol 177no 3-4 pp 289ndash295 2015
[26] G Borzacchiello V Russo C Spoleto et al ldquoBovine papillo-mavirus type-2 DNA and expression of E5 and E7 oncoproteinsin vascular tumours of the urinary bladder in cattlerdquo CancerLetters vol 250 no 1 pp 82ndash91 2007
[27] P Maiolino A Ozkul A Sepici-Dincel et al ldquoBovine papillo-mavirus type 2 infection and microscopic patterns of urothelialtumors of the urinary bladder in water buffaloesrdquo BioMedResearch International vol 2013 Article ID 937918 6 pages2013
[28] S Roperto G Borzacchiello R Brun et al ldquoMultiple glomustumors of the urinary bladder in a cow associated with bovinepapillomavirus type 2 (BPV-2) infectionrdquo Veterinary Pathologyvol 45 no 1 pp 39ndash42 2008
[29] S Duensing and K Munger ldquoThe human papillomavirus type16 E6 and E7 oncoproteins independently induce numerical andstructural chromosome instabilityrdquoCancer Research vol 62 no23 pp 7075ndash7082 2002
[30] S Duensing L Y Lee A Duensing et al ldquoThe human papil-lomavirus type 16 E6 and E7 oncoproteins cooperate to inducemitotic defects and genomic instability by uncoupling centro-some duplication from the cell division cyclerdquo Proceedings of theNational Academy of Sciences of theUnited States of America vol97 no 18 pp 10002ndash10007 2000
[31] C A Moody and L A Laimins ldquoHuman papillomavirus onco-proteins pathways to transformationrdquo Nature Reviews Cancervol 10 no 8 pp 550ndash560 2010
[32] C Mansur and E Androphy ldquoCellular transformation bypapillomavirus oncoproteinsrdquo Biochimica et Biophysica Actavol 1155 no 3 pp 323ndash345 1993
[33] J J Chen Y Hong and E J Androphy ldquoMutational analysis oftranscriptional activation by the bovine papillomavirus type 1E6rdquo Virology vol 236 no 1 pp 30ndash36 1997
[34] X Tong andPMHowley ldquoThebovine papillomavirus E6 onco-protein interacts with paxillin and disrupts the actin cytoskele-tonrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 94 no 9 pp 4412ndash4417 1997
[35] X TongW Boll T Kirchhausen andPMHowley ldquoInteractionof the bovine papillomavirus E6 protein with the clathrinadaptor complex AP-1rdquo Journal of Virology vol 72 no 1 pp476ndash482 1998
[36] Y Nomine M Masson S Charbonnier et al ldquoStructural andfunctional analysis of E6 oncoprotein insights in the molecularpathways of human papillomavirus-mediated pathogenesisrdquoMolecular Cell vol 21 no 5 pp 665ndash678 2006
[37] T Ristriani MMasson Y Nomine et al ldquoHPV oncoprotein E6is a structure-dependent DNA-binding protein that recognizesfour-way junctionsrdquo Journal of Molecular Biology vol 296 no5 pp 1189ndash1203 2000
[38] Y Liu and J D Baleja ldquoStructure and function of the papil-lomavirus E6 protein and its interacting proteinsrdquo Frontiers inBioscience vol 13 no 1 pp 121ndash134 2008
[39] F Lipari G A McGibbon E Wardrop and M G CordingleyldquoPurification and biophysical characterization of a minimalfunctional domain and of an N-terminal Zn2+-binding frag-ment from the human papillomavirus type 16 E6 proteinrdquoBiochemistry vol 40 no 5 pp 1196ndash1204 2001
[40] Y Nomine T Ristriani C Laurent J-F Lefevre E Weissand G Trave ldquoA strategy for optimizing the monodispersity offusion proteins application to purification of recombinantHPVE6 oncoproteinrdquo Protein Engineering vol 14 no 4 pp 297ndash3052001
[41] C G Ullman P I Haris D A Galloway V C Emery and SJ Perkins ldquoPredicted 120572-helix120573-sheet secondary structures forthe zinc-binding motifs of human papillomavirus E7 and E6proteins by consensus prediction averaging and spectroscopicstudies of E7rdquo Biochemical Journal vol 319 no 1 pp 229ndash2391996
[42] A Sidi K Babah N Brimer et al ldquoStrategies for bacterialexpression of protein-peptide complex application to solubi-lization of papillomavirus E6rdquo Protein Expression and Purifica-tion vol 29 no 6 pp 997ndash1003 2011
[43] K Zanier S Charbonnier A O M O Sidi et al ldquoStructuralbasis for hijacking of cellular LxxLL motifs by papillomavirusE6 oncoproteinsrdquo Science vol 339 no 6120 pp 694ndash698 2013
[44] J Mazzuchelli-de-Souza R F Carvalho R M Ruiz et alldquoExpression and in silico analysis of the recombinant bovinepapillomavirus e6 protein as a model for viral oncoproteinsstudiesrdquo BioMed Research International vol 2013 Article ID421398 9 pages 2013
[45] L Banks P Spence E Androphy et al ldquoIdentification of humanpapillomavirus type 18 E6 polypeptide in cells derived fromhuman cervical carcinomasrdquo Journal of General Virology vol68 no 5 pp 1351ndash1359 1987
[46] D Hanahan and R AWeinberg ldquoHallmarks of cancer the nextgenerationrdquo Cell vol 144 no 5 pp 646ndash674 2011
[47] G S Charames and B Bapat ldquoGenomic instability and cancerrdquoCurrent Molecular Medicine vol 3 no 7 pp 589ndash596 2003
[48] S Negrini V G Gorgoulis and T D Halazonetis ldquoGenomicinstabilitymdashan evolving hallmark of cancerrdquo Nature ReviewsMolecular Cell Biology vol 11 no 3 pp 220ndash228 2010
[49] K Zanier S Charbonnier M Baltzinger Y Nomine DAltschuh and G Trave ldquoKinetic analysis of the interactionsof human papillomavirus E6 oncoproteins with the ubiquitinligase E6AP using surface plasmon resonancerdquo Journal ofMolecular Biology vol 349 no 2 pp 401ndash412 2005
BioMed Research International 13
[50] M Scheffner J M Huibregtse R D Vierstra and PM HowleyldquoThe HPV-16 E6 and E6-AP complex functions as a ubiquitin-protein ligase in the ubiquitination of p53rdquo Cell vol 75 no 3pp 495ndash505 1993
[51] H Zimmermann C-H Koh R Degenkolbe et al ldquoInteractionwith CBPp300 enables the bovine papillomavirus type 1 E6oncoprotein to downregulate CBPp300-mediated transactiva-tion by p53rdquo Journal of General Virology vol 81 no 11 pp 2617ndash2623 2000
[52] M Fenech ldquoThe in vitro micronucleus techniquerdquo MutationResearchmdashFundamental and Molecular Mechanisms of Mutage-nesis vol 455 no 1-2 pp 81ndash95 2000
[53] R P Araldi T C de Melo T B Mendes et al ldquoUsing the cometand micronucleus assays for genotoxicity studies a reviewrdquoBiomedicine amp Pharmacotherapy vol 72 pp 74ndash82 2015
[54] P Thomas and M Fenech ldquoCytokinesis-block micronucleuscytome assay in lymphocytesrdquo Methods in Molecular Biologyvol 682 pp 217ndash234 2011
[55] M Kirsch-Volders T Sofuni M Aardema et al ldquoReport fromthe in vitro micronucleus assay working grouprdquo MutationResearch Genetic Toxicology and Environmental Mutagenesisvol 540 no 2 pp 153ndash163 2003
[56] S Samanta and P Dey ldquoMicronucleus and its applicationsrdquoDiagnostic Cytopathology vol 40 no 1 pp 84ndash90 2012
[57] M Hayashi J T MacGregor D G Gatehouse et al ldquoIn vivoerythrocyte micronucleus assay III Validation and regulatoryacceptance of automated scoring and the use of rat periph-eral blood reticulocytes with discussion of non-hematopoietictarget cells and a single dose-level limit testrdquo MutationResearchGenetic Toxicology and Environmental Mutagenesisvol 627 no 1 pp 10ndash30 2007
[58] A Azqueta and A R Collins ldquoThe essential comet assay acomprehensive guide to measuring DNA damage and repairrdquoArchives of Toxicology vol 87 no 6 pp 949ndash968 2013
[59] N P Singh M T McCoy R R Tice and E L Schneider ldquoAsimple technique for quantitation of low levels of DNA damagein individual cellsrdquo Experimental Cell Research vol 175 no 1pp 184ndash191 1988
[60] J H Calinisan S R Chan A King and P J Chan ldquoHumanpapillomavirus and blastocyst apoptosisrdquo Journal of AssistedReproduction and Genetics vol 19 no 3 pp 132ndash136 2002
[61] R Oliveira Studies on the biopathological actions of Pteridiumaquilinum [PhD thesis] nstituto de Ciencias Biomedicas AbelSalazar 2012
[62] D M Potter and M S Baird ldquoCarcinogenic effects of ptaquilo-side in bracken fern and related compoundsrdquo British Journal ofCancer vol 83 no 7 pp 914ndash920 2000
[63] R B Lucena D R Rissi G D Kommers et al ldquoA retrospectivestudy of 586 tumours in Brazilian cattlerdquo Journal of ComparativePathology vol 145 no 1 pp 20ndash24 2011
[64] A Yaguiu M L Z Dagli E H Birgel Jr et al ldquoSimultaneouspresence of bovine papillomavirus and bovine leukemia virusin different bovine tissues in situ hybridization and cytogeneticanalysisrdquoGenetics andMolecular Research vol 7 no 2 pp 487ndash497 2008
[65] T C Melo R F Carvalho J Mazzucchelli-de-Souza et alldquoPhylogenetic classification and clinical aspects of a new puta-tive Deltapapillomavirus associated with skin lesions in cattlerdquoGenetics and Molecular Research vol 13 no 2 pp 2458ndash24692014
[66] R P Araldi D N S Giovanni T CMelo et al ldquoBovine papillo-mavirus isolation by ultracentrifugationrdquo Journal of VirologicalMethods vol 208 pp 119ndash124 2014
[67] L M Chandrachud B W OrsquoNeil W F H Jarrett G J Grind-lay G M McGarvie and M S Campo ldquoHumoral immuneresponse to the E7 protein of bovine papillomavirus type 4 andidentification of B-cell epitopesrdquoVirology vol 200 no 1 pp 98ndash104 1994
[68] M Ayres M J Ayres D L Ayres and A A Santos BioEstatmdashAplicacoes Estatısticas nas Areas de Ciencias Bio-Medicas Insti-tuto Mamiraua Belem Brazil 2007
[69] K Das J Bohl and S B Vande Pol ldquoIdentification of a secondtransforming function in bovine papillomavirus type 1 E6 andthe role of E6 interactions with paxillin E6BP and E6APrdquoJournal of Virology vol 74 no 2 pp 812ndash816 2000
[70] O Danos E Georges G Orth and M Yaniv ldquoFine structureof the cottontail rabbit papillomavirus mRNAs expressed in thetransplantable VX2 carcinomardquo Journal of Virology vol 53 no3 pp 735ndash741 1985
[71] M Nasseri and F O Wettstein ldquoDifferences exist between viraltranscripts in cottontail rabbit papillomavirus-induced benignand malignant tumors as well as non-virus-producing andvirus-producing tumorsrdquo Journal of Virology vol 51 no 3 pp706ndash712 1984
[72] X Tong R Salgia J-L Li J D Griffin and P M Howley ldquoThebovine papillomavirus E6 protein binds to the LDmotif repeatsof paxillin and blocks its interaction with vinculin and the focaladhesion kinaserdquo Journal of Biological Chemistry vol 272 no52 pp 33373ndash33376 1997
[73] Y Maeda T Shibahara Y Wada et al ldquoAn outbreak of teatpapillomatosis in cattle caused by bovine papilloma virus (BPV)type 6 and unclassified BPVsrdquo Veterinary Microbiology vol 121no 3-4 pp 242ndash248 2007
[74] R P Araldi T C Melo A Neves et al ldquoHyperproliferativeaction of bovine papillomavirus genetic and histopathologicalaspectsrdquo Genetics and Molecular Research vol 14 no 4 pp12942ndash12954 2015
[75] NDiniz T CMelo J F Santos et al ldquoSimultaneous presence ofbovine papillomavirus in blood and in short-term lymphocytecultures from dairy cattle in Pernambuco Brazilrdquo Genetics andMolecular Research vol 8 no 4 pp 1474ndash1480 2009
[76] O M Brito M F Guimaraes and C C Lanna ldquoCiclofosfamidae funcao ovarianardquo Revista Brasileira de Reumatologia vol 48no 1 pp 39ndash45 2008
[77] D Anderson T-W Yu and D B McGregor ldquoComet assayresponses as indicators of carcinogen exposurerdquo Mutagenesisvol 13 no 6 pp 539ndash555 1998
[78] M L Adam C Pini S Tulio J C L L Cantalice R A Torresand M T Dos Santos Correia ldquoAssessment of the associationbetween micronuclei and the degree of uterine lesions and viralload in women with human papillomavirusrdquo Cancer Genomicsamp Proteomics vol 12 no 2 pp 67ndash72 2015
[79] E Cortes-Gutierrez M Davila-Rodrıguez J Vargas-VillarrealF Hernandez-Garza and R Cerda-Flores ldquoAssociationbetween human papilloma virus-type infections with micro-nuclei frequenciesrdquo Prague Medical Report vol 111 no 1 pp35ndash41 2010
[80] R N Chakrabarti and K Dutta ldquoMicronuclei test in routinesmears fromuterine cervixrdquo European Journal of GynaecologicalOncology vol 9 no 5 pp 370ndash372 1988
[81] E M M Cerqueira C L Santoro N F Donozo et al ldquoGeneticdamage in exfoliated cells of the uterine cervix Association
14 BioMed Research International
and interaction between cigarette smoking and progression tomalignant transformationrdquo Acta Cytologica vol 42 no 3 pp639ndash649 1998
[82] S M Germann V Schramke R T Pedersen et al ldquoTopBP1Dpb11 bindsDNA anaphase bridges to prevent genome instabil-ityrdquoThe Journal of Cell Biology vol 204 no 1 pp 45ndash59 2014
[83] D R Hoffelder L Luo N A Burke S CWatkins S M Gollinand W S Saunders ldquoResolution of anaphase bridges in cancercellsrdquo Chromosoma vol 112 no 8 pp 389ndash397 2004
[84] C Acilan D M Potter and W S Saunders ldquoDNA repairpathways involved in anaphase bridge formationrdquo Genes Chro-mosomes and Cancer vol 46 no 6 pp 522ndash531 2007
[85] J DeMasi M C Chao A S Kumar and P M Howley ldquoBovinepapillomavirus E7 oncoprotein inhibits anoikisrdquo Journal ofVirology vol 81 no 17 pp 9419ndash9425 2007
[86] J DeMasi K-W Huh Y Nakatani K Munger and P MHowley ldquoBovine papillomavirus E7 transformation functioncorrelates with cellular p600 protein bindingrdquo Proceedings of theNational Academy of Sciences of theUnited States of America vol102 no 32 pp 11486ndash11491 2005
[87] E A White and P M Howley ldquoProteomic approaches to thestudy of papillomavirusndashhost interactionsrdquo Virology vol 435no 1 pp 57ndash69 2013
[88] FMantovani and L Banks ldquoThe human papillomavirus E6 pro-tein and its contribution to malignant progressionrdquo Oncogenevol 20 no 54 pp 7874ndash7887 2001
[89] P Chen and H Lee ldquoFOXM1 induced by E6 oncoprotein pro-motes tumor invasion and chemoresistance in HPV-infectedlung cancerrdquo Cancer Cell amp Microenvironment vol 2 no 1 pp6ndash9 2015
[90] N Zhang P Wei A Gong et al ldquoFoxM1 promotes 120573-cateninnuclear localization and controls Wnt target-gene expressionand glioma tumorigenesisrdquo Cancer Cell vol 127 no 4 pp 358ndash366 2012
[91] Z Liu Y Liu Y Hong L Rapp E J Androphy and J JChen ldquoBovine papillomavirus type 1 E6-induced sensitizationto apoptosis is distinct from its transforming activityrdquo Virologyvol 295 no 2 pp 230ndash237 2002
[92] M Thomas P Massimi C Navarro J-P Borg and L BanksldquoThe hScribDlg apico-basal control complex is differentiallytargeted by HPV-16 and HPV-18 E6 proteinsrdquo Oncogene vol24 no 41 pp 6222ndash6230 2005
[93] C H Storrs and S J Silverstein ldquoPATJ a tight junction-associated PDZ protein is a novel degradation target of high-risk human papillomavirus E6 and the alternatively splicedisoform 18 E6rdquo Journal of Virology vol 81 no 8 pp 4080ndash40902007
[94] R Accardi R Rubino M Scalise et al ldquoE6 and E7 from humanpapillomavirus type 16 cooperate to target the PDZ proteinNaH exchange regulatory factor 1rdquo Journal of Virology vol 85no 16 pp 8208ndash8216 2011
[95] S Nakagawa and J M Huibregtse ldquoHuman scribble (Vartul)is targeted for ubiquitin-mediated degradation by the high-risk papillomavirus E6 proteins and the E6APubiquitin-proteinligaserdquoMolecular and Cellular Biology vol 20 no 21 pp 8244ndash8253 2000
[96] L Gewin H Myers T Kiyono and D A Galloway ldquoIdentifi-cation of a novel telomerase repressor that interacts with thehuman papillomavirus type-16 E6E6-AP complexrdquo Genes ampDevelopment vol 18 no 18 pp 2269ndash2282 2004
[97] M Sundaram D L GuernseyMM Rajaraman and R Rajara-man ldquoNeosis a novel type of cell division in cancerrdquo CancerBiology ampTherapy vol 3 no 2 pp 207ndash218 2004
[98] R D Snyder and J W Green ldquoA review of the genotoxicity ofmarketed pharmaceuticalsrdquo Mutation Research vol 488 no 2pp 151ndash169 2001
[99] T Ristriani Y Nomine C Laurent E Weiss and G TraveldquoProtein mutagenesis with monodispersity-based quality prob-ing selective inactivation of p53 degradation and DNA-bindingproperties of HPV E6 oncoproteinrdquo Protein Expression andPurification vol 26 no 3 pp 357ndash367 2002
[100] M Kadaja A Sumerina T Verst M Ojarand E Ustav and MUstav ldquoGenomic instability of the host cell induced by thehuman papillomavirus replicationmachineryrdquoTheEMBO Jour-nal vol 26 no 8 pp 2180ndash2191 2007
[101] PMDay D R Lowy and J T Schiller ldquoPapillomaviruses infectcells via a clathrin-dependent pathwayrdquo Virology vol 307 no 1pp 1ndash11 2003
[102] T D Tlsty A Briot A Gualberto et al ldquoGenomic instabilityand cancerrdquoMutation Research vol 337 no 1 pp 1ndash7 1995
[103] J E Eyfjord and S K Bodvarsdottir ldquoGenomic instabilityand cancer networks involved in response to DNA damagerdquoMutation Research vol 592 no 1-2 pp 18ndash28 2005
[104] Y Yao W Huang X Yang et al ldquoHPV-16 E6 and E7 protein Tcell epitopes prediction analysis based on distributions of HLA-A loci across populations an in silico approachrdquo Vaccine vol31 no 18 pp 2289ndash2294 2013
[105] C-F Hung B Ma A Monie S-W Tsen and T-C Wu ldquoTher-apeutic human papillomavirus vaccines current clinical trialsand future directionsrdquo Expert Opinion on Biological Therapyvol 8 no 4 pp 421ndash439 2008
[106] M J P Welters G G Kenter S J Piersma et al ldquoInductionof tumor-specific CD4+ and CD8+ T-cell immunity in cervicalcancer patients by a human papillomavirus type 16 E6 and E7long peptides vaccinerdquo Clinical Cancer Research vol 14 no 1pp 178ndash187 2008
[107] R Han N M Cladel C A Reed X Peng and N D Chris-tensen ldquoProtection of rabbits from viral challenge by gene gun-based intracutaneous vaccination with a combination of cot-tontail rabbit papillomavirus E1 E2 E6 and E7 genesrdquo Journalof Virology vol 73 no 8 pp 7039ndash7043 1999
[108] Z He A P Wlazlo D W Kowalczyk et al ldquoViral recombinantvaccines to the E6 and E7 antigens of HPV-16rdquo Virology vol270 no 1 pp 146ndash161 2000
[109] A Kaufmann P Stern E Rankin et al ldquoSafety and immuno-genicity of TA-HPV a recombinant vaccinia virus expressingmodified human papillomavirus (HPV)-16 and HPV-18 E6 andE7 genes in women with progressive cervical cancerrdquo ClinicalCancer Research vol 8 no 12 pp 3676ndash3685 2002
[110] L K Borysiewicz A Fiander M Nimako et al ldquoA recombinantvaccinia virus encoding human papillomavirus types 16 and 18E6 and E7 proteins as immunotherapy for cervical cancerrdquoTheLancet vol 347 no 9014 pp 1523ndash1527 1996
[111] A De Jong T OrsquoNeill A Y Khan et al ldquoEnhancement ofhuman papillomavirus (HPV) type 16 E6 and E7-specific T-cellimmunity in healthy volunteers through vaccination with TA-CIN an HPV16 L2E7E6 fusion protein vaccinerdquo Vaccine vol20 no 29-30 pp 3456ndash3464 2002
[112] S Chen C Liao Y Lai et al ldquoDe-oncogenicHPVE6E7 vaccinegets enhanced antigenicity and promotes tumoricidal synergywith cisplatinrdquo Acta Biochimica et Biophysica Sinica vol 46 no1 pp 6ndash14 2014
BioMed Research International 15
[113] J Bonhoeffer E B Imoukhuede G Aldrovandi et al ldquoTemplateprotocol for clinical trials investigating vaccines-focus on safetyelementsrdquo Vaccine vol 31 no 47 pp 5602ndash5620 2013
[114] S Jackson and A Storey ldquoE6 proteins from diverse cutaneonsHPV types inhibit apoptosis in response to UV damagerdquoOncogene vol 19 no 4 pp 592ndash598 2000
[115] M Thomas and L Banks ldquoPDZRN3LNX3 is a novel targetof human papillomavirus type 16 (HPV-16) and HPV-18 E6rdquoJournal of Virology vol 89 no 2 pp 1439ndash1444 2015
[116] P R Daniels C M Sanders P Coulson and N J MaitlandldquoMolecular analysis of the interaction between HPV type 16 E6and human E6-associated proteinrdquo FEBS Letters vol 416 no 1pp 6ndash10 1997
[117] W H Kao S L Beaudenon A L Talis J M Huibregtse andP M Howley ldquoHuman papillomavirus type 16 E6 induces self-ubiquitination of the E6AP ubiquitin-protein ligaserdquo Journal ofVirology vol 74 no 14 pp 6408ndash6417 2000
[118] T Iftner M Elbel B Schopp et al ldquoInterference of papillo-mavirus E6 protein with single-strand break repair by interac-tion with XRCC1rdquo The EMBO Journal vol 21 no 17 pp 4741ndash4748 2002
[119] Y Imai Y Tsunokawa T Sugimura and M Terada ldquoPurifi-cation and DNA-binding properties of human papillomavirustype 16 E6 protein expressed in Escherichia colirdquo Biochemicaland Biophysical Research Communications vol 164 no 3 pp1402ndash1410 1989
[120] M Lusky L Berg H Weiher and M Botchan ldquoBovinepapilloma virus contains an activator of gene expression at thedistal end of the early transcription unitrdquoMolecular andCellularBiology vol 3 no 6 pp 1108ndash1122 1983
[121] S Dalal Q Gao E J Androphy and V Band ldquoMutationalanalysis of human papillomavirus type 16 E6 demonstrates thatp53 degradation is necessary for immortalization of mammaryepithelial cellsrdquo Journal of Virology vol 70 no 2 pp 683ndash6881996
[122] J M Huibregtse M Scheffner and P M Howley ldquoA cellularprotein mediates association of p53 with the E6 oncoprotein ofhuman papillomavirus types 16 or 18rdquo The EMBO Journal vol10 no 13 pp 4129ndash4135 1991
[123] M Scheffner B A Werness J M Huibregtse A J Levine andP M Howley ldquoThe E6 oncoprotein encoded by human papillo-mavirus types 16 and 18 promotes the degradation of p53rdquo Cellvol 63 no 6 pp 1129ndash1136 1990
[124] M Elbel S Carl S Spaderna and T Iftner ldquoA comparativeanalysis of the interactions of the E6 proteins from cutaneousand genital papillomaviruses with p53 and E6AP in correlationto their transforming potentialrdquoVirology vol 239 no 1 pp 132ndash149 1997
[125] M C Thomas and C-M Chiang ldquoE6 oncoprotein repressesp53-dependent gene activation via inhibition of protein acety-lation independently of inducing p53 degradationrdquo MolecularCell vol 17 no 2 pp 251ndash264 2005
[126] H Zimmermann R Degenkolbe H-U Bernard and M JOrsquoConnor ldquoThe human papillomavirus type 16 E6 oncoproteincan down-regulate p53 activity by targeting the transcriptionalcoactivator CBPp300rdquo Journal of Virology vol 73 no 8 pp6209ndash6219 1999
[127] D A Thompson G Belinsky T H-T Chang D L Jones RSchlegel and K Munger ldquoThe human papillomavirus-16 E6oncoprotein decreases the vigilance of mitotic checkpointsrdquoOncogene vol 15 no 25 pp 3025ndash3035 1997
[128] J Bohl K Das B Dasgupta and S B Vande Pol ldquoCompetitivebinding to a charged leucine motif represses transformation bya papillomavirus E6 oncoproteinrdquo Virology vol 271 no 1 pp163ndash170 2000
[129] S Song H C Pitot and P F Lambert ldquoThe human papillo-mavirus type 16 E6 gene alone is sufficient to induce carcinomasin transgenic animalsrdquo Journal of Virology vol 73 no 7 pp5887ndash5893 1999
[130] S Yasumoto A L Burkhardt J Doniger and J A DiPaololdquoHumanpapillomavirus type 16DNA-inducedmalignant trans-formation of NIH 3T3 cellsrdquo Journal of Virology vol 57 no 2pp 572ndash577 1986
[131] S B Vande Pol M C Brown and C E Turner ldquoAssociation ofBovine Papillomavirus Type 1 E6 oncoprotein with the focaladhesion protein paxillin through a conserved protein interac-tion motifrdquo Oncogene vol 16 no 1 pp 43ndash52 1998
[132] R Wade N Brimer and S Vande Pol ldquoTransformation bybovine papillomavirus type 1 E6 requires paxillinrdquo Journal ofVirology vol 82 no 12 pp 5962ndash5966 2008
[133] A J Klingelhutz S A Foster and J KMcDougall ldquoTelomeraseactivation by the E6 gene product of humanpapillomavirus type16rdquo Nature vol 380 no 6569 pp 79ndash82 1996
[134] S T Oh S Kyo and L A Laimins ldquoTelomerase activation byhuman papillomavirus type 16 E6 protein Induction of humantelomerase reverse transcriptase expression through Myc andGC-rich Sp1 binding sitesrdquo Journal of Virology vol 75 no 12pp 5559ndash5566 2001
[135] L V Ronco A Y KarpovaMVidal and PMHowley ldquoHumanpapillomavirus 16 E6 oncoprotein binds to interferon regulatoryfactor-3 and inhibits its transcriptional activityrdquo Genes andDevelopment vol 12 no 13 pp 2061ndash2072 1998
performed based on the comet scores reinforced the CBMNAresults Comet assay allows detecting both simple and dou-ble DNA breaks being more sensitive than the CBMNAClastogenesis is considered the most serious type of DNAdamage [98] DNA breaks can be repaired by homologous ornonhomologous recombination However studies point outthat the E6 oncoprotein can link to the Holliday junctionduring the homologous repair avoiding the junction reso-lution [99] Moreover E6 oncoprotein promotes the TP53gene deacetylation resulting in p53 downregulation [51]Thisepigenetic effect results in cell cycle deregulation affectingthe checkpoints
Studies have shown that the HPV-induced DNA breaksare required for the virus integration into the host genome[100] Although there are no studies showing the BPV inte-gration to date the results observed in this work in additionto those already published [24] indicate the necessity ofmorestudies to evaluate the virus-host interaction
Studies also showed that the E6 oncoprotein inducesclathrin-transporter adapter protein (AP-1) increasing thelevel of this protein in the plasma membrane [76 77] Theincreasing quantity of AP-1 in membrane can facilitate theBPV virions infection This occurs because the infectionprocess is clathrin dependent In this scenario the E6 onco-protein can contribute to the virus infection [101]
In summary the CBMNA and CA results showed thatthe BPV E6 oncoprotein participates not only in cancerpromotion but also in initiation inducing DNA breaksThese DNA breaks represent mutagenic effects consideredthe first step in the oncogenesis process which are associatedwith genomic instability [47 48 102 103] Thus the E6recombinant oncoprotein has been suggested as a possiblevaccine candidate [104ndash112] due to its immunogenicity [44]However the mutagenic tests such as CBMNA and CA arerequired in drug validation process [113] In this scenariothis study pointed out that the BPV-1 E6 recombinant onco-protein in tested concentration showedmutagenic potentialThis is the first study that reports the mutagenic potential ofa possible therapeutic vaccine candidate On the one handthese results allowed better understanding the mechanism ofcancer promotion associatedwith the BPVE6oncoprotein aswell as revealing that this oncoprotein can induce carcinogen-esis per se on the other hand this data pointed out thatmaybeBPV E6 recombinant oncoprotein requires protein modifica-tions to be used as vaccine
Conflict of Interests
The authors declare that there is no conflict of interests
Acknowledgments
Theauthors thank Fundacao deAmparo a Pesquisa do Estadode Sao Paulo (FAPESP Process 201420617-5) and FundacaoButantan for the financial support
References
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experimental modelrdquo Journal of General Virology vol 79 no 9pp 2127ndash2135 1998
[2] A Antonsson and B G Hansson ldquoHealthy skin ofmany animalspecies harbors papillomaviruses which are closely related totheir human counterpartsrdquo Journal of Virology vol 76 no 24pp 12537ndash12542 2002
[3] P Rous and J W Beard ldquoA virus-induced mammalian growthwith the characters of a tumor (the Shope rabbit papilloma) IThe growth on implantationwithin favorable hostsrdquoThe Journalof Experimental Medicine vol 60 no 6 pp 701ndash722 1934
[4] R Shope and E Hurst ldquoInfectious papillomatosis of rabbitsrdquoThe Journal of Experimental Medicine vol 58 no 1 pp 607ndash624 1933
[5] H Delius M A Van Ranst A B Jenson H Zur Hausen and JP Sundberg ldquoCanine oral papillomavirus genomic sequence aunique 15-kb intervening sequence between the E2 andL2 openreading framesrdquo Virology vol 204 no 1 pp 447ndash452 1994
[6] W A Demonbreun and E Goodpasture ldquoInfectious oral papil-lomatosis of dogsrdquo The American Journal of Pathology vol 8no 1 pp 43ndash56 1932
[7] P K Nicholls B A Klaunberg R A Moore et al ldquoNaturallyoccurring nonregressing canine oral papillomavirus infectionhost immunity virus characterization and experimental infec-tionrdquo Virology vol 265 no 2 pp 365ndash374 1999
[8] A Alberti S Pirino F Pintore et al ldquoOvis aries Papillomavirus3 a prototype of a novel genus in the family Papillomaviridaeassociated with ovine squamous cell carcinomardquo Virology vol407 no 2 pp 352ndash359 2010
[9] H zur Hausen E-M de Villiers and L Gissmann ldquoPapil-lomavirus infections and human genital cancerrdquo GynecologicOncology vol 12 no 2 pp S124ndashS128 1981
[10] C Foresta A Bertoldo A Garolla et al ldquoHuman papillo-mavirus proteins are found in peripheral blood and semenCd20+ and Cd56+ cells during Hpv-16 semen infectionrdquo BMCInfectious Diseases vol 13 article 593 2013
[11] S Ljubojevic andM Skerlev ldquoHPV-associated diseasesrdquo Clinicsin Dermatology vol 32 no 2 pp 227ndash234 2014
[12] A P R Cassel R B Barcellos C M D da Silva S E de MatosAlmeida and M L R Rossetti ldquoAssociation between humanpapillomavirus (HPV) DNA and micronuclei in normal cervi-cal cytologyrdquo Genetics and Molecular Biology vol 37 no 2 pp360ndash363 2014
[13] W Jarrett M Campo B OrsquoNeil H Laird and L Coggins ldquoAnovel bovine papillomavirus (BPV-6) causing true epithelialpapillomas of the mammary gland skin a member of a pro-posed newBPV subgrouprdquoVirology vol 136 no 2 pp 255ndash2641984
[14] T CMelo N Diniz S R C Campos et al ldquoCytogenetic studiesin peripheral blood of bovines afflicted by papillomatosisrdquoVeterinary and Comparative Oncology vol 9 no 4 pp 269ndash2742011
[15] S R C Campos T C Melo S Assaf et al ldquoChromosome aber-rations in cells infected with bovine papillomavirus comparingcutaneous papilloma esophagus papilloma and urinary blad-der lesion cellsrdquo ISRN Oncology vol 2013 Article ID 910849 8pages 2013
[16] H zur Hausen ldquoThe search for infectious causes of humancancers where and whyrdquoVirology vol 392 no 1 pp 1ndash10 2009
[17] R P Araldi R F Carvalho T C Melo et al ldquoBovine papillo-mavirus in beef cattle first description of BPV-12 and putativetype BAPV8 in BrazilrdquoGenetics andMolecular Research vol 13no 3 pp 5644ndash5653 2014
12 BioMed Research International
[18] T Rampias C Sasaki and A Psyrri ldquoMolecular mechanisms ofHPV induced carcinogenesis in head and neckrdquoOral Oncologyvol 50 no 5 pp 356ndash363 2014
[19] S Roperto V Russo L Leonardi et al ldquoBovine papillomavirustype 13 expression in the urothelial bladder tumours of cattlerdquoTransboundary and Emerging Diseases 2015
[20] S RCCampos C TrindadeO P Ferraz et al ldquoCan establishedcultured papilloma cells harbor bovine papillomavirusrdquoGenet-ics and Molecular Research vol 7 no 4 pp 1119ndash1126 2008
[21] M S Campo ldquoAnimalmodels of papilloma virus pathogenesisrdquoVirus Research vol 89 no 2 pp 249ndash261 2002
[22] G Borzacchiello ldquoBovine papillomavirus infections in ani-malsrdquo in Communicating Current Research and EducationalTopics and Trends in AppliedMicrobiology pp 673ndash679 Forma-tex Badajoz Spain 2007
[23] F Bocaneti G Altamura A Corteggio E Velescu F RopertoandG Borzacchiello ldquoBovine papillomavirus new insights intoan old diseaserdquo Transboundary and Emerging Diseases 2014
[24] R P Araldi T C Melo N Diniz et al ldquoBovine papillomavirusclastogenic effect analyzed in comet assayrdquo BioMed ResearchInternational vol 2013 Article ID 630683 7 pages 2013
[25] J Munday N Thomson M Dunowska C Knight R Laurieand S Hills ldquoGenomic characterisation of the feline sarcoid-associated papillomavirus and proposed classification as Bostaurus papillomavirus type 14rdquoVeterinaryMicrobiology vol 177no 3-4 pp 289ndash295 2015
[26] G Borzacchiello V Russo C Spoleto et al ldquoBovine papillo-mavirus type-2 DNA and expression of E5 and E7 oncoproteinsin vascular tumours of the urinary bladder in cattlerdquo CancerLetters vol 250 no 1 pp 82ndash91 2007
[27] P Maiolino A Ozkul A Sepici-Dincel et al ldquoBovine papillo-mavirus type 2 infection and microscopic patterns of urothelialtumors of the urinary bladder in water buffaloesrdquo BioMedResearch International vol 2013 Article ID 937918 6 pages2013
[28] S Roperto G Borzacchiello R Brun et al ldquoMultiple glomustumors of the urinary bladder in a cow associated with bovinepapillomavirus type 2 (BPV-2) infectionrdquo Veterinary Pathologyvol 45 no 1 pp 39ndash42 2008
[29] S Duensing and K Munger ldquoThe human papillomavirus type16 E6 and E7 oncoproteins independently induce numerical andstructural chromosome instabilityrdquoCancer Research vol 62 no23 pp 7075ndash7082 2002
[30] S Duensing L Y Lee A Duensing et al ldquoThe human papil-lomavirus type 16 E6 and E7 oncoproteins cooperate to inducemitotic defects and genomic instability by uncoupling centro-some duplication from the cell division cyclerdquo Proceedings of theNational Academy of Sciences of theUnited States of America vol97 no 18 pp 10002ndash10007 2000
[31] C A Moody and L A Laimins ldquoHuman papillomavirus onco-proteins pathways to transformationrdquo Nature Reviews Cancervol 10 no 8 pp 550ndash560 2010
[32] C Mansur and E Androphy ldquoCellular transformation bypapillomavirus oncoproteinsrdquo Biochimica et Biophysica Actavol 1155 no 3 pp 323ndash345 1993
[33] J J Chen Y Hong and E J Androphy ldquoMutational analysis oftranscriptional activation by the bovine papillomavirus type 1E6rdquo Virology vol 236 no 1 pp 30ndash36 1997
[34] X Tong andPMHowley ldquoThebovine papillomavirus E6 onco-protein interacts with paxillin and disrupts the actin cytoskele-tonrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 94 no 9 pp 4412ndash4417 1997
[35] X TongW Boll T Kirchhausen andPMHowley ldquoInteractionof the bovine papillomavirus E6 protein with the clathrinadaptor complex AP-1rdquo Journal of Virology vol 72 no 1 pp476ndash482 1998
[36] Y Nomine M Masson S Charbonnier et al ldquoStructural andfunctional analysis of E6 oncoprotein insights in the molecularpathways of human papillomavirus-mediated pathogenesisrdquoMolecular Cell vol 21 no 5 pp 665ndash678 2006
[37] T Ristriani MMasson Y Nomine et al ldquoHPV oncoprotein E6is a structure-dependent DNA-binding protein that recognizesfour-way junctionsrdquo Journal of Molecular Biology vol 296 no5 pp 1189ndash1203 2000
[38] Y Liu and J D Baleja ldquoStructure and function of the papil-lomavirus E6 protein and its interacting proteinsrdquo Frontiers inBioscience vol 13 no 1 pp 121ndash134 2008
[39] F Lipari G A McGibbon E Wardrop and M G CordingleyldquoPurification and biophysical characterization of a minimalfunctional domain and of an N-terminal Zn2+-binding frag-ment from the human papillomavirus type 16 E6 proteinrdquoBiochemistry vol 40 no 5 pp 1196ndash1204 2001
[40] Y Nomine T Ristriani C Laurent J-F Lefevre E Weissand G Trave ldquoA strategy for optimizing the monodispersity offusion proteins application to purification of recombinantHPVE6 oncoproteinrdquo Protein Engineering vol 14 no 4 pp 297ndash3052001
[41] C G Ullman P I Haris D A Galloway V C Emery and SJ Perkins ldquoPredicted 120572-helix120573-sheet secondary structures forthe zinc-binding motifs of human papillomavirus E7 and E6proteins by consensus prediction averaging and spectroscopicstudies of E7rdquo Biochemical Journal vol 319 no 1 pp 229ndash2391996
[42] A Sidi K Babah N Brimer et al ldquoStrategies for bacterialexpression of protein-peptide complex application to solubi-lization of papillomavirus E6rdquo Protein Expression and Purifica-tion vol 29 no 6 pp 997ndash1003 2011
[43] K Zanier S Charbonnier A O M O Sidi et al ldquoStructuralbasis for hijacking of cellular LxxLL motifs by papillomavirusE6 oncoproteinsrdquo Science vol 339 no 6120 pp 694ndash698 2013
[44] J Mazzuchelli-de-Souza R F Carvalho R M Ruiz et alldquoExpression and in silico analysis of the recombinant bovinepapillomavirus e6 protein as a model for viral oncoproteinsstudiesrdquo BioMed Research International vol 2013 Article ID421398 9 pages 2013
[45] L Banks P Spence E Androphy et al ldquoIdentification of humanpapillomavirus type 18 E6 polypeptide in cells derived fromhuman cervical carcinomasrdquo Journal of General Virology vol68 no 5 pp 1351ndash1359 1987
[46] D Hanahan and R AWeinberg ldquoHallmarks of cancer the nextgenerationrdquo Cell vol 144 no 5 pp 646ndash674 2011
[47] G S Charames and B Bapat ldquoGenomic instability and cancerrdquoCurrent Molecular Medicine vol 3 no 7 pp 589ndash596 2003
[48] S Negrini V G Gorgoulis and T D Halazonetis ldquoGenomicinstabilitymdashan evolving hallmark of cancerrdquo Nature ReviewsMolecular Cell Biology vol 11 no 3 pp 220ndash228 2010
[49] K Zanier S Charbonnier M Baltzinger Y Nomine DAltschuh and G Trave ldquoKinetic analysis of the interactionsof human papillomavirus E6 oncoproteins with the ubiquitinligase E6AP using surface plasmon resonancerdquo Journal ofMolecular Biology vol 349 no 2 pp 401ndash412 2005
BioMed Research International 13
[50] M Scheffner J M Huibregtse R D Vierstra and PM HowleyldquoThe HPV-16 E6 and E6-AP complex functions as a ubiquitin-protein ligase in the ubiquitination of p53rdquo Cell vol 75 no 3pp 495ndash505 1993
[51] H Zimmermann C-H Koh R Degenkolbe et al ldquoInteractionwith CBPp300 enables the bovine papillomavirus type 1 E6oncoprotein to downregulate CBPp300-mediated transactiva-tion by p53rdquo Journal of General Virology vol 81 no 11 pp 2617ndash2623 2000
[52] M Fenech ldquoThe in vitro micronucleus techniquerdquo MutationResearchmdashFundamental and Molecular Mechanisms of Mutage-nesis vol 455 no 1-2 pp 81ndash95 2000
[53] R P Araldi T C de Melo T B Mendes et al ldquoUsing the cometand micronucleus assays for genotoxicity studies a reviewrdquoBiomedicine amp Pharmacotherapy vol 72 pp 74ndash82 2015
[54] P Thomas and M Fenech ldquoCytokinesis-block micronucleuscytome assay in lymphocytesrdquo Methods in Molecular Biologyvol 682 pp 217ndash234 2011
[55] M Kirsch-Volders T Sofuni M Aardema et al ldquoReport fromthe in vitro micronucleus assay working grouprdquo MutationResearch Genetic Toxicology and Environmental Mutagenesisvol 540 no 2 pp 153ndash163 2003
[56] S Samanta and P Dey ldquoMicronucleus and its applicationsrdquoDiagnostic Cytopathology vol 40 no 1 pp 84ndash90 2012
[57] M Hayashi J T MacGregor D G Gatehouse et al ldquoIn vivoerythrocyte micronucleus assay III Validation and regulatoryacceptance of automated scoring and the use of rat periph-eral blood reticulocytes with discussion of non-hematopoietictarget cells and a single dose-level limit testrdquo MutationResearchGenetic Toxicology and Environmental Mutagenesisvol 627 no 1 pp 10ndash30 2007
[58] A Azqueta and A R Collins ldquoThe essential comet assay acomprehensive guide to measuring DNA damage and repairrdquoArchives of Toxicology vol 87 no 6 pp 949ndash968 2013
[59] N P Singh M T McCoy R R Tice and E L Schneider ldquoAsimple technique for quantitation of low levels of DNA damagein individual cellsrdquo Experimental Cell Research vol 175 no 1pp 184ndash191 1988
[60] J H Calinisan S R Chan A King and P J Chan ldquoHumanpapillomavirus and blastocyst apoptosisrdquo Journal of AssistedReproduction and Genetics vol 19 no 3 pp 132ndash136 2002
[61] R Oliveira Studies on the biopathological actions of Pteridiumaquilinum [PhD thesis] nstituto de Ciencias Biomedicas AbelSalazar 2012
[62] D M Potter and M S Baird ldquoCarcinogenic effects of ptaquilo-side in bracken fern and related compoundsrdquo British Journal ofCancer vol 83 no 7 pp 914ndash920 2000
[63] R B Lucena D R Rissi G D Kommers et al ldquoA retrospectivestudy of 586 tumours in Brazilian cattlerdquo Journal of ComparativePathology vol 145 no 1 pp 20ndash24 2011
[64] A Yaguiu M L Z Dagli E H Birgel Jr et al ldquoSimultaneouspresence of bovine papillomavirus and bovine leukemia virusin different bovine tissues in situ hybridization and cytogeneticanalysisrdquoGenetics andMolecular Research vol 7 no 2 pp 487ndash497 2008
[65] T C Melo R F Carvalho J Mazzucchelli-de-Souza et alldquoPhylogenetic classification and clinical aspects of a new puta-tive Deltapapillomavirus associated with skin lesions in cattlerdquoGenetics and Molecular Research vol 13 no 2 pp 2458ndash24692014
[66] R P Araldi D N S Giovanni T CMelo et al ldquoBovine papillo-mavirus isolation by ultracentrifugationrdquo Journal of VirologicalMethods vol 208 pp 119ndash124 2014
[67] L M Chandrachud B W OrsquoNeil W F H Jarrett G J Grind-lay G M McGarvie and M S Campo ldquoHumoral immuneresponse to the E7 protein of bovine papillomavirus type 4 andidentification of B-cell epitopesrdquoVirology vol 200 no 1 pp 98ndash104 1994
[68] M Ayres M J Ayres D L Ayres and A A Santos BioEstatmdashAplicacoes Estatısticas nas Areas de Ciencias Bio-Medicas Insti-tuto Mamiraua Belem Brazil 2007
[69] K Das J Bohl and S B Vande Pol ldquoIdentification of a secondtransforming function in bovine papillomavirus type 1 E6 andthe role of E6 interactions with paxillin E6BP and E6APrdquoJournal of Virology vol 74 no 2 pp 812ndash816 2000
[70] O Danos E Georges G Orth and M Yaniv ldquoFine structureof the cottontail rabbit papillomavirus mRNAs expressed in thetransplantable VX2 carcinomardquo Journal of Virology vol 53 no3 pp 735ndash741 1985
[71] M Nasseri and F O Wettstein ldquoDifferences exist between viraltranscripts in cottontail rabbit papillomavirus-induced benignand malignant tumors as well as non-virus-producing andvirus-producing tumorsrdquo Journal of Virology vol 51 no 3 pp706ndash712 1984
[72] X Tong R Salgia J-L Li J D Griffin and P M Howley ldquoThebovine papillomavirus E6 protein binds to the LDmotif repeatsof paxillin and blocks its interaction with vinculin and the focaladhesion kinaserdquo Journal of Biological Chemistry vol 272 no52 pp 33373ndash33376 1997
[73] Y Maeda T Shibahara Y Wada et al ldquoAn outbreak of teatpapillomatosis in cattle caused by bovine papilloma virus (BPV)type 6 and unclassified BPVsrdquo Veterinary Microbiology vol 121no 3-4 pp 242ndash248 2007
[74] R P Araldi T C Melo A Neves et al ldquoHyperproliferativeaction of bovine papillomavirus genetic and histopathologicalaspectsrdquo Genetics and Molecular Research vol 14 no 4 pp12942ndash12954 2015
[75] NDiniz T CMelo J F Santos et al ldquoSimultaneous presence ofbovine papillomavirus in blood and in short-term lymphocytecultures from dairy cattle in Pernambuco Brazilrdquo Genetics andMolecular Research vol 8 no 4 pp 1474ndash1480 2009
[76] O M Brito M F Guimaraes and C C Lanna ldquoCiclofosfamidae funcao ovarianardquo Revista Brasileira de Reumatologia vol 48no 1 pp 39ndash45 2008
[77] D Anderson T-W Yu and D B McGregor ldquoComet assayresponses as indicators of carcinogen exposurerdquo Mutagenesisvol 13 no 6 pp 539ndash555 1998
[78] M L Adam C Pini S Tulio J C L L Cantalice R A Torresand M T Dos Santos Correia ldquoAssessment of the associationbetween micronuclei and the degree of uterine lesions and viralload in women with human papillomavirusrdquo Cancer Genomicsamp Proteomics vol 12 no 2 pp 67ndash72 2015
[79] E Cortes-Gutierrez M Davila-Rodrıguez J Vargas-VillarrealF Hernandez-Garza and R Cerda-Flores ldquoAssociationbetween human papilloma virus-type infections with micro-nuclei frequenciesrdquo Prague Medical Report vol 111 no 1 pp35ndash41 2010
[80] R N Chakrabarti and K Dutta ldquoMicronuclei test in routinesmears fromuterine cervixrdquo European Journal of GynaecologicalOncology vol 9 no 5 pp 370ndash372 1988
[81] E M M Cerqueira C L Santoro N F Donozo et al ldquoGeneticdamage in exfoliated cells of the uterine cervix Association
14 BioMed Research International
and interaction between cigarette smoking and progression tomalignant transformationrdquo Acta Cytologica vol 42 no 3 pp639ndash649 1998
[82] S M Germann V Schramke R T Pedersen et al ldquoTopBP1Dpb11 bindsDNA anaphase bridges to prevent genome instabil-ityrdquoThe Journal of Cell Biology vol 204 no 1 pp 45ndash59 2014
[83] D R Hoffelder L Luo N A Burke S CWatkins S M Gollinand W S Saunders ldquoResolution of anaphase bridges in cancercellsrdquo Chromosoma vol 112 no 8 pp 389ndash397 2004
[84] C Acilan D M Potter and W S Saunders ldquoDNA repairpathways involved in anaphase bridge formationrdquo Genes Chro-mosomes and Cancer vol 46 no 6 pp 522ndash531 2007
[85] J DeMasi M C Chao A S Kumar and P M Howley ldquoBovinepapillomavirus E7 oncoprotein inhibits anoikisrdquo Journal ofVirology vol 81 no 17 pp 9419ndash9425 2007
[86] J DeMasi K-W Huh Y Nakatani K Munger and P MHowley ldquoBovine papillomavirus E7 transformation functioncorrelates with cellular p600 protein bindingrdquo Proceedings of theNational Academy of Sciences of theUnited States of America vol102 no 32 pp 11486ndash11491 2005
[87] E A White and P M Howley ldquoProteomic approaches to thestudy of papillomavirusndashhost interactionsrdquo Virology vol 435no 1 pp 57ndash69 2013
[88] FMantovani and L Banks ldquoThe human papillomavirus E6 pro-tein and its contribution to malignant progressionrdquo Oncogenevol 20 no 54 pp 7874ndash7887 2001
[89] P Chen and H Lee ldquoFOXM1 induced by E6 oncoprotein pro-motes tumor invasion and chemoresistance in HPV-infectedlung cancerrdquo Cancer Cell amp Microenvironment vol 2 no 1 pp6ndash9 2015
[90] N Zhang P Wei A Gong et al ldquoFoxM1 promotes 120573-cateninnuclear localization and controls Wnt target-gene expressionand glioma tumorigenesisrdquo Cancer Cell vol 127 no 4 pp 358ndash366 2012
[91] Z Liu Y Liu Y Hong L Rapp E J Androphy and J JChen ldquoBovine papillomavirus type 1 E6-induced sensitizationto apoptosis is distinct from its transforming activityrdquo Virologyvol 295 no 2 pp 230ndash237 2002
[92] M Thomas P Massimi C Navarro J-P Borg and L BanksldquoThe hScribDlg apico-basal control complex is differentiallytargeted by HPV-16 and HPV-18 E6 proteinsrdquo Oncogene vol24 no 41 pp 6222ndash6230 2005
[93] C H Storrs and S J Silverstein ldquoPATJ a tight junction-associated PDZ protein is a novel degradation target of high-risk human papillomavirus E6 and the alternatively splicedisoform 18 E6rdquo Journal of Virology vol 81 no 8 pp 4080ndash40902007
[94] R Accardi R Rubino M Scalise et al ldquoE6 and E7 from humanpapillomavirus type 16 cooperate to target the PDZ proteinNaH exchange regulatory factor 1rdquo Journal of Virology vol 85no 16 pp 8208ndash8216 2011
[95] S Nakagawa and J M Huibregtse ldquoHuman scribble (Vartul)is targeted for ubiquitin-mediated degradation by the high-risk papillomavirus E6 proteins and the E6APubiquitin-proteinligaserdquoMolecular and Cellular Biology vol 20 no 21 pp 8244ndash8253 2000
[96] L Gewin H Myers T Kiyono and D A Galloway ldquoIdentifi-cation of a novel telomerase repressor that interacts with thehuman papillomavirus type-16 E6E6-AP complexrdquo Genes ampDevelopment vol 18 no 18 pp 2269ndash2282 2004
[97] M Sundaram D L GuernseyMM Rajaraman and R Rajara-man ldquoNeosis a novel type of cell division in cancerrdquo CancerBiology ampTherapy vol 3 no 2 pp 207ndash218 2004
[98] R D Snyder and J W Green ldquoA review of the genotoxicity ofmarketed pharmaceuticalsrdquo Mutation Research vol 488 no 2pp 151ndash169 2001
[99] T Ristriani Y Nomine C Laurent E Weiss and G TraveldquoProtein mutagenesis with monodispersity-based quality prob-ing selective inactivation of p53 degradation and DNA-bindingproperties of HPV E6 oncoproteinrdquo Protein Expression andPurification vol 26 no 3 pp 357ndash367 2002
[100] M Kadaja A Sumerina T Verst M Ojarand E Ustav and MUstav ldquoGenomic instability of the host cell induced by thehuman papillomavirus replicationmachineryrdquoTheEMBO Jour-nal vol 26 no 8 pp 2180ndash2191 2007
[101] PMDay D R Lowy and J T Schiller ldquoPapillomaviruses infectcells via a clathrin-dependent pathwayrdquo Virology vol 307 no 1pp 1ndash11 2003
[102] T D Tlsty A Briot A Gualberto et al ldquoGenomic instabilityand cancerrdquoMutation Research vol 337 no 1 pp 1ndash7 1995
[103] J E Eyfjord and S K Bodvarsdottir ldquoGenomic instabilityand cancer networks involved in response to DNA damagerdquoMutation Research vol 592 no 1-2 pp 18ndash28 2005
[104] Y Yao W Huang X Yang et al ldquoHPV-16 E6 and E7 protein Tcell epitopes prediction analysis based on distributions of HLA-A loci across populations an in silico approachrdquo Vaccine vol31 no 18 pp 2289ndash2294 2013
[105] C-F Hung B Ma A Monie S-W Tsen and T-C Wu ldquoTher-apeutic human papillomavirus vaccines current clinical trialsand future directionsrdquo Expert Opinion on Biological Therapyvol 8 no 4 pp 421ndash439 2008
[106] M J P Welters G G Kenter S J Piersma et al ldquoInductionof tumor-specific CD4+ and CD8+ T-cell immunity in cervicalcancer patients by a human papillomavirus type 16 E6 and E7long peptides vaccinerdquo Clinical Cancer Research vol 14 no 1pp 178ndash187 2008
[107] R Han N M Cladel C A Reed X Peng and N D Chris-tensen ldquoProtection of rabbits from viral challenge by gene gun-based intracutaneous vaccination with a combination of cot-tontail rabbit papillomavirus E1 E2 E6 and E7 genesrdquo Journalof Virology vol 73 no 8 pp 7039ndash7043 1999
[108] Z He A P Wlazlo D W Kowalczyk et al ldquoViral recombinantvaccines to the E6 and E7 antigens of HPV-16rdquo Virology vol270 no 1 pp 146ndash161 2000
[109] A Kaufmann P Stern E Rankin et al ldquoSafety and immuno-genicity of TA-HPV a recombinant vaccinia virus expressingmodified human papillomavirus (HPV)-16 and HPV-18 E6 andE7 genes in women with progressive cervical cancerrdquo ClinicalCancer Research vol 8 no 12 pp 3676ndash3685 2002
[110] L K Borysiewicz A Fiander M Nimako et al ldquoA recombinantvaccinia virus encoding human papillomavirus types 16 and 18E6 and E7 proteins as immunotherapy for cervical cancerrdquoTheLancet vol 347 no 9014 pp 1523ndash1527 1996
[111] A De Jong T OrsquoNeill A Y Khan et al ldquoEnhancement ofhuman papillomavirus (HPV) type 16 E6 and E7-specific T-cellimmunity in healthy volunteers through vaccination with TA-CIN an HPV16 L2E7E6 fusion protein vaccinerdquo Vaccine vol20 no 29-30 pp 3456ndash3464 2002
[112] S Chen C Liao Y Lai et al ldquoDe-oncogenicHPVE6E7 vaccinegets enhanced antigenicity and promotes tumoricidal synergywith cisplatinrdquo Acta Biochimica et Biophysica Sinica vol 46 no1 pp 6ndash14 2014
BioMed Research International 15
[113] J Bonhoeffer E B Imoukhuede G Aldrovandi et al ldquoTemplateprotocol for clinical trials investigating vaccines-focus on safetyelementsrdquo Vaccine vol 31 no 47 pp 5602ndash5620 2013
[114] S Jackson and A Storey ldquoE6 proteins from diverse cutaneonsHPV types inhibit apoptosis in response to UV damagerdquoOncogene vol 19 no 4 pp 592ndash598 2000
[115] M Thomas and L Banks ldquoPDZRN3LNX3 is a novel targetof human papillomavirus type 16 (HPV-16) and HPV-18 E6rdquoJournal of Virology vol 89 no 2 pp 1439ndash1444 2015
[116] P R Daniels C M Sanders P Coulson and N J MaitlandldquoMolecular analysis of the interaction between HPV type 16 E6and human E6-associated proteinrdquo FEBS Letters vol 416 no 1pp 6ndash10 1997
[117] W H Kao S L Beaudenon A L Talis J M Huibregtse andP M Howley ldquoHuman papillomavirus type 16 E6 induces self-ubiquitination of the E6AP ubiquitin-protein ligaserdquo Journal ofVirology vol 74 no 14 pp 6408ndash6417 2000
[118] T Iftner M Elbel B Schopp et al ldquoInterference of papillo-mavirus E6 protein with single-strand break repair by interac-tion with XRCC1rdquo The EMBO Journal vol 21 no 17 pp 4741ndash4748 2002
[119] Y Imai Y Tsunokawa T Sugimura and M Terada ldquoPurifi-cation and DNA-binding properties of human papillomavirustype 16 E6 protein expressed in Escherichia colirdquo Biochemicaland Biophysical Research Communications vol 164 no 3 pp1402ndash1410 1989
[120] M Lusky L Berg H Weiher and M Botchan ldquoBovinepapilloma virus contains an activator of gene expression at thedistal end of the early transcription unitrdquoMolecular andCellularBiology vol 3 no 6 pp 1108ndash1122 1983
[121] S Dalal Q Gao E J Androphy and V Band ldquoMutationalanalysis of human papillomavirus type 16 E6 demonstrates thatp53 degradation is necessary for immortalization of mammaryepithelial cellsrdquo Journal of Virology vol 70 no 2 pp 683ndash6881996
[122] J M Huibregtse M Scheffner and P M Howley ldquoA cellularprotein mediates association of p53 with the E6 oncoprotein ofhuman papillomavirus types 16 or 18rdquo The EMBO Journal vol10 no 13 pp 4129ndash4135 1991
[123] M Scheffner B A Werness J M Huibregtse A J Levine andP M Howley ldquoThe E6 oncoprotein encoded by human papillo-mavirus types 16 and 18 promotes the degradation of p53rdquo Cellvol 63 no 6 pp 1129ndash1136 1990
[124] M Elbel S Carl S Spaderna and T Iftner ldquoA comparativeanalysis of the interactions of the E6 proteins from cutaneousand genital papillomaviruses with p53 and E6AP in correlationto their transforming potentialrdquoVirology vol 239 no 1 pp 132ndash149 1997
[125] M C Thomas and C-M Chiang ldquoE6 oncoprotein repressesp53-dependent gene activation via inhibition of protein acety-lation independently of inducing p53 degradationrdquo MolecularCell vol 17 no 2 pp 251ndash264 2005
[126] H Zimmermann R Degenkolbe H-U Bernard and M JOrsquoConnor ldquoThe human papillomavirus type 16 E6 oncoproteincan down-regulate p53 activity by targeting the transcriptionalcoactivator CBPp300rdquo Journal of Virology vol 73 no 8 pp6209ndash6219 1999
[127] D A Thompson G Belinsky T H-T Chang D L Jones RSchlegel and K Munger ldquoThe human papillomavirus-16 E6oncoprotein decreases the vigilance of mitotic checkpointsrdquoOncogene vol 15 no 25 pp 3025ndash3035 1997
[128] J Bohl K Das B Dasgupta and S B Vande Pol ldquoCompetitivebinding to a charged leucine motif represses transformation bya papillomavirus E6 oncoproteinrdquo Virology vol 271 no 1 pp163ndash170 2000
[129] S Song H C Pitot and P F Lambert ldquoThe human papillo-mavirus type 16 E6 gene alone is sufficient to induce carcinomasin transgenic animalsrdquo Journal of Virology vol 73 no 7 pp5887ndash5893 1999
[130] S Yasumoto A L Burkhardt J Doniger and J A DiPaololdquoHumanpapillomavirus type 16DNA-inducedmalignant trans-formation of NIH 3T3 cellsrdquo Journal of Virology vol 57 no 2pp 572ndash577 1986
[131] S B Vande Pol M C Brown and C E Turner ldquoAssociation ofBovine Papillomavirus Type 1 E6 oncoprotein with the focaladhesion protein paxillin through a conserved protein interac-tion motifrdquo Oncogene vol 16 no 1 pp 43ndash52 1998
[132] R Wade N Brimer and S Vande Pol ldquoTransformation bybovine papillomavirus type 1 E6 requires paxillinrdquo Journal ofVirology vol 82 no 12 pp 5962ndash5966 2008
[133] A J Klingelhutz S A Foster and J KMcDougall ldquoTelomeraseactivation by the E6 gene product of humanpapillomavirus type16rdquo Nature vol 380 no 6569 pp 79ndash82 1996
[134] S T Oh S Kyo and L A Laimins ldquoTelomerase activation byhuman papillomavirus type 16 E6 protein Induction of humantelomerase reverse transcriptase expression through Myc andGC-rich Sp1 binding sitesrdquo Journal of Virology vol 75 no 12pp 5559ndash5566 2001
[135] L V Ronco A Y KarpovaMVidal and PMHowley ldquoHumanpapillomavirus 16 E6 oncoprotein binds to interferon regulatoryfactor-3 and inhibits its transcriptional activityrdquo Genes andDevelopment vol 12 no 13 pp 2061ndash2072 1998
[18] T Rampias C Sasaki and A Psyrri ldquoMolecular mechanisms ofHPV induced carcinogenesis in head and neckrdquoOral Oncologyvol 50 no 5 pp 356ndash363 2014
[19] S Roperto V Russo L Leonardi et al ldquoBovine papillomavirustype 13 expression in the urothelial bladder tumours of cattlerdquoTransboundary and Emerging Diseases 2015
[20] S RCCampos C TrindadeO P Ferraz et al ldquoCan establishedcultured papilloma cells harbor bovine papillomavirusrdquoGenet-ics and Molecular Research vol 7 no 4 pp 1119ndash1126 2008
[21] M S Campo ldquoAnimalmodels of papilloma virus pathogenesisrdquoVirus Research vol 89 no 2 pp 249ndash261 2002
[22] G Borzacchiello ldquoBovine papillomavirus infections in ani-malsrdquo in Communicating Current Research and EducationalTopics and Trends in AppliedMicrobiology pp 673ndash679 Forma-tex Badajoz Spain 2007
[23] F Bocaneti G Altamura A Corteggio E Velescu F RopertoandG Borzacchiello ldquoBovine papillomavirus new insights intoan old diseaserdquo Transboundary and Emerging Diseases 2014
[24] R P Araldi T C Melo N Diniz et al ldquoBovine papillomavirusclastogenic effect analyzed in comet assayrdquo BioMed ResearchInternational vol 2013 Article ID 630683 7 pages 2013
[25] J Munday N Thomson M Dunowska C Knight R Laurieand S Hills ldquoGenomic characterisation of the feline sarcoid-associated papillomavirus and proposed classification as Bostaurus papillomavirus type 14rdquoVeterinaryMicrobiology vol 177no 3-4 pp 289ndash295 2015
[26] G Borzacchiello V Russo C Spoleto et al ldquoBovine papillo-mavirus type-2 DNA and expression of E5 and E7 oncoproteinsin vascular tumours of the urinary bladder in cattlerdquo CancerLetters vol 250 no 1 pp 82ndash91 2007
[27] P Maiolino A Ozkul A Sepici-Dincel et al ldquoBovine papillo-mavirus type 2 infection and microscopic patterns of urothelialtumors of the urinary bladder in water buffaloesrdquo BioMedResearch International vol 2013 Article ID 937918 6 pages2013
[28] S Roperto G Borzacchiello R Brun et al ldquoMultiple glomustumors of the urinary bladder in a cow associated with bovinepapillomavirus type 2 (BPV-2) infectionrdquo Veterinary Pathologyvol 45 no 1 pp 39ndash42 2008
[29] S Duensing and K Munger ldquoThe human papillomavirus type16 E6 and E7 oncoproteins independently induce numerical andstructural chromosome instabilityrdquoCancer Research vol 62 no23 pp 7075ndash7082 2002
[30] S Duensing L Y Lee A Duensing et al ldquoThe human papil-lomavirus type 16 E6 and E7 oncoproteins cooperate to inducemitotic defects and genomic instability by uncoupling centro-some duplication from the cell division cyclerdquo Proceedings of theNational Academy of Sciences of theUnited States of America vol97 no 18 pp 10002ndash10007 2000
[31] C A Moody and L A Laimins ldquoHuman papillomavirus onco-proteins pathways to transformationrdquo Nature Reviews Cancervol 10 no 8 pp 550ndash560 2010
[32] C Mansur and E Androphy ldquoCellular transformation bypapillomavirus oncoproteinsrdquo Biochimica et Biophysica Actavol 1155 no 3 pp 323ndash345 1993
[33] J J Chen Y Hong and E J Androphy ldquoMutational analysis oftranscriptional activation by the bovine papillomavirus type 1E6rdquo Virology vol 236 no 1 pp 30ndash36 1997
[34] X Tong andPMHowley ldquoThebovine papillomavirus E6 onco-protein interacts with paxillin and disrupts the actin cytoskele-tonrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 94 no 9 pp 4412ndash4417 1997
[35] X TongW Boll T Kirchhausen andPMHowley ldquoInteractionof the bovine papillomavirus E6 protein with the clathrinadaptor complex AP-1rdquo Journal of Virology vol 72 no 1 pp476ndash482 1998
[36] Y Nomine M Masson S Charbonnier et al ldquoStructural andfunctional analysis of E6 oncoprotein insights in the molecularpathways of human papillomavirus-mediated pathogenesisrdquoMolecular Cell vol 21 no 5 pp 665ndash678 2006
[37] T Ristriani MMasson Y Nomine et al ldquoHPV oncoprotein E6is a structure-dependent DNA-binding protein that recognizesfour-way junctionsrdquo Journal of Molecular Biology vol 296 no5 pp 1189ndash1203 2000
[38] Y Liu and J D Baleja ldquoStructure and function of the papil-lomavirus E6 protein and its interacting proteinsrdquo Frontiers inBioscience vol 13 no 1 pp 121ndash134 2008
[39] F Lipari G A McGibbon E Wardrop and M G CordingleyldquoPurification and biophysical characterization of a minimalfunctional domain and of an N-terminal Zn2+-binding frag-ment from the human papillomavirus type 16 E6 proteinrdquoBiochemistry vol 40 no 5 pp 1196ndash1204 2001
[40] Y Nomine T Ristriani C Laurent J-F Lefevre E Weissand G Trave ldquoA strategy for optimizing the monodispersity offusion proteins application to purification of recombinantHPVE6 oncoproteinrdquo Protein Engineering vol 14 no 4 pp 297ndash3052001
[41] C G Ullman P I Haris D A Galloway V C Emery and SJ Perkins ldquoPredicted 120572-helix120573-sheet secondary structures forthe zinc-binding motifs of human papillomavirus E7 and E6proteins by consensus prediction averaging and spectroscopicstudies of E7rdquo Biochemical Journal vol 319 no 1 pp 229ndash2391996
[42] A Sidi K Babah N Brimer et al ldquoStrategies for bacterialexpression of protein-peptide complex application to solubi-lization of papillomavirus E6rdquo Protein Expression and Purifica-tion vol 29 no 6 pp 997ndash1003 2011
[43] K Zanier S Charbonnier A O M O Sidi et al ldquoStructuralbasis for hijacking of cellular LxxLL motifs by papillomavirusE6 oncoproteinsrdquo Science vol 339 no 6120 pp 694ndash698 2013
[44] J Mazzuchelli-de-Souza R F Carvalho R M Ruiz et alldquoExpression and in silico analysis of the recombinant bovinepapillomavirus e6 protein as a model for viral oncoproteinsstudiesrdquo BioMed Research International vol 2013 Article ID421398 9 pages 2013
[45] L Banks P Spence E Androphy et al ldquoIdentification of humanpapillomavirus type 18 E6 polypeptide in cells derived fromhuman cervical carcinomasrdquo Journal of General Virology vol68 no 5 pp 1351ndash1359 1987
[46] D Hanahan and R AWeinberg ldquoHallmarks of cancer the nextgenerationrdquo Cell vol 144 no 5 pp 646ndash674 2011
[47] G S Charames and B Bapat ldquoGenomic instability and cancerrdquoCurrent Molecular Medicine vol 3 no 7 pp 589ndash596 2003
[48] S Negrini V G Gorgoulis and T D Halazonetis ldquoGenomicinstabilitymdashan evolving hallmark of cancerrdquo Nature ReviewsMolecular Cell Biology vol 11 no 3 pp 220ndash228 2010
[49] K Zanier S Charbonnier M Baltzinger Y Nomine DAltschuh and G Trave ldquoKinetic analysis of the interactionsof human papillomavirus E6 oncoproteins with the ubiquitinligase E6AP using surface plasmon resonancerdquo Journal ofMolecular Biology vol 349 no 2 pp 401ndash412 2005
BioMed Research International 13
[50] M Scheffner J M Huibregtse R D Vierstra and PM HowleyldquoThe HPV-16 E6 and E6-AP complex functions as a ubiquitin-protein ligase in the ubiquitination of p53rdquo Cell vol 75 no 3pp 495ndash505 1993
[51] H Zimmermann C-H Koh R Degenkolbe et al ldquoInteractionwith CBPp300 enables the bovine papillomavirus type 1 E6oncoprotein to downregulate CBPp300-mediated transactiva-tion by p53rdquo Journal of General Virology vol 81 no 11 pp 2617ndash2623 2000
[52] M Fenech ldquoThe in vitro micronucleus techniquerdquo MutationResearchmdashFundamental and Molecular Mechanisms of Mutage-nesis vol 455 no 1-2 pp 81ndash95 2000
[53] R P Araldi T C de Melo T B Mendes et al ldquoUsing the cometand micronucleus assays for genotoxicity studies a reviewrdquoBiomedicine amp Pharmacotherapy vol 72 pp 74ndash82 2015
[54] P Thomas and M Fenech ldquoCytokinesis-block micronucleuscytome assay in lymphocytesrdquo Methods in Molecular Biologyvol 682 pp 217ndash234 2011
[55] M Kirsch-Volders T Sofuni M Aardema et al ldquoReport fromthe in vitro micronucleus assay working grouprdquo MutationResearch Genetic Toxicology and Environmental Mutagenesisvol 540 no 2 pp 153ndash163 2003
[56] S Samanta and P Dey ldquoMicronucleus and its applicationsrdquoDiagnostic Cytopathology vol 40 no 1 pp 84ndash90 2012
[57] M Hayashi J T MacGregor D G Gatehouse et al ldquoIn vivoerythrocyte micronucleus assay III Validation and regulatoryacceptance of automated scoring and the use of rat periph-eral blood reticulocytes with discussion of non-hematopoietictarget cells and a single dose-level limit testrdquo MutationResearchGenetic Toxicology and Environmental Mutagenesisvol 627 no 1 pp 10ndash30 2007
[58] A Azqueta and A R Collins ldquoThe essential comet assay acomprehensive guide to measuring DNA damage and repairrdquoArchives of Toxicology vol 87 no 6 pp 949ndash968 2013
[59] N P Singh M T McCoy R R Tice and E L Schneider ldquoAsimple technique for quantitation of low levels of DNA damagein individual cellsrdquo Experimental Cell Research vol 175 no 1pp 184ndash191 1988
[60] J H Calinisan S R Chan A King and P J Chan ldquoHumanpapillomavirus and blastocyst apoptosisrdquo Journal of AssistedReproduction and Genetics vol 19 no 3 pp 132ndash136 2002
[61] R Oliveira Studies on the biopathological actions of Pteridiumaquilinum [PhD thesis] nstituto de Ciencias Biomedicas AbelSalazar 2012
[62] D M Potter and M S Baird ldquoCarcinogenic effects of ptaquilo-side in bracken fern and related compoundsrdquo British Journal ofCancer vol 83 no 7 pp 914ndash920 2000
[63] R B Lucena D R Rissi G D Kommers et al ldquoA retrospectivestudy of 586 tumours in Brazilian cattlerdquo Journal of ComparativePathology vol 145 no 1 pp 20ndash24 2011
[64] A Yaguiu M L Z Dagli E H Birgel Jr et al ldquoSimultaneouspresence of bovine papillomavirus and bovine leukemia virusin different bovine tissues in situ hybridization and cytogeneticanalysisrdquoGenetics andMolecular Research vol 7 no 2 pp 487ndash497 2008
[65] T C Melo R F Carvalho J Mazzucchelli-de-Souza et alldquoPhylogenetic classification and clinical aspects of a new puta-tive Deltapapillomavirus associated with skin lesions in cattlerdquoGenetics and Molecular Research vol 13 no 2 pp 2458ndash24692014
[66] R P Araldi D N S Giovanni T CMelo et al ldquoBovine papillo-mavirus isolation by ultracentrifugationrdquo Journal of VirologicalMethods vol 208 pp 119ndash124 2014
[67] L M Chandrachud B W OrsquoNeil W F H Jarrett G J Grind-lay G M McGarvie and M S Campo ldquoHumoral immuneresponse to the E7 protein of bovine papillomavirus type 4 andidentification of B-cell epitopesrdquoVirology vol 200 no 1 pp 98ndash104 1994
[68] M Ayres M J Ayres D L Ayres and A A Santos BioEstatmdashAplicacoes Estatısticas nas Areas de Ciencias Bio-Medicas Insti-tuto Mamiraua Belem Brazil 2007
[69] K Das J Bohl and S B Vande Pol ldquoIdentification of a secondtransforming function in bovine papillomavirus type 1 E6 andthe role of E6 interactions with paxillin E6BP and E6APrdquoJournal of Virology vol 74 no 2 pp 812ndash816 2000
[70] O Danos E Georges G Orth and M Yaniv ldquoFine structureof the cottontail rabbit papillomavirus mRNAs expressed in thetransplantable VX2 carcinomardquo Journal of Virology vol 53 no3 pp 735ndash741 1985
[71] M Nasseri and F O Wettstein ldquoDifferences exist between viraltranscripts in cottontail rabbit papillomavirus-induced benignand malignant tumors as well as non-virus-producing andvirus-producing tumorsrdquo Journal of Virology vol 51 no 3 pp706ndash712 1984
[72] X Tong R Salgia J-L Li J D Griffin and P M Howley ldquoThebovine papillomavirus E6 protein binds to the LDmotif repeatsof paxillin and blocks its interaction with vinculin and the focaladhesion kinaserdquo Journal of Biological Chemistry vol 272 no52 pp 33373ndash33376 1997
[73] Y Maeda T Shibahara Y Wada et al ldquoAn outbreak of teatpapillomatosis in cattle caused by bovine papilloma virus (BPV)type 6 and unclassified BPVsrdquo Veterinary Microbiology vol 121no 3-4 pp 242ndash248 2007
[74] R P Araldi T C Melo A Neves et al ldquoHyperproliferativeaction of bovine papillomavirus genetic and histopathologicalaspectsrdquo Genetics and Molecular Research vol 14 no 4 pp12942ndash12954 2015
[75] NDiniz T CMelo J F Santos et al ldquoSimultaneous presence ofbovine papillomavirus in blood and in short-term lymphocytecultures from dairy cattle in Pernambuco Brazilrdquo Genetics andMolecular Research vol 8 no 4 pp 1474ndash1480 2009
[76] O M Brito M F Guimaraes and C C Lanna ldquoCiclofosfamidae funcao ovarianardquo Revista Brasileira de Reumatologia vol 48no 1 pp 39ndash45 2008
[77] D Anderson T-W Yu and D B McGregor ldquoComet assayresponses as indicators of carcinogen exposurerdquo Mutagenesisvol 13 no 6 pp 539ndash555 1998
[78] M L Adam C Pini S Tulio J C L L Cantalice R A Torresand M T Dos Santos Correia ldquoAssessment of the associationbetween micronuclei and the degree of uterine lesions and viralload in women with human papillomavirusrdquo Cancer Genomicsamp Proteomics vol 12 no 2 pp 67ndash72 2015
[79] E Cortes-Gutierrez M Davila-Rodrıguez J Vargas-VillarrealF Hernandez-Garza and R Cerda-Flores ldquoAssociationbetween human papilloma virus-type infections with micro-nuclei frequenciesrdquo Prague Medical Report vol 111 no 1 pp35ndash41 2010
[80] R N Chakrabarti and K Dutta ldquoMicronuclei test in routinesmears fromuterine cervixrdquo European Journal of GynaecologicalOncology vol 9 no 5 pp 370ndash372 1988
[81] E M M Cerqueira C L Santoro N F Donozo et al ldquoGeneticdamage in exfoliated cells of the uterine cervix Association
14 BioMed Research International
and interaction between cigarette smoking and progression tomalignant transformationrdquo Acta Cytologica vol 42 no 3 pp639ndash649 1998
[82] S M Germann V Schramke R T Pedersen et al ldquoTopBP1Dpb11 bindsDNA anaphase bridges to prevent genome instabil-ityrdquoThe Journal of Cell Biology vol 204 no 1 pp 45ndash59 2014
[83] D R Hoffelder L Luo N A Burke S CWatkins S M Gollinand W S Saunders ldquoResolution of anaphase bridges in cancercellsrdquo Chromosoma vol 112 no 8 pp 389ndash397 2004
[84] C Acilan D M Potter and W S Saunders ldquoDNA repairpathways involved in anaphase bridge formationrdquo Genes Chro-mosomes and Cancer vol 46 no 6 pp 522ndash531 2007
[85] J DeMasi M C Chao A S Kumar and P M Howley ldquoBovinepapillomavirus E7 oncoprotein inhibits anoikisrdquo Journal ofVirology vol 81 no 17 pp 9419ndash9425 2007
[86] J DeMasi K-W Huh Y Nakatani K Munger and P MHowley ldquoBovine papillomavirus E7 transformation functioncorrelates with cellular p600 protein bindingrdquo Proceedings of theNational Academy of Sciences of theUnited States of America vol102 no 32 pp 11486ndash11491 2005
[87] E A White and P M Howley ldquoProteomic approaches to thestudy of papillomavirusndashhost interactionsrdquo Virology vol 435no 1 pp 57ndash69 2013
[88] FMantovani and L Banks ldquoThe human papillomavirus E6 pro-tein and its contribution to malignant progressionrdquo Oncogenevol 20 no 54 pp 7874ndash7887 2001
[89] P Chen and H Lee ldquoFOXM1 induced by E6 oncoprotein pro-motes tumor invasion and chemoresistance in HPV-infectedlung cancerrdquo Cancer Cell amp Microenvironment vol 2 no 1 pp6ndash9 2015
[90] N Zhang P Wei A Gong et al ldquoFoxM1 promotes 120573-cateninnuclear localization and controls Wnt target-gene expressionand glioma tumorigenesisrdquo Cancer Cell vol 127 no 4 pp 358ndash366 2012
[91] Z Liu Y Liu Y Hong L Rapp E J Androphy and J JChen ldquoBovine papillomavirus type 1 E6-induced sensitizationto apoptosis is distinct from its transforming activityrdquo Virologyvol 295 no 2 pp 230ndash237 2002
[92] M Thomas P Massimi C Navarro J-P Borg and L BanksldquoThe hScribDlg apico-basal control complex is differentiallytargeted by HPV-16 and HPV-18 E6 proteinsrdquo Oncogene vol24 no 41 pp 6222ndash6230 2005
[93] C H Storrs and S J Silverstein ldquoPATJ a tight junction-associated PDZ protein is a novel degradation target of high-risk human papillomavirus E6 and the alternatively splicedisoform 18 E6rdquo Journal of Virology vol 81 no 8 pp 4080ndash40902007
[94] R Accardi R Rubino M Scalise et al ldquoE6 and E7 from humanpapillomavirus type 16 cooperate to target the PDZ proteinNaH exchange regulatory factor 1rdquo Journal of Virology vol 85no 16 pp 8208ndash8216 2011
[95] S Nakagawa and J M Huibregtse ldquoHuman scribble (Vartul)is targeted for ubiquitin-mediated degradation by the high-risk papillomavirus E6 proteins and the E6APubiquitin-proteinligaserdquoMolecular and Cellular Biology vol 20 no 21 pp 8244ndash8253 2000
[96] L Gewin H Myers T Kiyono and D A Galloway ldquoIdentifi-cation of a novel telomerase repressor that interacts with thehuman papillomavirus type-16 E6E6-AP complexrdquo Genes ampDevelopment vol 18 no 18 pp 2269ndash2282 2004
[97] M Sundaram D L GuernseyMM Rajaraman and R Rajara-man ldquoNeosis a novel type of cell division in cancerrdquo CancerBiology ampTherapy vol 3 no 2 pp 207ndash218 2004
[98] R D Snyder and J W Green ldquoA review of the genotoxicity ofmarketed pharmaceuticalsrdquo Mutation Research vol 488 no 2pp 151ndash169 2001
[99] T Ristriani Y Nomine C Laurent E Weiss and G TraveldquoProtein mutagenesis with monodispersity-based quality prob-ing selective inactivation of p53 degradation and DNA-bindingproperties of HPV E6 oncoproteinrdquo Protein Expression andPurification vol 26 no 3 pp 357ndash367 2002
[100] M Kadaja A Sumerina T Verst M Ojarand E Ustav and MUstav ldquoGenomic instability of the host cell induced by thehuman papillomavirus replicationmachineryrdquoTheEMBO Jour-nal vol 26 no 8 pp 2180ndash2191 2007
[101] PMDay D R Lowy and J T Schiller ldquoPapillomaviruses infectcells via a clathrin-dependent pathwayrdquo Virology vol 307 no 1pp 1ndash11 2003
[102] T D Tlsty A Briot A Gualberto et al ldquoGenomic instabilityand cancerrdquoMutation Research vol 337 no 1 pp 1ndash7 1995
[103] J E Eyfjord and S K Bodvarsdottir ldquoGenomic instabilityand cancer networks involved in response to DNA damagerdquoMutation Research vol 592 no 1-2 pp 18ndash28 2005
[104] Y Yao W Huang X Yang et al ldquoHPV-16 E6 and E7 protein Tcell epitopes prediction analysis based on distributions of HLA-A loci across populations an in silico approachrdquo Vaccine vol31 no 18 pp 2289ndash2294 2013
[105] C-F Hung B Ma A Monie S-W Tsen and T-C Wu ldquoTher-apeutic human papillomavirus vaccines current clinical trialsand future directionsrdquo Expert Opinion on Biological Therapyvol 8 no 4 pp 421ndash439 2008
[106] M J P Welters G G Kenter S J Piersma et al ldquoInductionof tumor-specific CD4+ and CD8+ T-cell immunity in cervicalcancer patients by a human papillomavirus type 16 E6 and E7long peptides vaccinerdquo Clinical Cancer Research vol 14 no 1pp 178ndash187 2008
[107] R Han N M Cladel C A Reed X Peng and N D Chris-tensen ldquoProtection of rabbits from viral challenge by gene gun-based intracutaneous vaccination with a combination of cot-tontail rabbit papillomavirus E1 E2 E6 and E7 genesrdquo Journalof Virology vol 73 no 8 pp 7039ndash7043 1999
[108] Z He A P Wlazlo D W Kowalczyk et al ldquoViral recombinantvaccines to the E6 and E7 antigens of HPV-16rdquo Virology vol270 no 1 pp 146ndash161 2000
[109] A Kaufmann P Stern E Rankin et al ldquoSafety and immuno-genicity of TA-HPV a recombinant vaccinia virus expressingmodified human papillomavirus (HPV)-16 and HPV-18 E6 andE7 genes in women with progressive cervical cancerrdquo ClinicalCancer Research vol 8 no 12 pp 3676ndash3685 2002
[110] L K Borysiewicz A Fiander M Nimako et al ldquoA recombinantvaccinia virus encoding human papillomavirus types 16 and 18E6 and E7 proteins as immunotherapy for cervical cancerrdquoTheLancet vol 347 no 9014 pp 1523ndash1527 1996
[111] A De Jong T OrsquoNeill A Y Khan et al ldquoEnhancement ofhuman papillomavirus (HPV) type 16 E6 and E7-specific T-cellimmunity in healthy volunteers through vaccination with TA-CIN an HPV16 L2E7E6 fusion protein vaccinerdquo Vaccine vol20 no 29-30 pp 3456ndash3464 2002
[112] S Chen C Liao Y Lai et al ldquoDe-oncogenicHPVE6E7 vaccinegets enhanced antigenicity and promotes tumoricidal synergywith cisplatinrdquo Acta Biochimica et Biophysica Sinica vol 46 no1 pp 6ndash14 2014
BioMed Research International 15
[113] J Bonhoeffer E B Imoukhuede G Aldrovandi et al ldquoTemplateprotocol for clinical trials investigating vaccines-focus on safetyelementsrdquo Vaccine vol 31 no 47 pp 5602ndash5620 2013
[114] S Jackson and A Storey ldquoE6 proteins from diverse cutaneonsHPV types inhibit apoptosis in response to UV damagerdquoOncogene vol 19 no 4 pp 592ndash598 2000
[115] M Thomas and L Banks ldquoPDZRN3LNX3 is a novel targetof human papillomavirus type 16 (HPV-16) and HPV-18 E6rdquoJournal of Virology vol 89 no 2 pp 1439ndash1444 2015
[116] P R Daniels C M Sanders P Coulson and N J MaitlandldquoMolecular analysis of the interaction between HPV type 16 E6and human E6-associated proteinrdquo FEBS Letters vol 416 no 1pp 6ndash10 1997
[117] W H Kao S L Beaudenon A L Talis J M Huibregtse andP M Howley ldquoHuman papillomavirus type 16 E6 induces self-ubiquitination of the E6AP ubiquitin-protein ligaserdquo Journal ofVirology vol 74 no 14 pp 6408ndash6417 2000
[118] T Iftner M Elbel B Schopp et al ldquoInterference of papillo-mavirus E6 protein with single-strand break repair by interac-tion with XRCC1rdquo The EMBO Journal vol 21 no 17 pp 4741ndash4748 2002
[119] Y Imai Y Tsunokawa T Sugimura and M Terada ldquoPurifi-cation and DNA-binding properties of human papillomavirustype 16 E6 protein expressed in Escherichia colirdquo Biochemicaland Biophysical Research Communications vol 164 no 3 pp1402ndash1410 1989
[120] M Lusky L Berg H Weiher and M Botchan ldquoBovinepapilloma virus contains an activator of gene expression at thedistal end of the early transcription unitrdquoMolecular andCellularBiology vol 3 no 6 pp 1108ndash1122 1983
[121] S Dalal Q Gao E J Androphy and V Band ldquoMutationalanalysis of human papillomavirus type 16 E6 demonstrates thatp53 degradation is necessary for immortalization of mammaryepithelial cellsrdquo Journal of Virology vol 70 no 2 pp 683ndash6881996
[122] J M Huibregtse M Scheffner and P M Howley ldquoA cellularprotein mediates association of p53 with the E6 oncoprotein ofhuman papillomavirus types 16 or 18rdquo The EMBO Journal vol10 no 13 pp 4129ndash4135 1991
[123] M Scheffner B A Werness J M Huibregtse A J Levine andP M Howley ldquoThe E6 oncoprotein encoded by human papillo-mavirus types 16 and 18 promotes the degradation of p53rdquo Cellvol 63 no 6 pp 1129ndash1136 1990
[124] M Elbel S Carl S Spaderna and T Iftner ldquoA comparativeanalysis of the interactions of the E6 proteins from cutaneousand genital papillomaviruses with p53 and E6AP in correlationto their transforming potentialrdquoVirology vol 239 no 1 pp 132ndash149 1997
[125] M C Thomas and C-M Chiang ldquoE6 oncoprotein repressesp53-dependent gene activation via inhibition of protein acety-lation independently of inducing p53 degradationrdquo MolecularCell vol 17 no 2 pp 251ndash264 2005
[126] H Zimmermann R Degenkolbe H-U Bernard and M JOrsquoConnor ldquoThe human papillomavirus type 16 E6 oncoproteincan down-regulate p53 activity by targeting the transcriptionalcoactivator CBPp300rdquo Journal of Virology vol 73 no 8 pp6209ndash6219 1999
[127] D A Thompson G Belinsky T H-T Chang D L Jones RSchlegel and K Munger ldquoThe human papillomavirus-16 E6oncoprotein decreases the vigilance of mitotic checkpointsrdquoOncogene vol 15 no 25 pp 3025ndash3035 1997
[128] J Bohl K Das B Dasgupta and S B Vande Pol ldquoCompetitivebinding to a charged leucine motif represses transformation bya papillomavirus E6 oncoproteinrdquo Virology vol 271 no 1 pp163ndash170 2000
[129] S Song H C Pitot and P F Lambert ldquoThe human papillo-mavirus type 16 E6 gene alone is sufficient to induce carcinomasin transgenic animalsrdquo Journal of Virology vol 73 no 7 pp5887ndash5893 1999
[130] S Yasumoto A L Burkhardt J Doniger and J A DiPaololdquoHumanpapillomavirus type 16DNA-inducedmalignant trans-formation of NIH 3T3 cellsrdquo Journal of Virology vol 57 no 2pp 572ndash577 1986
[131] S B Vande Pol M C Brown and C E Turner ldquoAssociation ofBovine Papillomavirus Type 1 E6 oncoprotein with the focaladhesion protein paxillin through a conserved protein interac-tion motifrdquo Oncogene vol 16 no 1 pp 43ndash52 1998
[132] R Wade N Brimer and S Vande Pol ldquoTransformation bybovine papillomavirus type 1 E6 requires paxillinrdquo Journal ofVirology vol 82 no 12 pp 5962ndash5966 2008
[133] A J Klingelhutz S A Foster and J KMcDougall ldquoTelomeraseactivation by the E6 gene product of humanpapillomavirus type16rdquo Nature vol 380 no 6569 pp 79ndash82 1996
[134] S T Oh S Kyo and L A Laimins ldquoTelomerase activation byhuman papillomavirus type 16 E6 protein Induction of humantelomerase reverse transcriptase expression through Myc andGC-rich Sp1 binding sitesrdquo Journal of Virology vol 75 no 12pp 5559ndash5566 2001
[135] L V Ronco A Y KarpovaMVidal and PMHowley ldquoHumanpapillomavirus 16 E6 oncoprotein binds to interferon regulatoryfactor-3 and inhibits its transcriptional activityrdquo Genes andDevelopment vol 12 no 13 pp 2061ndash2072 1998
[50] M Scheffner J M Huibregtse R D Vierstra and PM HowleyldquoThe HPV-16 E6 and E6-AP complex functions as a ubiquitin-protein ligase in the ubiquitination of p53rdquo Cell vol 75 no 3pp 495ndash505 1993
[51] H Zimmermann C-H Koh R Degenkolbe et al ldquoInteractionwith CBPp300 enables the bovine papillomavirus type 1 E6oncoprotein to downregulate CBPp300-mediated transactiva-tion by p53rdquo Journal of General Virology vol 81 no 11 pp 2617ndash2623 2000
[52] M Fenech ldquoThe in vitro micronucleus techniquerdquo MutationResearchmdashFundamental and Molecular Mechanisms of Mutage-nesis vol 455 no 1-2 pp 81ndash95 2000
[53] R P Araldi T C de Melo T B Mendes et al ldquoUsing the cometand micronucleus assays for genotoxicity studies a reviewrdquoBiomedicine amp Pharmacotherapy vol 72 pp 74ndash82 2015
[54] P Thomas and M Fenech ldquoCytokinesis-block micronucleuscytome assay in lymphocytesrdquo Methods in Molecular Biologyvol 682 pp 217ndash234 2011
[55] M Kirsch-Volders T Sofuni M Aardema et al ldquoReport fromthe in vitro micronucleus assay working grouprdquo MutationResearch Genetic Toxicology and Environmental Mutagenesisvol 540 no 2 pp 153ndash163 2003
[56] S Samanta and P Dey ldquoMicronucleus and its applicationsrdquoDiagnostic Cytopathology vol 40 no 1 pp 84ndash90 2012
[57] M Hayashi J T MacGregor D G Gatehouse et al ldquoIn vivoerythrocyte micronucleus assay III Validation and regulatoryacceptance of automated scoring and the use of rat periph-eral blood reticulocytes with discussion of non-hematopoietictarget cells and a single dose-level limit testrdquo MutationResearchGenetic Toxicology and Environmental Mutagenesisvol 627 no 1 pp 10ndash30 2007
[58] A Azqueta and A R Collins ldquoThe essential comet assay acomprehensive guide to measuring DNA damage and repairrdquoArchives of Toxicology vol 87 no 6 pp 949ndash968 2013
[59] N P Singh M T McCoy R R Tice and E L Schneider ldquoAsimple technique for quantitation of low levels of DNA damagein individual cellsrdquo Experimental Cell Research vol 175 no 1pp 184ndash191 1988
[60] J H Calinisan S R Chan A King and P J Chan ldquoHumanpapillomavirus and blastocyst apoptosisrdquo Journal of AssistedReproduction and Genetics vol 19 no 3 pp 132ndash136 2002
[61] R Oliveira Studies on the biopathological actions of Pteridiumaquilinum [PhD thesis] nstituto de Ciencias Biomedicas AbelSalazar 2012
[62] D M Potter and M S Baird ldquoCarcinogenic effects of ptaquilo-side in bracken fern and related compoundsrdquo British Journal ofCancer vol 83 no 7 pp 914ndash920 2000
[63] R B Lucena D R Rissi G D Kommers et al ldquoA retrospectivestudy of 586 tumours in Brazilian cattlerdquo Journal of ComparativePathology vol 145 no 1 pp 20ndash24 2011
[64] A Yaguiu M L Z Dagli E H Birgel Jr et al ldquoSimultaneouspresence of bovine papillomavirus and bovine leukemia virusin different bovine tissues in situ hybridization and cytogeneticanalysisrdquoGenetics andMolecular Research vol 7 no 2 pp 487ndash497 2008
[65] T C Melo R F Carvalho J Mazzucchelli-de-Souza et alldquoPhylogenetic classification and clinical aspects of a new puta-tive Deltapapillomavirus associated with skin lesions in cattlerdquoGenetics and Molecular Research vol 13 no 2 pp 2458ndash24692014
[66] R P Araldi D N S Giovanni T CMelo et al ldquoBovine papillo-mavirus isolation by ultracentrifugationrdquo Journal of VirologicalMethods vol 208 pp 119ndash124 2014
[67] L M Chandrachud B W OrsquoNeil W F H Jarrett G J Grind-lay G M McGarvie and M S Campo ldquoHumoral immuneresponse to the E7 protein of bovine papillomavirus type 4 andidentification of B-cell epitopesrdquoVirology vol 200 no 1 pp 98ndash104 1994
[68] M Ayres M J Ayres D L Ayres and A A Santos BioEstatmdashAplicacoes Estatısticas nas Areas de Ciencias Bio-Medicas Insti-tuto Mamiraua Belem Brazil 2007
[69] K Das J Bohl and S B Vande Pol ldquoIdentification of a secondtransforming function in bovine papillomavirus type 1 E6 andthe role of E6 interactions with paxillin E6BP and E6APrdquoJournal of Virology vol 74 no 2 pp 812ndash816 2000
[70] O Danos E Georges G Orth and M Yaniv ldquoFine structureof the cottontail rabbit papillomavirus mRNAs expressed in thetransplantable VX2 carcinomardquo Journal of Virology vol 53 no3 pp 735ndash741 1985
[71] M Nasseri and F O Wettstein ldquoDifferences exist between viraltranscripts in cottontail rabbit papillomavirus-induced benignand malignant tumors as well as non-virus-producing andvirus-producing tumorsrdquo Journal of Virology vol 51 no 3 pp706ndash712 1984
[72] X Tong R Salgia J-L Li J D Griffin and P M Howley ldquoThebovine papillomavirus E6 protein binds to the LDmotif repeatsof paxillin and blocks its interaction with vinculin and the focaladhesion kinaserdquo Journal of Biological Chemistry vol 272 no52 pp 33373ndash33376 1997
[73] Y Maeda T Shibahara Y Wada et al ldquoAn outbreak of teatpapillomatosis in cattle caused by bovine papilloma virus (BPV)type 6 and unclassified BPVsrdquo Veterinary Microbiology vol 121no 3-4 pp 242ndash248 2007
[74] R P Araldi T C Melo A Neves et al ldquoHyperproliferativeaction of bovine papillomavirus genetic and histopathologicalaspectsrdquo Genetics and Molecular Research vol 14 no 4 pp12942ndash12954 2015
[75] NDiniz T CMelo J F Santos et al ldquoSimultaneous presence ofbovine papillomavirus in blood and in short-term lymphocytecultures from dairy cattle in Pernambuco Brazilrdquo Genetics andMolecular Research vol 8 no 4 pp 1474ndash1480 2009
[76] O M Brito M F Guimaraes and C C Lanna ldquoCiclofosfamidae funcao ovarianardquo Revista Brasileira de Reumatologia vol 48no 1 pp 39ndash45 2008
[77] D Anderson T-W Yu and D B McGregor ldquoComet assayresponses as indicators of carcinogen exposurerdquo Mutagenesisvol 13 no 6 pp 539ndash555 1998
[78] M L Adam C Pini S Tulio J C L L Cantalice R A Torresand M T Dos Santos Correia ldquoAssessment of the associationbetween micronuclei and the degree of uterine lesions and viralload in women with human papillomavirusrdquo Cancer Genomicsamp Proteomics vol 12 no 2 pp 67ndash72 2015
[79] E Cortes-Gutierrez M Davila-Rodrıguez J Vargas-VillarrealF Hernandez-Garza and R Cerda-Flores ldquoAssociationbetween human papilloma virus-type infections with micro-nuclei frequenciesrdquo Prague Medical Report vol 111 no 1 pp35ndash41 2010
[80] R N Chakrabarti and K Dutta ldquoMicronuclei test in routinesmears fromuterine cervixrdquo European Journal of GynaecologicalOncology vol 9 no 5 pp 370ndash372 1988
[81] E M M Cerqueira C L Santoro N F Donozo et al ldquoGeneticdamage in exfoliated cells of the uterine cervix Association
14 BioMed Research International
and interaction between cigarette smoking and progression tomalignant transformationrdquo Acta Cytologica vol 42 no 3 pp639ndash649 1998
[82] S M Germann V Schramke R T Pedersen et al ldquoTopBP1Dpb11 bindsDNA anaphase bridges to prevent genome instabil-ityrdquoThe Journal of Cell Biology vol 204 no 1 pp 45ndash59 2014
[83] D R Hoffelder L Luo N A Burke S CWatkins S M Gollinand W S Saunders ldquoResolution of anaphase bridges in cancercellsrdquo Chromosoma vol 112 no 8 pp 389ndash397 2004
[84] C Acilan D M Potter and W S Saunders ldquoDNA repairpathways involved in anaphase bridge formationrdquo Genes Chro-mosomes and Cancer vol 46 no 6 pp 522ndash531 2007
[85] J DeMasi M C Chao A S Kumar and P M Howley ldquoBovinepapillomavirus E7 oncoprotein inhibits anoikisrdquo Journal ofVirology vol 81 no 17 pp 9419ndash9425 2007
[86] J DeMasi K-W Huh Y Nakatani K Munger and P MHowley ldquoBovine papillomavirus E7 transformation functioncorrelates with cellular p600 protein bindingrdquo Proceedings of theNational Academy of Sciences of theUnited States of America vol102 no 32 pp 11486ndash11491 2005
[87] E A White and P M Howley ldquoProteomic approaches to thestudy of papillomavirusndashhost interactionsrdquo Virology vol 435no 1 pp 57ndash69 2013
[88] FMantovani and L Banks ldquoThe human papillomavirus E6 pro-tein and its contribution to malignant progressionrdquo Oncogenevol 20 no 54 pp 7874ndash7887 2001
[89] P Chen and H Lee ldquoFOXM1 induced by E6 oncoprotein pro-motes tumor invasion and chemoresistance in HPV-infectedlung cancerrdquo Cancer Cell amp Microenvironment vol 2 no 1 pp6ndash9 2015
[90] N Zhang P Wei A Gong et al ldquoFoxM1 promotes 120573-cateninnuclear localization and controls Wnt target-gene expressionand glioma tumorigenesisrdquo Cancer Cell vol 127 no 4 pp 358ndash366 2012
[91] Z Liu Y Liu Y Hong L Rapp E J Androphy and J JChen ldquoBovine papillomavirus type 1 E6-induced sensitizationto apoptosis is distinct from its transforming activityrdquo Virologyvol 295 no 2 pp 230ndash237 2002
[92] M Thomas P Massimi C Navarro J-P Borg and L BanksldquoThe hScribDlg apico-basal control complex is differentiallytargeted by HPV-16 and HPV-18 E6 proteinsrdquo Oncogene vol24 no 41 pp 6222ndash6230 2005
[93] C H Storrs and S J Silverstein ldquoPATJ a tight junction-associated PDZ protein is a novel degradation target of high-risk human papillomavirus E6 and the alternatively splicedisoform 18 E6rdquo Journal of Virology vol 81 no 8 pp 4080ndash40902007
[94] R Accardi R Rubino M Scalise et al ldquoE6 and E7 from humanpapillomavirus type 16 cooperate to target the PDZ proteinNaH exchange regulatory factor 1rdquo Journal of Virology vol 85no 16 pp 8208ndash8216 2011
[95] S Nakagawa and J M Huibregtse ldquoHuman scribble (Vartul)is targeted for ubiquitin-mediated degradation by the high-risk papillomavirus E6 proteins and the E6APubiquitin-proteinligaserdquoMolecular and Cellular Biology vol 20 no 21 pp 8244ndash8253 2000
[96] L Gewin H Myers T Kiyono and D A Galloway ldquoIdentifi-cation of a novel telomerase repressor that interacts with thehuman papillomavirus type-16 E6E6-AP complexrdquo Genes ampDevelopment vol 18 no 18 pp 2269ndash2282 2004
[97] M Sundaram D L GuernseyMM Rajaraman and R Rajara-man ldquoNeosis a novel type of cell division in cancerrdquo CancerBiology ampTherapy vol 3 no 2 pp 207ndash218 2004
[98] R D Snyder and J W Green ldquoA review of the genotoxicity ofmarketed pharmaceuticalsrdquo Mutation Research vol 488 no 2pp 151ndash169 2001
[99] T Ristriani Y Nomine C Laurent E Weiss and G TraveldquoProtein mutagenesis with monodispersity-based quality prob-ing selective inactivation of p53 degradation and DNA-bindingproperties of HPV E6 oncoproteinrdquo Protein Expression andPurification vol 26 no 3 pp 357ndash367 2002
[100] M Kadaja A Sumerina T Verst M Ojarand E Ustav and MUstav ldquoGenomic instability of the host cell induced by thehuman papillomavirus replicationmachineryrdquoTheEMBO Jour-nal vol 26 no 8 pp 2180ndash2191 2007
[101] PMDay D R Lowy and J T Schiller ldquoPapillomaviruses infectcells via a clathrin-dependent pathwayrdquo Virology vol 307 no 1pp 1ndash11 2003
[102] T D Tlsty A Briot A Gualberto et al ldquoGenomic instabilityand cancerrdquoMutation Research vol 337 no 1 pp 1ndash7 1995
[103] J E Eyfjord and S K Bodvarsdottir ldquoGenomic instabilityand cancer networks involved in response to DNA damagerdquoMutation Research vol 592 no 1-2 pp 18ndash28 2005
[104] Y Yao W Huang X Yang et al ldquoHPV-16 E6 and E7 protein Tcell epitopes prediction analysis based on distributions of HLA-A loci across populations an in silico approachrdquo Vaccine vol31 no 18 pp 2289ndash2294 2013
[105] C-F Hung B Ma A Monie S-W Tsen and T-C Wu ldquoTher-apeutic human papillomavirus vaccines current clinical trialsand future directionsrdquo Expert Opinion on Biological Therapyvol 8 no 4 pp 421ndash439 2008
[106] M J P Welters G G Kenter S J Piersma et al ldquoInductionof tumor-specific CD4+ and CD8+ T-cell immunity in cervicalcancer patients by a human papillomavirus type 16 E6 and E7long peptides vaccinerdquo Clinical Cancer Research vol 14 no 1pp 178ndash187 2008
[107] R Han N M Cladel C A Reed X Peng and N D Chris-tensen ldquoProtection of rabbits from viral challenge by gene gun-based intracutaneous vaccination with a combination of cot-tontail rabbit papillomavirus E1 E2 E6 and E7 genesrdquo Journalof Virology vol 73 no 8 pp 7039ndash7043 1999
[108] Z He A P Wlazlo D W Kowalczyk et al ldquoViral recombinantvaccines to the E6 and E7 antigens of HPV-16rdquo Virology vol270 no 1 pp 146ndash161 2000
[109] A Kaufmann P Stern E Rankin et al ldquoSafety and immuno-genicity of TA-HPV a recombinant vaccinia virus expressingmodified human papillomavirus (HPV)-16 and HPV-18 E6 andE7 genes in women with progressive cervical cancerrdquo ClinicalCancer Research vol 8 no 12 pp 3676ndash3685 2002
[110] L K Borysiewicz A Fiander M Nimako et al ldquoA recombinantvaccinia virus encoding human papillomavirus types 16 and 18E6 and E7 proteins as immunotherapy for cervical cancerrdquoTheLancet vol 347 no 9014 pp 1523ndash1527 1996
[111] A De Jong T OrsquoNeill A Y Khan et al ldquoEnhancement ofhuman papillomavirus (HPV) type 16 E6 and E7-specific T-cellimmunity in healthy volunteers through vaccination with TA-CIN an HPV16 L2E7E6 fusion protein vaccinerdquo Vaccine vol20 no 29-30 pp 3456ndash3464 2002
[112] S Chen C Liao Y Lai et al ldquoDe-oncogenicHPVE6E7 vaccinegets enhanced antigenicity and promotes tumoricidal synergywith cisplatinrdquo Acta Biochimica et Biophysica Sinica vol 46 no1 pp 6ndash14 2014
BioMed Research International 15
[113] J Bonhoeffer E B Imoukhuede G Aldrovandi et al ldquoTemplateprotocol for clinical trials investigating vaccines-focus on safetyelementsrdquo Vaccine vol 31 no 47 pp 5602ndash5620 2013
[114] S Jackson and A Storey ldquoE6 proteins from diverse cutaneonsHPV types inhibit apoptosis in response to UV damagerdquoOncogene vol 19 no 4 pp 592ndash598 2000
[115] M Thomas and L Banks ldquoPDZRN3LNX3 is a novel targetof human papillomavirus type 16 (HPV-16) and HPV-18 E6rdquoJournal of Virology vol 89 no 2 pp 1439ndash1444 2015
[116] P R Daniels C M Sanders P Coulson and N J MaitlandldquoMolecular analysis of the interaction between HPV type 16 E6and human E6-associated proteinrdquo FEBS Letters vol 416 no 1pp 6ndash10 1997
[117] W H Kao S L Beaudenon A L Talis J M Huibregtse andP M Howley ldquoHuman papillomavirus type 16 E6 induces self-ubiquitination of the E6AP ubiquitin-protein ligaserdquo Journal ofVirology vol 74 no 14 pp 6408ndash6417 2000
[118] T Iftner M Elbel B Schopp et al ldquoInterference of papillo-mavirus E6 protein with single-strand break repair by interac-tion with XRCC1rdquo The EMBO Journal vol 21 no 17 pp 4741ndash4748 2002
[119] Y Imai Y Tsunokawa T Sugimura and M Terada ldquoPurifi-cation and DNA-binding properties of human papillomavirustype 16 E6 protein expressed in Escherichia colirdquo Biochemicaland Biophysical Research Communications vol 164 no 3 pp1402ndash1410 1989
[120] M Lusky L Berg H Weiher and M Botchan ldquoBovinepapilloma virus contains an activator of gene expression at thedistal end of the early transcription unitrdquoMolecular andCellularBiology vol 3 no 6 pp 1108ndash1122 1983
[121] S Dalal Q Gao E J Androphy and V Band ldquoMutationalanalysis of human papillomavirus type 16 E6 demonstrates thatp53 degradation is necessary for immortalization of mammaryepithelial cellsrdquo Journal of Virology vol 70 no 2 pp 683ndash6881996
[122] J M Huibregtse M Scheffner and P M Howley ldquoA cellularprotein mediates association of p53 with the E6 oncoprotein ofhuman papillomavirus types 16 or 18rdquo The EMBO Journal vol10 no 13 pp 4129ndash4135 1991
[123] M Scheffner B A Werness J M Huibregtse A J Levine andP M Howley ldquoThe E6 oncoprotein encoded by human papillo-mavirus types 16 and 18 promotes the degradation of p53rdquo Cellvol 63 no 6 pp 1129ndash1136 1990
[124] M Elbel S Carl S Spaderna and T Iftner ldquoA comparativeanalysis of the interactions of the E6 proteins from cutaneousand genital papillomaviruses with p53 and E6AP in correlationto their transforming potentialrdquoVirology vol 239 no 1 pp 132ndash149 1997
[125] M C Thomas and C-M Chiang ldquoE6 oncoprotein repressesp53-dependent gene activation via inhibition of protein acety-lation independently of inducing p53 degradationrdquo MolecularCell vol 17 no 2 pp 251ndash264 2005
[126] H Zimmermann R Degenkolbe H-U Bernard and M JOrsquoConnor ldquoThe human papillomavirus type 16 E6 oncoproteincan down-regulate p53 activity by targeting the transcriptionalcoactivator CBPp300rdquo Journal of Virology vol 73 no 8 pp6209ndash6219 1999
[127] D A Thompson G Belinsky T H-T Chang D L Jones RSchlegel and K Munger ldquoThe human papillomavirus-16 E6oncoprotein decreases the vigilance of mitotic checkpointsrdquoOncogene vol 15 no 25 pp 3025ndash3035 1997
[128] J Bohl K Das B Dasgupta and S B Vande Pol ldquoCompetitivebinding to a charged leucine motif represses transformation bya papillomavirus E6 oncoproteinrdquo Virology vol 271 no 1 pp163ndash170 2000
[129] S Song H C Pitot and P F Lambert ldquoThe human papillo-mavirus type 16 E6 gene alone is sufficient to induce carcinomasin transgenic animalsrdquo Journal of Virology vol 73 no 7 pp5887ndash5893 1999
[130] S Yasumoto A L Burkhardt J Doniger and J A DiPaololdquoHumanpapillomavirus type 16DNA-inducedmalignant trans-formation of NIH 3T3 cellsrdquo Journal of Virology vol 57 no 2pp 572ndash577 1986
[131] S B Vande Pol M C Brown and C E Turner ldquoAssociation ofBovine Papillomavirus Type 1 E6 oncoprotein with the focaladhesion protein paxillin through a conserved protein interac-tion motifrdquo Oncogene vol 16 no 1 pp 43ndash52 1998
[132] R Wade N Brimer and S Vande Pol ldquoTransformation bybovine papillomavirus type 1 E6 requires paxillinrdquo Journal ofVirology vol 82 no 12 pp 5962ndash5966 2008
[133] A J Klingelhutz S A Foster and J KMcDougall ldquoTelomeraseactivation by the E6 gene product of humanpapillomavirus type16rdquo Nature vol 380 no 6569 pp 79ndash82 1996
[134] S T Oh S Kyo and L A Laimins ldquoTelomerase activation byhuman papillomavirus type 16 E6 protein Induction of humantelomerase reverse transcriptase expression through Myc andGC-rich Sp1 binding sitesrdquo Journal of Virology vol 75 no 12pp 5559ndash5566 2001
[135] L V Ronco A Y KarpovaMVidal and PMHowley ldquoHumanpapillomavirus 16 E6 oncoprotein binds to interferon regulatoryfactor-3 and inhibits its transcriptional activityrdquo Genes andDevelopment vol 12 no 13 pp 2061ndash2072 1998
and interaction between cigarette smoking and progression tomalignant transformationrdquo Acta Cytologica vol 42 no 3 pp639ndash649 1998
[82] S M Germann V Schramke R T Pedersen et al ldquoTopBP1Dpb11 bindsDNA anaphase bridges to prevent genome instabil-ityrdquoThe Journal of Cell Biology vol 204 no 1 pp 45ndash59 2014
[83] D R Hoffelder L Luo N A Burke S CWatkins S M Gollinand W S Saunders ldquoResolution of anaphase bridges in cancercellsrdquo Chromosoma vol 112 no 8 pp 389ndash397 2004
[84] C Acilan D M Potter and W S Saunders ldquoDNA repairpathways involved in anaphase bridge formationrdquo Genes Chro-mosomes and Cancer vol 46 no 6 pp 522ndash531 2007
[85] J DeMasi M C Chao A S Kumar and P M Howley ldquoBovinepapillomavirus E7 oncoprotein inhibits anoikisrdquo Journal ofVirology vol 81 no 17 pp 9419ndash9425 2007
[86] J DeMasi K-W Huh Y Nakatani K Munger and P MHowley ldquoBovine papillomavirus E7 transformation functioncorrelates with cellular p600 protein bindingrdquo Proceedings of theNational Academy of Sciences of theUnited States of America vol102 no 32 pp 11486ndash11491 2005
[87] E A White and P M Howley ldquoProteomic approaches to thestudy of papillomavirusndashhost interactionsrdquo Virology vol 435no 1 pp 57ndash69 2013
[88] FMantovani and L Banks ldquoThe human papillomavirus E6 pro-tein and its contribution to malignant progressionrdquo Oncogenevol 20 no 54 pp 7874ndash7887 2001
[89] P Chen and H Lee ldquoFOXM1 induced by E6 oncoprotein pro-motes tumor invasion and chemoresistance in HPV-infectedlung cancerrdquo Cancer Cell amp Microenvironment vol 2 no 1 pp6ndash9 2015
[90] N Zhang P Wei A Gong et al ldquoFoxM1 promotes 120573-cateninnuclear localization and controls Wnt target-gene expressionand glioma tumorigenesisrdquo Cancer Cell vol 127 no 4 pp 358ndash366 2012
[91] Z Liu Y Liu Y Hong L Rapp E J Androphy and J JChen ldquoBovine papillomavirus type 1 E6-induced sensitizationto apoptosis is distinct from its transforming activityrdquo Virologyvol 295 no 2 pp 230ndash237 2002
[92] M Thomas P Massimi C Navarro J-P Borg and L BanksldquoThe hScribDlg apico-basal control complex is differentiallytargeted by HPV-16 and HPV-18 E6 proteinsrdquo Oncogene vol24 no 41 pp 6222ndash6230 2005
[93] C H Storrs and S J Silverstein ldquoPATJ a tight junction-associated PDZ protein is a novel degradation target of high-risk human papillomavirus E6 and the alternatively splicedisoform 18 E6rdquo Journal of Virology vol 81 no 8 pp 4080ndash40902007
[94] R Accardi R Rubino M Scalise et al ldquoE6 and E7 from humanpapillomavirus type 16 cooperate to target the PDZ proteinNaH exchange regulatory factor 1rdquo Journal of Virology vol 85no 16 pp 8208ndash8216 2011
[95] S Nakagawa and J M Huibregtse ldquoHuman scribble (Vartul)is targeted for ubiquitin-mediated degradation by the high-risk papillomavirus E6 proteins and the E6APubiquitin-proteinligaserdquoMolecular and Cellular Biology vol 20 no 21 pp 8244ndash8253 2000
[96] L Gewin H Myers T Kiyono and D A Galloway ldquoIdentifi-cation of a novel telomerase repressor that interacts with thehuman papillomavirus type-16 E6E6-AP complexrdquo Genes ampDevelopment vol 18 no 18 pp 2269ndash2282 2004
[97] M Sundaram D L GuernseyMM Rajaraman and R Rajara-man ldquoNeosis a novel type of cell division in cancerrdquo CancerBiology ampTherapy vol 3 no 2 pp 207ndash218 2004
[98] R D Snyder and J W Green ldquoA review of the genotoxicity ofmarketed pharmaceuticalsrdquo Mutation Research vol 488 no 2pp 151ndash169 2001
[99] T Ristriani Y Nomine C Laurent E Weiss and G TraveldquoProtein mutagenesis with monodispersity-based quality prob-ing selective inactivation of p53 degradation and DNA-bindingproperties of HPV E6 oncoproteinrdquo Protein Expression andPurification vol 26 no 3 pp 357ndash367 2002
[100] M Kadaja A Sumerina T Verst M Ojarand E Ustav and MUstav ldquoGenomic instability of the host cell induced by thehuman papillomavirus replicationmachineryrdquoTheEMBO Jour-nal vol 26 no 8 pp 2180ndash2191 2007
[101] PMDay D R Lowy and J T Schiller ldquoPapillomaviruses infectcells via a clathrin-dependent pathwayrdquo Virology vol 307 no 1pp 1ndash11 2003
[102] T D Tlsty A Briot A Gualberto et al ldquoGenomic instabilityand cancerrdquoMutation Research vol 337 no 1 pp 1ndash7 1995
[103] J E Eyfjord and S K Bodvarsdottir ldquoGenomic instabilityand cancer networks involved in response to DNA damagerdquoMutation Research vol 592 no 1-2 pp 18ndash28 2005
[104] Y Yao W Huang X Yang et al ldquoHPV-16 E6 and E7 protein Tcell epitopes prediction analysis based on distributions of HLA-A loci across populations an in silico approachrdquo Vaccine vol31 no 18 pp 2289ndash2294 2013
[105] C-F Hung B Ma A Monie S-W Tsen and T-C Wu ldquoTher-apeutic human papillomavirus vaccines current clinical trialsand future directionsrdquo Expert Opinion on Biological Therapyvol 8 no 4 pp 421ndash439 2008
[106] M J P Welters G G Kenter S J Piersma et al ldquoInductionof tumor-specific CD4+ and CD8+ T-cell immunity in cervicalcancer patients by a human papillomavirus type 16 E6 and E7long peptides vaccinerdquo Clinical Cancer Research vol 14 no 1pp 178ndash187 2008
[107] R Han N M Cladel C A Reed X Peng and N D Chris-tensen ldquoProtection of rabbits from viral challenge by gene gun-based intracutaneous vaccination with a combination of cot-tontail rabbit papillomavirus E1 E2 E6 and E7 genesrdquo Journalof Virology vol 73 no 8 pp 7039ndash7043 1999
[108] Z He A P Wlazlo D W Kowalczyk et al ldquoViral recombinantvaccines to the E6 and E7 antigens of HPV-16rdquo Virology vol270 no 1 pp 146ndash161 2000
[109] A Kaufmann P Stern E Rankin et al ldquoSafety and immuno-genicity of TA-HPV a recombinant vaccinia virus expressingmodified human papillomavirus (HPV)-16 and HPV-18 E6 andE7 genes in women with progressive cervical cancerrdquo ClinicalCancer Research vol 8 no 12 pp 3676ndash3685 2002
[110] L K Borysiewicz A Fiander M Nimako et al ldquoA recombinantvaccinia virus encoding human papillomavirus types 16 and 18E6 and E7 proteins as immunotherapy for cervical cancerrdquoTheLancet vol 347 no 9014 pp 1523ndash1527 1996
[111] A De Jong T OrsquoNeill A Y Khan et al ldquoEnhancement ofhuman papillomavirus (HPV) type 16 E6 and E7-specific T-cellimmunity in healthy volunteers through vaccination with TA-CIN an HPV16 L2E7E6 fusion protein vaccinerdquo Vaccine vol20 no 29-30 pp 3456ndash3464 2002
[112] S Chen C Liao Y Lai et al ldquoDe-oncogenicHPVE6E7 vaccinegets enhanced antigenicity and promotes tumoricidal synergywith cisplatinrdquo Acta Biochimica et Biophysica Sinica vol 46 no1 pp 6ndash14 2014
BioMed Research International 15
[113] J Bonhoeffer E B Imoukhuede G Aldrovandi et al ldquoTemplateprotocol for clinical trials investigating vaccines-focus on safetyelementsrdquo Vaccine vol 31 no 47 pp 5602ndash5620 2013
[114] S Jackson and A Storey ldquoE6 proteins from diverse cutaneonsHPV types inhibit apoptosis in response to UV damagerdquoOncogene vol 19 no 4 pp 592ndash598 2000
[115] M Thomas and L Banks ldquoPDZRN3LNX3 is a novel targetof human papillomavirus type 16 (HPV-16) and HPV-18 E6rdquoJournal of Virology vol 89 no 2 pp 1439ndash1444 2015
[116] P R Daniels C M Sanders P Coulson and N J MaitlandldquoMolecular analysis of the interaction between HPV type 16 E6and human E6-associated proteinrdquo FEBS Letters vol 416 no 1pp 6ndash10 1997
[117] W H Kao S L Beaudenon A L Talis J M Huibregtse andP M Howley ldquoHuman papillomavirus type 16 E6 induces self-ubiquitination of the E6AP ubiquitin-protein ligaserdquo Journal ofVirology vol 74 no 14 pp 6408ndash6417 2000
[118] T Iftner M Elbel B Schopp et al ldquoInterference of papillo-mavirus E6 protein with single-strand break repair by interac-tion with XRCC1rdquo The EMBO Journal vol 21 no 17 pp 4741ndash4748 2002
[119] Y Imai Y Tsunokawa T Sugimura and M Terada ldquoPurifi-cation and DNA-binding properties of human papillomavirustype 16 E6 protein expressed in Escherichia colirdquo Biochemicaland Biophysical Research Communications vol 164 no 3 pp1402ndash1410 1989
[120] M Lusky L Berg H Weiher and M Botchan ldquoBovinepapilloma virus contains an activator of gene expression at thedistal end of the early transcription unitrdquoMolecular andCellularBiology vol 3 no 6 pp 1108ndash1122 1983
[121] S Dalal Q Gao E J Androphy and V Band ldquoMutationalanalysis of human papillomavirus type 16 E6 demonstrates thatp53 degradation is necessary for immortalization of mammaryepithelial cellsrdquo Journal of Virology vol 70 no 2 pp 683ndash6881996
[122] J M Huibregtse M Scheffner and P M Howley ldquoA cellularprotein mediates association of p53 with the E6 oncoprotein ofhuman papillomavirus types 16 or 18rdquo The EMBO Journal vol10 no 13 pp 4129ndash4135 1991
[123] M Scheffner B A Werness J M Huibregtse A J Levine andP M Howley ldquoThe E6 oncoprotein encoded by human papillo-mavirus types 16 and 18 promotes the degradation of p53rdquo Cellvol 63 no 6 pp 1129ndash1136 1990
[124] M Elbel S Carl S Spaderna and T Iftner ldquoA comparativeanalysis of the interactions of the E6 proteins from cutaneousand genital papillomaviruses with p53 and E6AP in correlationto their transforming potentialrdquoVirology vol 239 no 1 pp 132ndash149 1997
[125] M C Thomas and C-M Chiang ldquoE6 oncoprotein repressesp53-dependent gene activation via inhibition of protein acety-lation independently of inducing p53 degradationrdquo MolecularCell vol 17 no 2 pp 251ndash264 2005
[126] H Zimmermann R Degenkolbe H-U Bernard and M JOrsquoConnor ldquoThe human papillomavirus type 16 E6 oncoproteincan down-regulate p53 activity by targeting the transcriptionalcoactivator CBPp300rdquo Journal of Virology vol 73 no 8 pp6209ndash6219 1999
[127] D A Thompson G Belinsky T H-T Chang D L Jones RSchlegel and K Munger ldquoThe human papillomavirus-16 E6oncoprotein decreases the vigilance of mitotic checkpointsrdquoOncogene vol 15 no 25 pp 3025ndash3035 1997
[128] J Bohl K Das B Dasgupta and S B Vande Pol ldquoCompetitivebinding to a charged leucine motif represses transformation bya papillomavirus E6 oncoproteinrdquo Virology vol 271 no 1 pp163ndash170 2000
[129] S Song H C Pitot and P F Lambert ldquoThe human papillo-mavirus type 16 E6 gene alone is sufficient to induce carcinomasin transgenic animalsrdquo Journal of Virology vol 73 no 7 pp5887ndash5893 1999
[130] S Yasumoto A L Burkhardt J Doniger and J A DiPaololdquoHumanpapillomavirus type 16DNA-inducedmalignant trans-formation of NIH 3T3 cellsrdquo Journal of Virology vol 57 no 2pp 572ndash577 1986
[131] S B Vande Pol M C Brown and C E Turner ldquoAssociation ofBovine Papillomavirus Type 1 E6 oncoprotein with the focaladhesion protein paxillin through a conserved protein interac-tion motifrdquo Oncogene vol 16 no 1 pp 43ndash52 1998
[132] R Wade N Brimer and S Vande Pol ldquoTransformation bybovine papillomavirus type 1 E6 requires paxillinrdquo Journal ofVirology vol 82 no 12 pp 5962ndash5966 2008
[133] A J Klingelhutz S A Foster and J KMcDougall ldquoTelomeraseactivation by the E6 gene product of humanpapillomavirus type16rdquo Nature vol 380 no 6569 pp 79ndash82 1996
[134] S T Oh S Kyo and L A Laimins ldquoTelomerase activation byhuman papillomavirus type 16 E6 protein Induction of humantelomerase reverse transcriptase expression through Myc andGC-rich Sp1 binding sitesrdquo Journal of Virology vol 75 no 12pp 5559ndash5566 2001
[135] L V Ronco A Y KarpovaMVidal and PMHowley ldquoHumanpapillomavirus 16 E6 oncoprotein binds to interferon regulatoryfactor-3 and inhibits its transcriptional activityrdquo Genes andDevelopment vol 12 no 13 pp 2061ndash2072 1998
[113] J Bonhoeffer E B Imoukhuede G Aldrovandi et al ldquoTemplateprotocol for clinical trials investigating vaccines-focus on safetyelementsrdquo Vaccine vol 31 no 47 pp 5602ndash5620 2013
[114] S Jackson and A Storey ldquoE6 proteins from diverse cutaneonsHPV types inhibit apoptosis in response to UV damagerdquoOncogene vol 19 no 4 pp 592ndash598 2000
[115] M Thomas and L Banks ldquoPDZRN3LNX3 is a novel targetof human papillomavirus type 16 (HPV-16) and HPV-18 E6rdquoJournal of Virology vol 89 no 2 pp 1439ndash1444 2015
[116] P R Daniels C M Sanders P Coulson and N J MaitlandldquoMolecular analysis of the interaction between HPV type 16 E6and human E6-associated proteinrdquo FEBS Letters vol 416 no 1pp 6ndash10 1997
[117] W H Kao S L Beaudenon A L Talis J M Huibregtse andP M Howley ldquoHuman papillomavirus type 16 E6 induces self-ubiquitination of the E6AP ubiquitin-protein ligaserdquo Journal ofVirology vol 74 no 14 pp 6408ndash6417 2000
[118] T Iftner M Elbel B Schopp et al ldquoInterference of papillo-mavirus E6 protein with single-strand break repair by interac-tion with XRCC1rdquo The EMBO Journal vol 21 no 17 pp 4741ndash4748 2002
[119] Y Imai Y Tsunokawa T Sugimura and M Terada ldquoPurifi-cation and DNA-binding properties of human papillomavirustype 16 E6 protein expressed in Escherichia colirdquo Biochemicaland Biophysical Research Communications vol 164 no 3 pp1402ndash1410 1989
[120] M Lusky L Berg H Weiher and M Botchan ldquoBovinepapilloma virus contains an activator of gene expression at thedistal end of the early transcription unitrdquoMolecular andCellularBiology vol 3 no 6 pp 1108ndash1122 1983
[121] S Dalal Q Gao E J Androphy and V Band ldquoMutationalanalysis of human papillomavirus type 16 E6 demonstrates thatp53 degradation is necessary for immortalization of mammaryepithelial cellsrdquo Journal of Virology vol 70 no 2 pp 683ndash6881996
[122] J M Huibregtse M Scheffner and P M Howley ldquoA cellularprotein mediates association of p53 with the E6 oncoprotein ofhuman papillomavirus types 16 or 18rdquo The EMBO Journal vol10 no 13 pp 4129ndash4135 1991
[123] M Scheffner B A Werness J M Huibregtse A J Levine andP M Howley ldquoThe E6 oncoprotein encoded by human papillo-mavirus types 16 and 18 promotes the degradation of p53rdquo Cellvol 63 no 6 pp 1129ndash1136 1990
[124] M Elbel S Carl S Spaderna and T Iftner ldquoA comparativeanalysis of the interactions of the E6 proteins from cutaneousand genital papillomaviruses with p53 and E6AP in correlationto their transforming potentialrdquoVirology vol 239 no 1 pp 132ndash149 1997
[125] M C Thomas and C-M Chiang ldquoE6 oncoprotein repressesp53-dependent gene activation via inhibition of protein acety-lation independently of inducing p53 degradationrdquo MolecularCell vol 17 no 2 pp 251ndash264 2005
[126] H Zimmermann R Degenkolbe H-U Bernard and M JOrsquoConnor ldquoThe human papillomavirus type 16 E6 oncoproteincan down-regulate p53 activity by targeting the transcriptionalcoactivator CBPp300rdquo Journal of Virology vol 73 no 8 pp6209ndash6219 1999
[127] D A Thompson G Belinsky T H-T Chang D L Jones RSchlegel and K Munger ldquoThe human papillomavirus-16 E6oncoprotein decreases the vigilance of mitotic checkpointsrdquoOncogene vol 15 no 25 pp 3025ndash3035 1997
[128] J Bohl K Das B Dasgupta and S B Vande Pol ldquoCompetitivebinding to a charged leucine motif represses transformation bya papillomavirus E6 oncoproteinrdquo Virology vol 271 no 1 pp163ndash170 2000
[129] S Song H C Pitot and P F Lambert ldquoThe human papillo-mavirus type 16 E6 gene alone is sufficient to induce carcinomasin transgenic animalsrdquo Journal of Virology vol 73 no 7 pp5887ndash5893 1999
[130] S Yasumoto A L Burkhardt J Doniger and J A DiPaololdquoHumanpapillomavirus type 16DNA-inducedmalignant trans-formation of NIH 3T3 cellsrdquo Journal of Virology vol 57 no 2pp 572ndash577 1986
[131] S B Vande Pol M C Brown and C E Turner ldquoAssociation ofBovine Papillomavirus Type 1 E6 oncoprotein with the focaladhesion protein paxillin through a conserved protein interac-tion motifrdquo Oncogene vol 16 no 1 pp 43ndash52 1998
[132] R Wade N Brimer and S Vande Pol ldquoTransformation bybovine papillomavirus type 1 E6 requires paxillinrdquo Journal ofVirology vol 82 no 12 pp 5962ndash5966 2008
[133] A J Klingelhutz S A Foster and J KMcDougall ldquoTelomeraseactivation by the E6 gene product of humanpapillomavirus type16rdquo Nature vol 380 no 6569 pp 79ndash82 1996
[134] S T Oh S Kyo and L A Laimins ldquoTelomerase activation byhuman papillomavirus type 16 E6 protein Induction of humantelomerase reverse transcriptase expression through Myc andGC-rich Sp1 binding sitesrdquo Journal of Virology vol 75 no 12pp 5559ndash5566 2001
[135] L V Ronco A Y KarpovaMVidal and PMHowley ldquoHumanpapillomavirus 16 E6 oncoprotein binds to interferon regulatoryfactor-3 and inhibits its transcriptional activityrdquo Genes andDevelopment vol 12 no 13 pp 2061ndash2072 1998
