Hindawi Publishing CorporationISRN OncologyVolume 2013 Article ID 910849 8 pageshttpdxdoiorg1011552013910849
Research ArticleChromosome Aberrations in Cells Infected withBovine Papillomavirus Comparing Cutaneous PapillomaEsophagus Papilloma and Urinary Bladder Lesion Cells
S R C Campos1 T C Melo12 S Assaf1 R P Araldi13 J Mazzuchelli-de-Souza13
M P Sircili13 R F Carvalho13 F Roperto4 W Beccedilak15 and R C Stocco13
1 Laboratorio de Genetica Instituto Butantan Avenida Vital Brasil 1500 Butanta 05503-900 Sao Paulo SP Brazil2 Programa de Pos-graduacao em Biologia Estrutural e Funcional Universidade Federal de Sao Paulo Rua Botucatu740 04023-900 Sao Paulo SP Brazil
3 Programa de Pos-graduacao Interunidades em Biotecnologia Instituto de Ciencias Biomedicas Universidade de Sao PauloAvenida Prof Lineu Prestes 2415 Edifıcio ICB-III-Cidade Universitaria 05508-900 Sao Paulo SP Brazil
4Department of Biology Naples University Federico II Via Mezzocannone 16 80134 Naples Italy5 Departamento de Biologia Universidade Federal da Integracao Latino-Americana (UNILA) Avenida Tancredo Neves6731 bloco 4 85867-970 Foz do Iguacu PR Brazil
Correspondence should be addressed to R C Stocco ritastoccobutantangovbr
Received 3 June 2013 Accepted 2 July 2013
Academic Editors K Sonoda M Stracke and K van Golen
Copyright copy 2013 S R C Campos et alThis is an open access article distributed under theCreative CommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited
Themajority of malignant cells present genetic instability with chromosome number changes plus segmental defects these changesinvolve intact chromosomes and breakage-induced alterations Some pathways of chromosomal instability have been proposedas random breakage telomere fusion and centromere fission Chromosome alterations in tumor cells have been described inanimal models and in vitro experiments One important question is about possible discrepancies between animal models in vitrostudies and the real events in cancer cells in vivo Papillomaviruses are relevant agents in oncogenic processes related to action onhost genome Recently many reports have discussed the presence of virus DNA in peripheral blood in humans and in animalsinfected by papillomaviruses The meaning of this event is of controversy possible product of apoptosis occurring in cancer cellsmetastasized cancer cells or active DNA sequences circulating in bloodstream This study compares chromosome aberrationsdetected in bovine cells in peripheral blood cells and in BPV lesion cells the literature is poor in this type of study Comparingchromosome aberrations described in the different cells a common mechanism in their origin can be suggested Furthermoreblood cells can be evaluated as an effective way of virus transmission
1 Introduction
The papillomaviruses (PVs) are viruses that require the envi-ronment of a differentiating epithelium for their replicationcycle [1] PVs infectmammals includingman and are relatedto development of benign lesions that can progress to cancer[2] Uterine cervical cancer the second most frequentlyoccurring cancer in women worldwide is causal related tohuman papillomavirus infection (HPV) [3 4]
The Papillomavirus genome comprises three regionsLCR ldquolong control regionrdquo responsible for genome transcrip-tion control L region encoding the capsid major and minor
proteins (L1 and L2 resp) presenting late transcription invirus replication cycle and E region with early transcriptionin virus cycle which codifies the proteins related to car-cinogenic action [5 6] The virus transmission is recognizedas occurring through direct contact the abrasion of theskin or sexual intercourse leads to PV infection [7] PVoncoproteins are the source for the alterations related tocarcinogenesis they interfere with the host cell cycle controlthrough interactions with specific proteins as p53 p RBp21 and p27 [8] As examples of viral oncoprotein actionsE6 induces accelerated degradation of p53 [9] E7 binds anddegrades p RB and interacts with p21 and p27 [10]
2 ISRN Oncology
Papillomaviruses maintain their genomes in episomalcondition linked to host cell chromosomes during celldivision and being distributed in dividing cells [11] Thepapillomavirus E2 protein simultaneously associates the hostchromatin and the viral genomeduringmitosis [12]The virusin its episomal form is found in benign lesions and E2 alsoacts in the integration of virus sequences in host chromatinof neoplasic cells These cells are tightly associated with theexpression of high-risk human papillomavirus (HPV) onco-genes E6 and E7 and exhibit chromosomal instability [13]
The virus is specific to epithelium but recently manyreports have described virus DNA sequences in blood stream[14ndash21] The exact source of these sequences is not clearcirculating virus DNA has been discussed as product of lysesof circulating cancer cells or micrometastasis from the tumor[18] Bovine Papillomavirus (BPV) DNA was simultaneouslydetected in different tissues of the same animals includingblood thus suggesting a blood stream virus spread [17 22ndash24]
It has been speculated that blood mononuclear cells thatmigrate to sites of tissue inflammation could act as a sourceof virus (BPV eg) in the infection of epithelial cells andcould become involved in tumor development independentlyor jointly with several biological andor chemical cofactors[14 20]
The seminar questions are as follows could thesesequences represent virus replicate status in the bloodstreamin particular in the lymphocytes acting as a reservoir of viralinfection Can the viruses present oncogene expression inother tissues
Papillomavirus DNA sequences have been identified inblood stream of specifically bovines in recent reports [16ndash19 25 26] The studies analyzed whole blood plasmaisolated lymphocytes and short-term peripheral lymphocytecultures the BPV DNA was detected in all systems [15 1718 22 23] Besides BPV DNA was detected also in semenuterus urine milk and seminal fluid [17] Furthermorethe cytogenetic analysis performed in short-term peripherallymphocyte cultures revealed significant increase of chromo-some aberrations in samples from infected animals [15ndash18]The mechanisms of virus action on host chromatin becameimportant issue of study
In this study we analyzed the levels and the types ofchromosome aberrations verified in cultured cells obtainedin samples collected from peripheral blood cutaneous papil-loma esophagus papilloma and urinary bladder cells ofanimal presenting enzootic hematuria The samples werecollected from a bovine infected with bovine papillomavirusand normal controls Short-term lymphocyte cultures wereperformed with the blood samples and the lesion fragmentswere used to obtain primary cell cultures
2 Material and Methods
21 Ethical Statements The protocols used in this studywere approved by the Ethical Committee in Research of theUniversidade Federal de Sao Paulo (number 083507) assignedby the president of this committee All efforts are made tominimize any suffering in the animals
22 Selection of Animals Nine animals (Bos taurus) wereselected for this study 2males and 7 females comparable ageThe animals were submitted to clinical evaluation performedby a veterinary All procedures followed the ethical principlesThe animal group included 2 animals clinically normal 3presented severe cutaneous papillomatosis 2 had esophaguspapillomas and 2 were affected by enzootic hematuriapresenting papilloma like lesion in the bladder
23 Samples Blood samples were collected in two sterilesyringes with heparin or EDTA respectively for cultures andDNA extraction Tissue fragments were obtained by surgicalprocedures performed by a veterinary with local anesthesia
24Histological Analysis For histological analysis fragmentsof the samples obtained from the solid lesions were fixed in10 neutral buffered formalin and embedded in paraffinwax5 120583m ticked sections were stained with hematoxylineosin
25 Short-Term Lymphocyte Cultures Eighteen drops ofblood were incubated in RPMI medium supplied with 10fetal serum and 2 phytohemagglutinin during 72 hours at37∘C
26 Primary Cell Cultures The tissue fragments were washedin PBS submitted to collagenase 001 30minutes 37∘C andincubated in Dulbecco MEM supplied with 10 fetal serumin CO
2atmosphere at 37∘C
27 Cytogenetic Preparations Colchicine 16 120583gmL 01mLwas added to culture cells for 1 hour at 37∘C The cells weresubmitted to hypotonic solution KCl 0075M at 37∘C 10minutes After that the cells were fixed in three baths with3 1 methanol acetic acid The slides were stained in Giemsa3 5 minutes
28 Cytogenetic Analysis The cells of each culture typewere analyzed and recorded in Photomicroscopy AxiophotZeiss Each type of chromosome aberration was recordedaccording to Melo et al [19]
29 Statistical Analysis The cytogenetic data were statisti-cally analyzed using Students 119905-test [26]
210 Virus Identification
2101 Tissue and Blood DNA Extraction The DNA extrac-tion was performed using the Cell amp Tissue Kit IllustraGenomicPrep Mini Spin (GE Healthcare BuckinghamshireUK) and the extracted DNA was kept at minus20∘C The qualityof obtained DNA samples was verified using the polymerasechain reaction (PCR) technique by means of the amplifi-cation of a bovine 120573-globin gene fragment (450 bp) withspecific primers (F 51015840-aacctctttgttcacaac cag-31015840 and R 51015840-cagatgcttaacccactgagc-31015840) according to Yaguiu et al [24]
211 Viral Identification Viral molecular identification wasperformed using the degenerate primers FAP59 (forward
ISRN Oncology 3
Table 1 Chromosome aberration in peripheral blood cells and primary culture cells C control group 1 cutaneous papillomatosis 2esophagus papillomas 3 enzootic hematuria
Group Total cells Numerical aberrations(mean plusmn SE)
119875 ge 005 lowastlowast119875 ge 001 curren119875 ge 01 sectcells presenting only gaps were evaluated isolate
51015840-TAACWGTIGGICAYCCWTATT-31015840) and FAP64 (reverse51015840-CCWATATCWVHCATITCICCATC-31015840) which promoteL1 gene amplification resulting in a fragment of 478 bpOgawa et al 2004 [27] In detail the amplification reac-tions were performed in a PTC-100TM (MJ Research Inc)thermo cycler with PCR Master Mix (Promega MadisonUSA) under the following conditions 10min at 94∘Cfollowed by 45 cycles of 1min and 30 s at 94∘C 2minat 52∘C and 1min and 30 s at 72∘C and a final exten-sion step of 5min at 72∘C for primer FAP59FAP64For specific BPV1 2 and 4 BPV-1 (5-ggagcgcctgctaactat agg a-3101584051015840-atctgttgtttgggtggtgac-31015840) BPV-2 (51015840-gttataccaccc aaagaagaccct-3101584051015840-ctggttgcaacagctctctttctc-31015840) and BPV-4 (51015840-gctgaccttccagtctta atmdash3101584051015840-cag tttcaatctcctcttca-31015840) indetail the amplification reactions were performed in aPTC-100TM (MJ Research Inc) thermo cycler with PCRMaster Mix (Promega Madison USA) under the followingconditions 3min at 94∘C followed by 35 cycles of 50 s at94∘C 1min at 60∘C and 1min at 72∘C and a final extensionstep of 5min at 72∘C The PCR products were analyzed in2 agarose gel electrophoresis stained with GelRed in TAEbuffer visualized under UV light
3 Results and Discussion
31 Results The histological analysis confirmed the identifi-cation of the fragments collected for the studies (Figure 1)Specifically a segment of a normal skin obtained by surgicalprocedure from a normal bovine was selected as control Bysimilar procedures biopsies were obtained from cutaneouspapilloma esophagus papilloma and papilloma fromurinarybladder of an animal affected with enzootic hematuria
The PCR technique using primers FAP 5964 amplifieda DNA fragment 474 bp long indicating virus sequencepresence in the biopsies collected frompapilloma lesionsThespecific primers allowed the identification of BPV1 (301 bp)and BPV2 (164 bp) (Figure 2) Only one animal presented
BPV4 (170 bp) in cutaneous papilloma Also the same virussequences were detected in the obtained primary cell culturesand in their first five passages (Figures 2 and 3)The samePCRprocedures with the same primers were used to investigatethe presence of virus sequences in blood samples but it wasnot possible to detect any virus sequence in any of the bloodsamples normal or affected animals
The Figure 3 presents cells in primary cultures The cellmorphology did not differ among the different lines
For cytogenetic analysis four groups of animals werefixed according to the clinical features C control (notaffected and without detection of virus DNA in culture cells)Group 1 cutaneous papillomaGroup 2 esophagus papillomaGroup 3 enzootic hematuria A total of 1068 cells wereevaluated 501 derived from short-term lymphocyte culturesand 567 obtained from biopsy primary cell cultures (Tables 1and 2)
The results observed in culture blood cells make itpossible to verify that the affected animals showed higherlevels of chromosome aberrations compared to not affectedanimals As the PCR procedures were unable to detect BPVsequences in blood cells the data were analyzed as notaffected animals compared to affected bovines However thePCR technique showed BPV DNA sequences in the biopsiescollected from the affected animals such as the fragmentsfrom not affected animals that were negative for BPV inPCR procedures So the data were analyzed comparing BPVpositive and BPV negative biopsies and respective primaryculture cells It is important to emphasize that the BPV nega-tive biopsies were obtained from the not affected animals andso these animals were our controls A specific analysis wasperformed considering the different chromosome aberrationtypes comparing the previous groups (C 1 2 and 3) In bloodcells comparing to control the aberration types presentingsignificant higher levels in cells from affected animals wereadditiondeletion chromatid breaks acentric fragments andcentromere associations (Table 2)
4 ISRN Oncology
(a) (b)
(c)
10 120583m
(d)
Figure 1 Histological sections (a) normal skin fragment (b) cutaneous papilloma fragment (c) esophagus papilloma fragment and (d)fragment of papilloma collected from an urinary bladder of an animal affected by enzootic hematuria (HE) (10x)
474 pb
1 kb Cminus C+ A B C D E F G H I J
(a)
164 pb
1 kbCminus C+ A B C D E F G H I J L M
(b)
Figure 2 Example of BPV DNA sequences detected in cutaneous lesions and in respective primary culture cells the samples were collectedfrom one of the animals I primers FAP5964 II BPV2 specific primers Cminus negative control C+ positive control A-B PCR with DNA frompapilloma lesion CndashE primary cell culture using fragments from BPV positive lesion in passage 1 FndashH primary cell culture using fragmentsfrom BPV positive lesion in passage 2 I-J primary cell culture using fragments from BPV positive lesion in passage 3 L primary cell cultureusing fragments from BPV positive lesion in passage 4 and M primary cell culture using fragments from BPV positive lesion in passage 5 Itis important to pay attention in the different amplicons suggesting possible different virus load
Considering the primary culture cells the more fre-quent chromosome aberrationswere additiondeletion chro-matid break chromosome breaks acentric fragments cen-tromere association and association of telomeres (Table 2and Figure 4) It was possible to verify that the cells obtainedfrom the BPV infected animals presented significant higherlevels of chromosome aberrations
32 Discussion As far it was possible to verify this is the firstreport describing chromosome aberrations in cells derivedfrom bovine papillomavirus lesions
As we have previously described in peripheral lympho-cytes [15] the chromosome aberrations occur in significantincreased levels in short-term lymphocyte cultures and inprimary cell cultures established from samples obtainedfrom BPV affected animals
A very important point has to be discussed the factthat it was not possible to detect virus DNA sequencesin peripheral blood of affected or not affected animalsDespite this fact the level of chromosome aberrations wasverified higher in the affected animal samples As it was notpossible to identify virus DNA in blood either in control or
ISRN Oncology 5
(a) (b)
(c) (d)
Figure 3 Primary cell cultures (a) normal skin (b) cutaneous papilloma (c) esophagus papilloma (d) papilloma of urinary bladder fromanimal affected with enzootic hematuria (10x) The arrow indicates a cell with a different morphology
affected animals it could be discussed that the chromosomeaberrations were not caused by virus action However thesame types of aberrations were detected in cells derived fromlesions positive for virus presence Furthermore there wasa significant difference between the chromosome aberrationlevels comparing affected and not affected animals
Inmatter of fact considering the similarity of the detectedchromosome aberrations the virus action on host chromatinwas verified as effective either in blood cells or in cells derivedfrom lesions We could argue that the virus load in theperipheral blood of the affected animals was too low to bedetected in conventional PCR The same argument could beused for the not affected animals leading to the need tocompare affected to nonaffected animals and emphasizing thedifferences observed in the levels of chromosomal aberra-tions
The virus action was verified rising numerical and struc-tural chromosome aberrations This fact indicates that thevirus acts in different ways in its interaction with host
chromatin It was previously described the virus oncoproteinaction on telomeres leading to centric fusion [28] Besidesthe virus acts on mitotic spindle changing the chromosomeset and rising aneuploidy [29]
Although BPV is described as nonintegrated in host cellits action produces different types of chromosome alterationssuggesting a large interaction with the chromatin and alsowith DNA repair mechanisms [30]
We have already described the presence of virussequences in primary culture cells in different passages [31]but now we demonstrate that these sequences are activeleading to chromosome alterations
4 Conclusions
We compare for the first time the action of bovine papillo-mavirus on host cell chromatin in cultured lymphocytes andprimary culture cells describing the increase of chromosomalaberrations in both cell types The primary cells cultures
6 ISRN Oncology
(a) (b) (c)
(d) (e) (f)
(g) (h)
Figure 4 Chromosome aberrations in primary culture cells ((a) (c) (e) and (g)) and peripheral lymphocytes ((b) (d) (f) and (h)) acentricfragments ((a) (b)) chromosome break (c) chromatid break (d) chromosome rearrangement with additiondeletion ((e) (f)) telomericassociation (g) centromeric association ((e) (h)) early chromatid separation (thin arrow in (a) (e))
not only presented BPV DNA sequences but also keep thesesequences throughout different passages These data plusrecent reports [18] emphasize possible active BPV infectionin blood as well as in in vitro cultured cells
Conflict of Interests
All authors declare that there is no conflict of interestsregarding the research authorship andor publication of thispaper The authors inform that all materials and trademarks
mentioned were used just as part of experimental protocoland there are no financial gains or favoritism of business
Acknowledgments
The authors would like to thank Ministerio de CienciaTecnologia e InovacaoConselho Nacional de DesenvolvimentoCientıfico e Tecnologico (CNPq Proc 5548162006-7 and4025392011-7) FAPESP (Proc 200602439-6) Coordenacaode Aperfeicoamento Pessoal de Nıvel Superior (CAPES) and
ISRN Oncology 7
Table 2 Frequencies of different aberrations detected in blood and primary culture cells of animals affected by cutaneous papillomasesophagus papilloma and enzootic hematuria
BloodGroup CA AT AF CB CrBC 088 plusmn 124 248 plusmn 146 233 plusmn 081 175 plusmn 248 160 plusmn 022
119875 gt 005 lowastlowast119875 gt 001 and curren119875 ge 01
CA centromere association AT association of telomere AF acentric fragments CB chromosome break CrB chromatid break AdDel additiondeletionEcrS early chromatid separation Aneu aneuploidy Polip polyploidy and gaps
Fundacao Butantan for financial support and Carolina da PazSabino for editorial support
References
[1] G Borzacchiello and F Roperto ldquoBovine papillomavirusespapillomas and cancer in cattlerdquoVeterinary Research vol 39 no5 article 45 2008
[2] M S Campo ldquoBovine papillomavirus and cancerrdquo VeterinaryJournal vol 154 no 3 pp 175ndash188 1997
[3] 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
[4] S Duensing L Y Lee A Duensing et al ldquoThe human papillo-mavirus 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
[5] M Kadaja H Isok-Paas T Laos E Ustav and M UstavldquoMechanism of genomic instability in cells infected with thehigh-risk human papillomavirusesrdquo PLoS Pathogens vol 5 no4 2009
[6] N A Hamid C Brown and K Gaston ldquoThe regulation of cellproliferation by the papillomavirus early proteinsrdquo Cellular andMolecular Life Sciences vol 66 no 10 pp 1700ndash1717 2009
[7] V Kashyap and B C Das ldquoDNA aneuploidy and infectionof human papillomavirus type 16 in preneoplastic lesions ofthe uterine cervix correlation with progression to malignancyrdquoCancer Letters vol 123 no 1 pp 47ndash52 1998
[8] D Patel A Incassati N Wang and D J McCance ldquoHumanpapillomavirus type 16 E6 and E7 cause polyploidy in humankeratinocytes and up-regulation of G2-M-phase proteinsrdquoCan-cer Research vol 64 no 4 pp 1299ndash1306 2004
[9] G Boulet C Horvath D V Broeck S Sahebali and J BogersldquoHuman papillomavirus E6 and E7 oncogenesrdquo InternationalJournal of Biochemistry andCell Biology vol 39 no 11 pp 2006ndash2011 2007
[10] V OrsquoBrien G J Grindlay and M S Campo ldquoCell transfor-mation by the E5E8 protein of bovine papillomavirus type4 p27Kip1 elevated through increased protein synthesis issequestered by cyclin D1-CDK4 complexesrdquo Journal of Biolog-ical Chemistry vol 276 no 36 pp 33861ndash33868 2001
[11] A A McBride J G Oliveira and M G McPhillips ldquoPartition-ing viral genomes in mitosis same idea different targetsrdquo CellCycle vol 5 no 14 pp 1499ndash1502 2006
[12] J G Oliveira L A Colf and A A McBride ldquoVariationsin the association of papillomavirus E2 proteins with mitoticchromosomesrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 103 no 4 pp 1047ndash10522006
[13] S Duensing and K Munger ldquoHuman papillomaviruses andcentrosomeduplication errorsmodeling the origins of genomicinstabilityrdquo Oncogene vol 21 no 40 pp 6241ndash6248 2002
8 ISRN Oncology
[14] R C Recouso R C Stocco dos Santos R Freitas et al ldquoClasto-genic effect of bracken fern (Pteridium aquilinum aracnoideum)diet in peripheral lymphocytes of human consumers prelimi-nary datardquo Veterinary and Comparative Oncology vol 1 no 1pp 22ndash29 2003
[15] R C StoccoDos Santos C J Lindsey O P Ferraz et al ldquoBovinepapillomavirus transmission and chromosomal aberrations anexperimental modelrdquo Journal of General Virology vol 79 no 9pp 2127ndash2135 1998
[16] A C De Freitas C De Carvalho O Brunner et al ldquoViralDNA sequences in peripheral blood and vertical transmissionof the virus a discussion about BPV-1rdquo Brazilian Journal ofMicrobiology vol 34 no 1 pp 76ndash78 2003
[17] C J Lindsey M E Almeida C F Vicari et al ldquoBovine papillo-mavirus DNA inmilk blood urine semen and spermatozoa ofbovine papillomavirus-infected animalsrdquo Genetics and Molecu-lar Research vol 8 no 1 pp 310ndash318 2009
[18] S Roperto R Brun F Paolini et al ldquoDetection of bovinepapillomavirus type 2 in the peripheral blood of cattle withurinary bladder tumours possible biological rolerdquo Journal ofGeneral Virology vol 89 no 12 pp 3027ndash3033 2008
[19] 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
[20] J W Moura R C Stocco Dos Santos M L Z Dagli J LDrsquoAngelino E H Birgel and W Becak ldquoChromosome aberra-tions in cattle raised on bracken fern pasturerdquo Experientia vol44 no 9 pp 785ndash788 1988
[21] M B Lioi R Barbieri G Borzacchiello et al ldquoChromosomeaberrations in cattle with chronic enzootic haematuriardquo Journalof Comparative Pathology vol 131 no 2-3 pp 233ndash236 2004
[22] C De Carvalho A C De Freitas O Brunner et al ldquoBovinepapillomavirus type 2 in reproductive tract and gametes ofslaughtered bovine femalesrdquo Brazilian Journal of Microbiologyvol 34 no 1 pp 82ndash84 2003
[23] A Yaguiu C Carvalho A C Freitas et al ldquoPapilomatosis in cat-tle in situ detection of bovine papillomavirus DNA sequencesin reproductive tissuesrdquo Brazilian Journal of MorphologicalSciences vol 23 no 3-4 pp 525ndash529 2006
[24] 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
[25] M S Campo W F H Jarrett W OrsquoNeil and R J BarronldquoLatent papillomavirus infection in cattlerdquo Research in Veteri-nary Science vol 56 no 2 pp 151ndash157 1994
[26] V Peretti F Ciotola S Albarella et al ldquoChromosome fragilityin cattle with chronic enzootic haematuriardquo Mutagenesis vol22 no 5 pp 317ndash320 2007
[27] T Ogawa Y Tomita M Okada et al ldquoBroad-spectrum detec-tion of papillomaviruses in bovine teat papillomas and healthyteat skinrdquo Journal of General Virology vol 85 no 8 pp 2191ndash2197 2004
[28] A M Leal O P Ferraz C Carvalho et al ldquoQuercetin inducesstructural chromosome aberrations and uncommon rearrange-ments in bovine cells transformed by the E7 protein of bovinepapillomavirus type 4rdquo Veterinary and Comparative Oncologyvol 1 no 1 pp 15ndash21 2003
[29] A Duensing A Chin L Wang S-F Kuan and S DuensingldquoAnalysis of centrosome overduplication in correlation to cell
division errors in high-risk human papillomavirus (HPV)-asso-ciated anal neoplasmsrdquo Virology vol 372 no 1 pp 157ndash1642008
[30] Z Liu Y Liu Y Hong L Rapp E J Androphy and J J ChenldquoBovine papillomavirus type 1 E6-induced sensitization to apo-ptosis is distinct from its transforming activityrdquo Virology vol295 no 2 pp 230ndash237 2002
[31] 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
Papillomaviruses maintain their genomes in episomalcondition linked to host cell chromosomes during celldivision and being distributed in dividing cells [11] Thepapillomavirus E2 protein simultaneously associates the hostchromatin and the viral genomeduringmitosis [12]The virusin its episomal form is found in benign lesions and E2 alsoacts in the integration of virus sequences in host chromatinof neoplasic cells These cells are tightly associated with theexpression of high-risk human papillomavirus (HPV) onco-genes E6 and E7 and exhibit chromosomal instability [13]
The virus is specific to epithelium but recently manyreports have described virus DNA sequences in blood stream[14ndash21] The exact source of these sequences is not clearcirculating virus DNA has been discussed as product of lysesof circulating cancer cells or micrometastasis from the tumor[18] Bovine Papillomavirus (BPV) DNA was simultaneouslydetected in different tissues of the same animals includingblood thus suggesting a blood stream virus spread [17 22ndash24]
It has been speculated that blood mononuclear cells thatmigrate to sites of tissue inflammation could act as a sourceof virus (BPV eg) in the infection of epithelial cells andcould become involved in tumor development independentlyor jointly with several biological andor chemical cofactors[14 20]
The seminar questions are as follows could thesesequences represent virus replicate status in the bloodstreamin particular in the lymphocytes acting as a reservoir of viralinfection Can the viruses present oncogene expression inother tissues
Papillomavirus DNA sequences have been identified inblood stream of specifically bovines in recent reports [16ndash19 25 26] The studies analyzed whole blood plasmaisolated lymphocytes and short-term peripheral lymphocytecultures the BPV DNA was detected in all systems [15 1718 22 23] Besides BPV DNA was detected also in semenuterus urine milk and seminal fluid [17] Furthermorethe cytogenetic analysis performed in short-term peripherallymphocyte cultures revealed significant increase of chromo-some aberrations in samples from infected animals [15ndash18]The mechanisms of virus action on host chromatin becameimportant issue of study
In this study we analyzed the levels and the types ofchromosome aberrations verified in cultured cells obtainedin samples collected from peripheral blood cutaneous papil-loma esophagus papilloma and urinary bladder cells ofanimal presenting enzootic hematuria The samples werecollected from a bovine infected with bovine papillomavirusand normal controls Short-term lymphocyte cultures wereperformed with the blood samples and the lesion fragmentswere used to obtain primary cell cultures
2 Material and Methods
21 Ethical Statements The protocols used in this studywere approved by the Ethical Committee in Research of theUniversidade Federal de Sao Paulo (number 083507) assignedby the president of this committee All efforts are made tominimize any suffering in the animals
22 Selection of Animals Nine animals (Bos taurus) wereselected for this study 2males and 7 females comparable ageThe animals were submitted to clinical evaluation performedby a veterinary All procedures followed the ethical principlesThe animal group included 2 animals clinically normal 3presented severe cutaneous papillomatosis 2 had esophaguspapillomas and 2 were affected by enzootic hematuriapresenting papilloma like lesion in the bladder
23 Samples Blood samples were collected in two sterilesyringes with heparin or EDTA respectively for cultures andDNA extraction Tissue fragments were obtained by surgicalprocedures performed by a veterinary with local anesthesia
24Histological Analysis For histological analysis fragmentsof the samples obtained from the solid lesions were fixed in10 neutral buffered formalin and embedded in paraffinwax5 120583m ticked sections were stained with hematoxylineosin
25 Short-Term Lymphocyte Cultures Eighteen drops ofblood were incubated in RPMI medium supplied with 10fetal serum and 2 phytohemagglutinin during 72 hours at37∘C
26 Primary Cell Cultures The tissue fragments were washedin PBS submitted to collagenase 001 30minutes 37∘C andincubated in Dulbecco MEM supplied with 10 fetal serumin CO
2atmosphere at 37∘C
27 Cytogenetic Preparations Colchicine 16 120583gmL 01mLwas added to culture cells for 1 hour at 37∘C The cells weresubmitted to hypotonic solution KCl 0075M at 37∘C 10minutes After that the cells were fixed in three baths with3 1 methanol acetic acid The slides were stained in Giemsa3 5 minutes
28 Cytogenetic Analysis The cells of each culture typewere analyzed and recorded in Photomicroscopy AxiophotZeiss Each type of chromosome aberration was recordedaccording to Melo et al [19]
29 Statistical Analysis The cytogenetic data were statisti-cally analyzed using Students 119905-test [26]
210 Virus Identification
2101 Tissue and Blood DNA Extraction The DNA extrac-tion was performed using the Cell amp Tissue Kit IllustraGenomicPrep Mini Spin (GE Healthcare BuckinghamshireUK) and the extracted DNA was kept at minus20∘C The qualityof obtained DNA samples was verified using the polymerasechain reaction (PCR) technique by means of the amplifi-cation of a bovine 120573-globin gene fragment (450 bp) withspecific primers (F 51015840-aacctctttgttcacaac cag-31015840 and R 51015840-cagatgcttaacccactgagc-31015840) according to Yaguiu et al [24]
211 Viral Identification Viral molecular identification wasperformed using the degenerate primers FAP59 (forward
ISRN Oncology 3
Table 1 Chromosome aberration in peripheral blood cells and primary culture cells C control group 1 cutaneous papillomatosis 2esophagus papillomas 3 enzootic hematuria
Group Total cells Numerical aberrations(mean plusmn SE)
119875 ge 005 lowastlowast119875 ge 001 curren119875 ge 01 sectcells presenting only gaps were evaluated isolate
51015840-TAACWGTIGGICAYCCWTATT-31015840) and FAP64 (reverse51015840-CCWATATCWVHCATITCICCATC-31015840) which promoteL1 gene amplification resulting in a fragment of 478 bpOgawa et al 2004 [27] In detail the amplification reac-tions were performed in a PTC-100TM (MJ Research Inc)thermo cycler with PCR Master Mix (Promega MadisonUSA) under the following conditions 10min at 94∘Cfollowed by 45 cycles of 1min and 30 s at 94∘C 2minat 52∘C and 1min and 30 s at 72∘C and a final exten-sion step of 5min at 72∘C for primer FAP59FAP64For specific BPV1 2 and 4 BPV-1 (5-ggagcgcctgctaactat agg a-3101584051015840-atctgttgtttgggtggtgac-31015840) BPV-2 (51015840-gttataccaccc aaagaagaccct-3101584051015840-ctggttgcaacagctctctttctc-31015840) and BPV-4 (51015840-gctgaccttccagtctta atmdash3101584051015840-cag tttcaatctcctcttca-31015840) indetail the amplification reactions were performed in aPTC-100TM (MJ Research Inc) thermo cycler with PCRMaster Mix (Promega Madison USA) under the followingconditions 3min at 94∘C followed by 35 cycles of 50 s at94∘C 1min at 60∘C and 1min at 72∘C and a final extensionstep of 5min at 72∘C The PCR products were analyzed in2 agarose gel electrophoresis stained with GelRed in TAEbuffer visualized under UV light
3 Results and Discussion
31 Results The histological analysis confirmed the identifi-cation of the fragments collected for the studies (Figure 1)Specifically a segment of a normal skin obtained by surgicalprocedure from a normal bovine was selected as control Bysimilar procedures biopsies were obtained from cutaneouspapilloma esophagus papilloma and papilloma fromurinarybladder of an animal affected with enzootic hematuria
The PCR technique using primers FAP 5964 amplifieda DNA fragment 474 bp long indicating virus sequencepresence in the biopsies collected frompapilloma lesionsThespecific primers allowed the identification of BPV1 (301 bp)and BPV2 (164 bp) (Figure 2) Only one animal presented
BPV4 (170 bp) in cutaneous papilloma Also the same virussequences were detected in the obtained primary cell culturesand in their first five passages (Figures 2 and 3)The samePCRprocedures with the same primers were used to investigatethe presence of virus sequences in blood samples but it wasnot possible to detect any virus sequence in any of the bloodsamples normal or affected animals
The Figure 3 presents cells in primary cultures The cellmorphology did not differ among the different lines
For cytogenetic analysis four groups of animals werefixed according to the clinical features C control (notaffected and without detection of virus DNA in culture cells)Group 1 cutaneous papillomaGroup 2 esophagus papillomaGroup 3 enzootic hematuria A total of 1068 cells wereevaluated 501 derived from short-term lymphocyte culturesand 567 obtained from biopsy primary cell cultures (Tables 1and 2)
The results observed in culture blood cells make itpossible to verify that the affected animals showed higherlevels of chromosome aberrations compared to not affectedanimals As the PCR procedures were unable to detect BPVsequences in blood cells the data were analyzed as notaffected animals compared to affected bovines However thePCR technique showed BPV DNA sequences in the biopsiescollected from the affected animals such as the fragmentsfrom not affected animals that were negative for BPV inPCR procedures So the data were analyzed comparing BPVpositive and BPV negative biopsies and respective primaryculture cells It is important to emphasize that the BPV nega-tive biopsies were obtained from the not affected animals andso these animals were our controls A specific analysis wasperformed considering the different chromosome aberrationtypes comparing the previous groups (C 1 2 and 3) In bloodcells comparing to control the aberration types presentingsignificant higher levels in cells from affected animals wereadditiondeletion chromatid breaks acentric fragments andcentromere associations (Table 2)
4 ISRN Oncology
(a) (b)
(c)
10 120583m
(d)
Figure 1 Histological sections (a) normal skin fragment (b) cutaneous papilloma fragment (c) esophagus papilloma fragment and (d)fragment of papilloma collected from an urinary bladder of an animal affected by enzootic hematuria (HE) (10x)
474 pb
1 kb Cminus C+ A B C D E F G H I J
(a)
164 pb
1 kbCminus C+ A B C D E F G H I J L M
(b)
Figure 2 Example of BPV DNA sequences detected in cutaneous lesions and in respective primary culture cells the samples were collectedfrom one of the animals I primers FAP5964 II BPV2 specific primers Cminus negative control C+ positive control A-B PCR with DNA frompapilloma lesion CndashE primary cell culture using fragments from BPV positive lesion in passage 1 FndashH primary cell culture using fragmentsfrom BPV positive lesion in passage 2 I-J primary cell culture using fragments from BPV positive lesion in passage 3 L primary cell cultureusing fragments from BPV positive lesion in passage 4 and M primary cell culture using fragments from BPV positive lesion in passage 5 Itis important to pay attention in the different amplicons suggesting possible different virus load
Considering the primary culture cells the more fre-quent chromosome aberrationswere additiondeletion chro-matid break chromosome breaks acentric fragments cen-tromere association and association of telomeres (Table 2and Figure 4) It was possible to verify that the cells obtainedfrom the BPV infected animals presented significant higherlevels of chromosome aberrations
32 Discussion As far it was possible to verify this is the firstreport describing chromosome aberrations in cells derivedfrom bovine papillomavirus lesions
As we have previously described in peripheral lympho-cytes [15] the chromosome aberrations occur in significantincreased levels in short-term lymphocyte cultures and inprimary cell cultures established from samples obtainedfrom BPV affected animals
A very important point has to be discussed the factthat it was not possible to detect virus DNA sequencesin peripheral blood of affected or not affected animalsDespite this fact the level of chromosome aberrations wasverified higher in the affected animal samples As it was notpossible to identify virus DNA in blood either in control or
ISRN Oncology 5
(a) (b)
(c) (d)
Figure 3 Primary cell cultures (a) normal skin (b) cutaneous papilloma (c) esophagus papilloma (d) papilloma of urinary bladder fromanimal affected with enzootic hematuria (10x) The arrow indicates a cell with a different morphology
affected animals it could be discussed that the chromosomeaberrations were not caused by virus action However thesame types of aberrations were detected in cells derived fromlesions positive for virus presence Furthermore there wasa significant difference between the chromosome aberrationlevels comparing affected and not affected animals
Inmatter of fact considering the similarity of the detectedchromosome aberrations the virus action on host chromatinwas verified as effective either in blood cells or in cells derivedfrom lesions We could argue that the virus load in theperipheral blood of the affected animals was too low to bedetected in conventional PCR The same argument could beused for the not affected animals leading to the need tocompare affected to nonaffected animals and emphasizing thedifferences observed in the levels of chromosomal aberra-tions
The virus action was verified rising numerical and struc-tural chromosome aberrations This fact indicates that thevirus acts in different ways in its interaction with host
chromatin It was previously described the virus oncoproteinaction on telomeres leading to centric fusion [28] Besidesthe virus acts on mitotic spindle changing the chromosomeset and rising aneuploidy [29]
Although BPV is described as nonintegrated in host cellits action produces different types of chromosome alterationssuggesting a large interaction with the chromatin and alsowith DNA repair mechanisms [30]
We have already described the presence of virussequences in primary culture cells in different passages [31]but now we demonstrate that these sequences are activeleading to chromosome alterations
4 Conclusions
We compare for the first time the action of bovine papillo-mavirus on host cell chromatin in cultured lymphocytes andprimary culture cells describing the increase of chromosomalaberrations in both cell types The primary cells cultures
6 ISRN Oncology
(a) (b) (c)
(d) (e) (f)
(g) (h)
Figure 4 Chromosome aberrations in primary culture cells ((a) (c) (e) and (g)) and peripheral lymphocytes ((b) (d) (f) and (h)) acentricfragments ((a) (b)) chromosome break (c) chromatid break (d) chromosome rearrangement with additiondeletion ((e) (f)) telomericassociation (g) centromeric association ((e) (h)) early chromatid separation (thin arrow in (a) (e))
not only presented BPV DNA sequences but also keep thesesequences throughout different passages These data plusrecent reports [18] emphasize possible active BPV infectionin blood as well as in in vitro cultured cells
Conflict of Interests
All authors declare that there is no conflict of interestsregarding the research authorship andor publication of thispaper The authors inform that all materials and trademarks
mentioned were used just as part of experimental protocoland there are no financial gains or favoritism of business
Acknowledgments
The authors would like to thank Ministerio de CienciaTecnologia e InovacaoConselho Nacional de DesenvolvimentoCientıfico e Tecnologico (CNPq Proc 5548162006-7 and4025392011-7) FAPESP (Proc 200602439-6) Coordenacaode Aperfeicoamento Pessoal de Nıvel Superior (CAPES) and
ISRN Oncology 7
Table 2 Frequencies of different aberrations detected in blood and primary culture cells of animals affected by cutaneous papillomasesophagus papilloma and enzootic hematuria
BloodGroup CA AT AF CB CrBC 088 plusmn 124 248 plusmn 146 233 plusmn 081 175 plusmn 248 160 plusmn 022
119875 gt 005 lowastlowast119875 gt 001 and curren119875 ge 01
CA centromere association AT association of telomere AF acentric fragments CB chromosome break CrB chromatid break AdDel additiondeletionEcrS early chromatid separation Aneu aneuploidy Polip polyploidy and gaps
Fundacao Butantan for financial support and Carolina da PazSabino for editorial support
References
[1] G Borzacchiello and F Roperto ldquoBovine papillomavirusespapillomas and cancer in cattlerdquoVeterinary Research vol 39 no5 article 45 2008
[2] M S Campo ldquoBovine papillomavirus and cancerrdquo VeterinaryJournal vol 154 no 3 pp 175ndash188 1997
[3] 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
[4] S Duensing L Y Lee A Duensing et al ldquoThe human papillo-mavirus 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
[5] M Kadaja H Isok-Paas T Laos E Ustav and M UstavldquoMechanism of genomic instability in cells infected with thehigh-risk human papillomavirusesrdquo PLoS Pathogens vol 5 no4 2009
[6] N A Hamid C Brown and K Gaston ldquoThe regulation of cellproliferation by the papillomavirus early proteinsrdquo Cellular andMolecular Life Sciences vol 66 no 10 pp 1700ndash1717 2009
[7] V Kashyap and B C Das ldquoDNA aneuploidy and infectionof human papillomavirus type 16 in preneoplastic lesions ofthe uterine cervix correlation with progression to malignancyrdquoCancer Letters vol 123 no 1 pp 47ndash52 1998
[8] D Patel A Incassati N Wang and D J McCance ldquoHumanpapillomavirus type 16 E6 and E7 cause polyploidy in humankeratinocytes and up-regulation of G2-M-phase proteinsrdquoCan-cer Research vol 64 no 4 pp 1299ndash1306 2004
[9] G Boulet C Horvath D V Broeck S Sahebali and J BogersldquoHuman papillomavirus E6 and E7 oncogenesrdquo InternationalJournal of Biochemistry andCell Biology vol 39 no 11 pp 2006ndash2011 2007
[10] V OrsquoBrien G J Grindlay and M S Campo ldquoCell transfor-mation by the E5E8 protein of bovine papillomavirus type4 p27Kip1 elevated through increased protein synthesis issequestered by cyclin D1-CDK4 complexesrdquo Journal of Biolog-ical Chemistry vol 276 no 36 pp 33861ndash33868 2001
[11] A A McBride J G Oliveira and M G McPhillips ldquoPartition-ing viral genomes in mitosis same idea different targetsrdquo CellCycle vol 5 no 14 pp 1499ndash1502 2006
[12] J G Oliveira L A Colf and A A McBride ldquoVariationsin the association of papillomavirus E2 proteins with mitoticchromosomesrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 103 no 4 pp 1047ndash10522006
[13] S Duensing and K Munger ldquoHuman papillomaviruses andcentrosomeduplication errorsmodeling the origins of genomicinstabilityrdquo Oncogene vol 21 no 40 pp 6241ndash6248 2002
8 ISRN Oncology
[14] R C Recouso R C Stocco dos Santos R Freitas et al ldquoClasto-genic effect of bracken fern (Pteridium aquilinum aracnoideum)diet in peripheral lymphocytes of human consumers prelimi-nary datardquo Veterinary and Comparative Oncology vol 1 no 1pp 22ndash29 2003
[15] R C StoccoDos Santos C J Lindsey O P Ferraz et al ldquoBovinepapillomavirus transmission and chromosomal aberrations anexperimental modelrdquo Journal of General Virology vol 79 no 9pp 2127ndash2135 1998
[16] A C De Freitas C De Carvalho O Brunner et al ldquoViralDNA sequences in peripheral blood and vertical transmissionof the virus a discussion about BPV-1rdquo Brazilian Journal ofMicrobiology vol 34 no 1 pp 76ndash78 2003
[17] C J Lindsey M E Almeida C F Vicari et al ldquoBovine papillo-mavirus DNA inmilk blood urine semen and spermatozoa ofbovine papillomavirus-infected animalsrdquo Genetics and Molecu-lar Research vol 8 no 1 pp 310ndash318 2009
[18] S Roperto R Brun F Paolini et al ldquoDetection of bovinepapillomavirus type 2 in the peripheral blood of cattle withurinary bladder tumours possible biological rolerdquo Journal ofGeneral Virology vol 89 no 12 pp 3027ndash3033 2008
[19] 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
[20] J W Moura R C Stocco Dos Santos M L Z Dagli J LDrsquoAngelino E H Birgel and W Becak ldquoChromosome aberra-tions in cattle raised on bracken fern pasturerdquo Experientia vol44 no 9 pp 785ndash788 1988
[21] M B Lioi R Barbieri G Borzacchiello et al ldquoChromosomeaberrations in cattle with chronic enzootic haematuriardquo Journalof Comparative Pathology vol 131 no 2-3 pp 233ndash236 2004
[22] C De Carvalho A C De Freitas O Brunner et al ldquoBovinepapillomavirus type 2 in reproductive tract and gametes ofslaughtered bovine femalesrdquo Brazilian Journal of Microbiologyvol 34 no 1 pp 82ndash84 2003
[23] A Yaguiu C Carvalho A C Freitas et al ldquoPapilomatosis in cat-tle in situ detection of bovine papillomavirus DNA sequencesin reproductive tissuesrdquo Brazilian Journal of MorphologicalSciences vol 23 no 3-4 pp 525ndash529 2006
[24] 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
[25] M S Campo W F H Jarrett W OrsquoNeil and R J BarronldquoLatent papillomavirus infection in cattlerdquo Research in Veteri-nary Science vol 56 no 2 pp 151ndash157 1994
[26] V Peretti F Ciotola S Albarella et al ldquoChromosome fragilityin cattle with chronic enzootic haematuriardquo Mutagenesis vol22 no 5 pp 317ndash320 2007
[27] T Ogawa Y Tomita M Okada et al ldquoBroad-spectrum detec-tion of papillomaviruses in bovine teat papillomas and healthyteat skinrdquo Journal of General Virology vol 85 no 8 pp 2191ndash2197 2004
[28] A M Leal O P Ferraz C Carvalho et al ldquoQuercetin inducesstructural chromosome aberrations and uncommon rearrange-ments in bovine cells transformed by the E7 protein of bovinepapillomavirus type 4rdquo Veterinary and Comparative Oncologyvol 1 no 1 pp 15ndash21 2003
[29] A Duensing A Chin L Wang S-F Kuan and S DuensingldquoAnalysis of centrosome overduplication in correlation to cell
division errors in high-risk human papillomavirus (HPV)-asso-ciated anal neoplasmsrdquo Virology vol 372 no 1 pp 157ndash1642008
[30] Z Liu Y Liu Y Hong L Rapp E J Androphy and J J ChenldquoBovine papillomavirus type 1 E6-induced sensitization to apo-ptosis is distinct from its transforming activityrdquo Virology vol295 no 2 pp 230ndash237 2002
[31] 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
Table 1 Chromosome aberration in peripheral blood cells and primary culture cells C control group 1 cutaneous papillomatosis 2esophagus papillomas 3 enzootic hematuria
Group Total cells Numerical aberrations(mean plusmn SE)
119875 ge 005 lowastlowast119875 ge 001 curren119875 ge 01 sectcells presenting only gaps were evaluated isolate
51015840-TAACWGTIGGICAYCCWTATT-31015840) and FAP64 (reverse51015840-CCWATATCWVHCATITCICCATC-31015840) which promoteL1 gene amplification resulting in a fragment of 478 bpOgawa et al 2004 [27] In detail the amplification reac-tions were performed in a PTC-100TM (MJ Research Inc)thermo cycler with PCR Master Mix (Promega MadisonUSA) under the following conditions 10min at 94∘Cfollowed by 45 cycles of 1min and 30 s at 94∘C 2minat 52∘C and 1min and 30 s at 72∘C and a final exten-sion step of 5min at 72∘C for primer FAP59FAP64For specific BPV1 2 and 4 BPV-1 (5-ggagcgcctgctaactat agg a-3101584051015840-atctgttgtttgggtggtgac-31015840) BPV-2 (51015840-gttataccaccc aaagaagaccct-3101584051015840-ctggttgcaacagctctctttctc-31015840) and BPV-4 (51015840-gctgaccttccagtctta atmdash3101584051015840-cag tttcaatctcctcttca-31015840) indetail the amplification reactions were performed in aPTC-100TM (MJ Research Inc) thermo cycler with PCRMaster Mix (Promega Madison USA) under the followingconditions 3min at 94∘C followed by 35 cycles of 50 s at94∘C 1min at 60∘C and 1min at 72∘C and a final extensionstep of 5min at 72∘C The PCR products were analyzed in2 agarose gel electrophoresis stained with GelRed in TAEbuffer visualized under UV light
3 Results and Discussion
31 Results The histological analysis confirmed the identifi-cation of the fragments collected for the studies (Figure 1)Specifically a segment of a normal skin obtained by surgicalprocedure from a normal bovine was selected as control Bysimilar procedures biopsies were obtained from cutaneouspapilloma esophagus papilloma and papilloma fromurinarybladder of an animal affected with enzootic hematuria
The PCR technique using primers FAP 5964 amplifieda DNA fragment 474 bp long indicating virus sequencepresence in the biopsies collected frompapilloma lesionsThespecific primers allowed the identification of BPV1 (301 bp)and BPV2 (164 bp) (Figure 2) Only one animal presented
BPV4 (170 bp) in cutaneous papilloma Also the same virussequences were detected in the obtained primary cell culturesand in their first five passages (Figures 2 and 3)The samePCRprocedures with the same primers were used to investigatethe presence of virus sequences in blood samples but it wasnot possible to detect any virus sequence in any of the bloodsamples normal or affected animals
The Figure 3 presents cells in primary cultures The cellmorphology did not differ among the different lines
For cytogenetic analysis four groups of animals werefixed according to the clinical features C control (notaffected and without detection of virus DNA in culture cells)Group 1 cutaneous papillomaGroup 2 esophagus papillomaGroup 3 enzootic hematuria A total of 1068 cells wereevaluated 501 derived from short-term lymphocyte culturesand 567 obtained from biopsy primary cell cultures (Tables 1and 2)
The results observed in culture blood cells make itpossible to verify that the affected animals showed higherlevels of chromosome aberrations compared to not affectedanimals As the PCR procedures were unable to detect BPVsequences in blood cells the data were analyzed as notaffected animals compared to affected bovines However thePCR technique showed BPV DNA sequences in the biopsiescollected from the affected animals such as the fragmentsfrom not affected animals that were negative for BPV inPCR procedures So the data were analyzed comparing BPVpositive and BPV negative biopsies and respective primaryculture cells It is important to emphasize that the BPV nega-tive biopsies were obtained from the not affected animals andso these animals were our controls A specific analysis wasperformed considering the different chromosome aberrationtypes comparing the previous groups (C 1 2 and 3) In bloodcells comparing to control the aberration types presentingsignificant higher levels in cells from affected animals wereadditiondeletion chromatid breaks acentric fragments andcentromere associations (Table 2)
4 ISRN Oncology
(a) (b)
(c)
10 120583m
(d)
Figure 1 Histological sections (a) normal skin fragment (b) cutaneous papilloma fragment (c) esophagus papilloma fragment and (d)fragment of papilloma collected from an urinary bladder of an animal affected by enzootic hematuria (HE) (10x)
474 pb
1 kb Cminus C+ A B C D E F G H I J
(a)
164 pb
1 kbCminus C+ A B C D E F G H I J L M
(b)
Figure 2 Example of BPV DNA sequences detected in cutaneous lesions and in respective primary culture cells the samples were collectedfrom one of the animals I primers FAP5964 II BPV2 specific primers Cminus negative control C+ positive control A-B PCR with DNA frompapilloma lesion CndashE primary cell culture using fragments from BPV positive lesion in passage 1 FndashH primary cell culture using fragmentsfrom BPV positive lesion in passage 2 I-J primary cell culture using fragments from BPV positive lesion in passage 3 L primary cell cultureusing fragments from BPV positive lesion in passage 4 and M primary cell culture using fragments from BPV positive lesion in passage 5 Itis important to pay attention in the different amplicons suggesting possible different virus load
Considering the primary culture cells the more fre-quent chromosome aberrationswere additiondeletion chro-matid break chromosome breaks acentric fragments cen-tromere association and association of telomeres (Table 2and Figure 4) It was possible to verify that the cells obtainedfrom the BPV infected animals presented significant higherlevels of chromosome aberrations
32 Discussion As far it was possible to verify this is the firstreport describing chromosome aberrations in cells derivedfrom bovine papillomavirus lesions
As we have previously described in peripheral lympho-cytes [15] the chromosome aberrations occur in significantincreased levels in short-term lymphocyte cultures and inprimary cell cultures established from samples obtainedfrom BPV affected animals
A very important point has to be discussed the factthat it was not possible to detect virus DNA sequencesin peripheral blood of affected or not affected animalsDespite this fact the level of chromosome aberrations wasverified higher in the affected animal samples As it was notpossible to identify virus DNA in blood either in control or
ISRN Oncology 5
(a) (b)
(c) (d)
Figure 3 Primary cell cultures (a) normal skin (b) cutaneous papilloma (c) esophagus papilloma (d) papilloma of urinary bladder fromanimal affected with enzootic hematuria (10x) The arrow indicates a cell with a different morphology
affected animals it could be discussed that the chromosomeaberrations were not caused by virus action However thesame types of aberrations were detected in cells derived fromlesions positive for virus presence Furthermore there wasa significant difference between the chromosome aberrationlevels comparing affected and not affected animals
Inmatter of fact considering the similarity of the detectedchromosome aberrations the virus action on host chromatinwas verified as effective either in blood cells or in cells derivedfrom lesions We could argue that the virus load in theperipheral blood of the affected animals was too low to bedetected in conventional PCR The same argument could beused for the not affected animals leading to the need tocompare affected to nonaffected animals and emphasizing thedifferences observed in the levels of chromosomal aberra-tions
The virus action was verified rising numerical and struc-tural chromosome aberrations This fact indicates that thevirus acts in different ways in its interaction with host
chromatin It was previously described the virus oncoproteinaction on telomeres leading to centric fusion [28] Besidesthe virus acts on mitotic spindle changing the chromosomeset and rising aneuploidy [29]
Although BPV is described as nonintegrated in host cellits action produces different types of chromosome alterationssuggesting a large interaction with the chromatin and alsowith DNA repair mechanisms [30]
We have already described the presence of virussequences in primary culture cells in different passages [31]but now we demonstrate that these sequences are activeleading to chromosome alterations
4 Conclusions
We compare for the first time the action of bovine papillo-mavirus on host cell chromatin in cultured lymphocytes andprimary culture cells describing the increase of chromosomalaberrations in both cell types The primary cells cultures
6 ISRN Oncology
(a) (b) (c)
(d) (e) (f)
(g) (h)
Figure 4 Chromosome aberrations in primary culture cells ((a) (c) (e) and (g)) and peripheral lymphocytes ((b) (d) (f) and (h)) acentricfragments ((a) (b)) chromosome break (c) chromatid break (d) chromosome rearrangement with additiondeletion ((e) (f)) telomericassociation (g) centromeric association ((e) (h)) early chromatid separation (thin arrow in (a) (e))
not only presented BPV DNA sequences but also keep thesesequences throughout different passages These data plusrecent reports [18] emphasize possible active BPV infectionin blood as well as in in vitro cultured cells
Conflict of Interests
All authors declare that there is no conflict of interestsregarding the research authorship andor publication of thispaper The authors inform that all materials and trademarks
mentioned were used just as part of experimental protocoland there are no financial gains or favoritism of business
Acknowledgments
The authors would like to thank Ministerio de CienciaTecnologia e InovacaoConselho Nacional de DesenvolvimentoCientıfico e Tecnologico (CNPq Proc 5548162006-7 and4025392011-7) FAPESP (Proc 200602439-6) Coordenacaode Aperfeicoamento Pessoal de Nıvel Superior (CAPES) and
ISRN Oncology 7
Table 2 Frequencies of different aberrations detected in blood and primary culture cells of animals affected by cutaneous papillomasesophagus papilloma and enzootic hematuria
BloodGroup CA AT AF CB CrBC 088 plusmn 124 248 plusmn 146 233 plusmn 081 175 plusmn 248 160 plusmn 022
119875 gt 005 lowastlowast119875 gt 001 and curren119875 ge 01
CA centromere association AT association of telomere AF acentric fragments CB chromosome break CrB chromatid break AdDel additiondeletionEcrS early chromatid separation Aneu aneuploidy Polip polyploidy and gaps
Fundacao Butantan for financial support and Carolina da PazSabino for editorial support
References
[1] G Borzacchiello and F Roperto ldquoBovine papillomavirusespapillomas and cancer in cattlerdquoVeterinary Research vol 39 no5 article 45 2008
[2] M S Campo ldquoBovine papillomavirus and cancerrdquo VeterinaryJournal vol 154 no 3 pp 175ndash188 1997
[3] 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
[4] S Duensing L Y Lee A Duensing et al ldquoThe human papillo-mavirus 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
[5] M Kadaja H Isok-Paas T Laos E Ustav and M UstavldquoMechanism of genomic instability in cells infected with thehigh-risk human papillomavirusesrdquo PLoS Pathogens vol 5 no4 2009
[6] N A Hamid C Brown and K Gaston ldquoThe regulation of cellproliferation by the papillomavirus early proteinsrdquo Cellular andMolecular Life Sciences vol 66 no 10 pp 1700ndash1717 2009
[7] V Kashyap and B C Das ldquoDNA aneuploidy and infectionof human papillomavirus type 16 in preneoplastic lesions ofthe uterine cervix correlation with progression to malignancyrdquoCancer Letters vol 123 no 1 pp 47ndash52 1998
[8] D Patel A Incassati N Wang and D J McCance ldquoHumanpapillomavirus type 16 E6 and E7 cause polyploidy in humankeratinocytes and up-regulation of G2-M-phase proteinsrdquoCan-cer Research vol 64 no 4 pp 1299ndash1306 2004
[9] G Boulet C Horvath D V Broeck S Sahebali and J BogersldquoHuman papillomavirus E6 and E7 oncogenesrdquo InternationalJournal of Biochemistry andCell Biology vol 39 no 11 pp 2006ndash2011 2007
[10] V OrsquoBrien G J Grindlay and M S Campo ldquoCell transfor-mation by the E5E8 protein of bovine papillomavirus type4 p27Kip1 elevated through increased protein synthesis issequestered by cyclin D1-CDK4 complexesrdquo Journal of Biolog-ical Chemistry vol 276 no 36 pp 33861ndash33868 2001
[11] A A McBride J G Oliveira and M G McPhillips ldquoPartition-ing viral genomes in mitosis same idea different targetsrdquo CellCycle vol 5 no 14 pp 1499ndash1502 2006
[12] J G Oliveira L A Colf and A A McBride ldquoVariationsin the association of papillomavirus E2 proteins with mitoticchromosomesrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 103 no 4 pp 1047ndash10522006
[13] S Duensing and K Munger ldquoHuman papillomaviruses andcentrosomeduplication errorsmodeling the origins of genomicinstabilityrdquo Oncogene vol 21 no 40 pp 6241ndash6248 2002
8 ISRN Oncology
[14] R C Recouso R C Stocco dos Santos R Freitas et al ldquoClasto-genic effect of bracken fern (Pteridium aquilinum aracnoideum)diet in peripheral lymphocytes of human consumers prelimi-nary datardquo Veterinary and Comparative Oncology vol 1 no 1pp 22ndash29 2003
[15] R C StoccoDos Santos C J Lindsey O P Ferraz et al ldquoBovinepapillomavirus transmission and chromosomal aberrations anexperimental modelrdquo Journal of General Virology vol 79 no 9pp 2127ndash2135 1998
[16] A C De Freitas C De Carvalho O Brunner et al ldquoViralDNA sequences in peripheral blood and vertical transmissionof the virus a discussion about BPV-1rdquo Brazilian Journal ofMicrobiology vol 34 no 1 pp 76ndash78 2003
[17] C J Lindsey M E Almeida C F Vicari et al ldquoBovine papillo-mavirus DNA inmilk blood urine semen and spermatozoa ofbovine papillomavirus-infected animalsrdquo Genetics and Molecu-lar Research vol 8 no 1 pp 310ndash318 2009
[18] S Roperto R Brun F Paolini et al ldquoDetection of bovinepapillomavirus type 2 in the peripheral blood of cattle withurinary bladder tumours possible biological rolerdquo Journal ofGeneral Virology vol 89 no 12 pp 3027ndash3033 2008
[19] 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
[20] J W Moura R C Stocco Dos Santos M L Z Dagli J LDrsquoAngelino E H Birgel and W Becak ldquoChromosome aberra-tions in cattle raised on bracken fern pasturerdquo Experientia vol44 no 9 pp 785ndash788 1988
[21] M B Lioi R Barbieri G Borzacchiello et al ldquoChromosomeaberrations in cattle with chronic enzootic haematuriardquo Journalof Comparative Pathology vol 131 no 2-3 pp 233ndash236 2004
[22] C De Carvalho A C De Freitas O Brunner et al ldquoBovinepapillomavirus type 2 in reproductive tract and gametes ofslaughtered bovine femalesrdquo Brazilian Journal of Microbiologyvol 34 no 1 pp 82ndash84 2003
[23] A Yaguiu C Carvalho A C Freitas et al ldquoPapilomatosis in cat-tle in situ detection of bovine papillomavirus DNA sequencesin reproductive tissuesrdquo Brazilian Journal of MorphologicalSciences vol 23 no 3-4 pp 525ndash529 2006
[24] 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
[25] M S Campo W F H Jarrett W OrsquoNeil and R J BarronldquoLatent papillomavirus infection in cattlerdquo Research in Veteri-nary Science vol 56 no 2 pp 151ndash157 1994
[26] V Peretti F Ciotola S Albarella et al ldquoChromosome fragilityin cattle with chronic enzootic haematuriardquo Mutagenesis vol22 no 5 pp 317ndash320 2007
[27] T Ogawa Y Tomita M Okada et al ldquoBroad-spectrum detec-tion of papillomaviruses in bovine teat papillomas and healthyteat skinrdquo Journal of General Virology vol 85 no 8 pp 2191ndash2197 2004
[28] A M Leal O P Ferraz C Carvalho et al ldquoQuercetin inducesstructural chromosome aberrations and uncommon rearrange-ments in bovine cells transformed by the E7 protein of bovinepapillomavirus type 4rdquo Veterinary and Comparative Oncologyvol 1 no 1 pp 15ndash21 2003
[29] A Duensing A Chin L Wang S-F Kuan and S DuensingldquoAnalysis of centrosome overduplication in correlation to cell
division errors in high-risk human papillomavirus (HPV)-asso-ciated anal neoplasmsrdquo Virology vol 372 no 1 pp 157ndash1642008
[30] Z Liu Y Liu Y Hong L Rapp E J Androphy and J J ChenldquoBovine papillomavirus type 1 E6-induced sensitization to apo-ptosis is distinct from its transforming activityrdquo Virology vol295 no 2 pp 230ndash237 2002
[31] 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
Figure 1 Histological sections (a) normal skin fragment (b) cutaneous papilloma fragment (c) esophagus papilloma fragment and (d)fragment of papilloma collected from an urinary bladder of an animal affected by enzootic hematuria (HE) (10x)
474 pb
1 kb Cminus C+ A B C D E F G H I J
(a)
164 pb
1 kbCminus C+ A B C D E F G H I J L M
(b)
Figure 2 Example of BPV DNA sequences detected in cutaneous lesions and in respective primary culture cells the samples were collectedfrom one of the animals I primers FAP5964 II BPV2 specific primers Cminus negative control C+ positive control A-B PCR with DNA frompapilloma lesion CndashE primary cell culture using fragments from BPV positive lesion in passage 1 FndashH primary cell culture using fragmentsfrom BPV positive lesion in passage 2 I-J primary cell culture using fragments from BPV positive lesion in passage 3 L primary cell cultureusing fragments from BPV positive lesion in passage 4 and M primary cell culture using fragments from BPV positive lesion in passage 5 Itis important to pay attention in the different amplicons suggesting possible different virus load
Considering the primary culture cells the more fre-quent chromosome aberrationswere additiondeletion chro-matid break chromosome breaks acentric fragments cen-tromere association and association of telomeres (Table 2and Figure 4) It was possible to verify that the cells obtainedfrom the BPV infected animals presented significant higherlevels of chromosome aberrations
32 Discussion As far it was possible to verify this is the firstreport describing chromosome aberrations in cells derivedfrom bovine papillomavirus lesions
As we have previously described in peripheral lympho-cytes [15] the chromosome aberrations occur in significantincreased levels in short-term lymphocyte cultures and inprimary cell cultures established from samples obtainedfrom BPV affected animals
A very important point has to be discussed the factthat it was not possible to detect virus DNA sequencesin peripheral blood of affected or not affected animalsDespite this fact the level of chromosome aberrations wasverified higher in the affected animal samples As it was notpossible to identify virus DNA in blood either in control or
ISRN Oncology 5
(a) (b)
(c) (d)
Figure 3 Primary cell cultures (a) normal skin (b) cutaneous papilloma (c) esophagus papilloma (d) papilloma of urinary bladder fromanimal affected with enzootic hematuria (10x) The arrow indicates a cell with a different morphology
affected animals it could be discussed that the chromosomeaberrations were not caused by virus action However thesame types of aberrations were detected in cells derived fromlesions positive for virus presence Furthermore there wasa significant difference between the chromosome aberrationlevels comparing affected and not affected animals
Inmatter of fact considering the similarity of the detectedchromosome aberrations the virus action on host chromatinwas verified as effective either in blood cells or in cells derivedfrom lesions We could argue that the virus load in theperipheral blood of the affected animals was too low to bedetected in conventional PCR The same argument could beused for the not affected animals leading to the need tocompare affected to nonaffected animals and emphasizing thedifferences observed in the levels of chromosomal aberra-tions
The virus action was verified rising numerical and struc-tural chromosome aberrations This fact indicates that thevirus acts in different ways in its interaction with host
chromatin It was previously described the virus oncoproteinaction on telomeres leading to centric fusion [28] Besidesthe virus acts on mitotic spindle changing the chromosomeset and rising aneuploidy [29]
Although BPV is described as nonintegrated in host cellits action produces different types of chromosome alterationssuggesting a large interaction with the chromatin and alsowith DNA repair mechanisms [30]
We have already described the presence of virussequences in primary culture cells in different passages [31]but now we demonstrate that these sequences are activeleading to chromosome alterations
4 Conclusions
We compare for the first time the action of bovine papillo-mavirus on host cell chromatin in cultured lymphocytes andprimary culture cells describing the increase of chromosomalaberrations in both cell types The primary cells cultures
6 ISRN Oncology
(a) (b) (c)
(d) (e) (f)
(g) (h)
Figure 4 Chromosome aberrations in primary culture cells ((a) (c) (e) and (g)) and peripheral lymphocytes ((b) (d) (f) and (h)) acentricfragments ((a) (b)) chromosome break (c) chromatid break (d) chromosome rearrangement with additiondeletion ((e) (f)) telomericassociation (g) centromeric association ((e) (h)) early chromatid separation (thin arrow in (a) (e))
not only presented BPV DNA sequences but also keep thesesequences throughout different passages These data plusrecent reports [18] emphasize possible active BPV infectionin blood as well as in in vitro cultured cells
Conflict of Interests
All authors declare that there is no conflict of interestsregarding the research authorship andor publication of thispaper The authors inform that all materials and trademarks
mentioned were used just as part of experimental protocoland there are no financial gains or favoritism of business
Acknowledgments
The authors would like to thank Ministerio de CienciaTecnologia e InovacaoConselho Nacional de DesenvolvimentoCientıfico e Tecnologico (CNPq Proc 5548162006-7 and4025392011-7) FAPESP (Proc 200602439-6) Coordenacaode Aperfeicoamento Pessoal de Nıvel Superior (CAPES) and
ISRN Oncology 7
Table 2 Frequencies of different aberrations detected in blood and primary culture cells of animals affected by cutaneous papillomasesophagus papilloma and enzootic hematuria
BloodGroup CA AT AF CB CrBC 088 plusmn 124 248 plusmn 146 233 plusmn 081 175 plusmn 248 160 plusmn 022
119875 gt 005 lowastlowast119875 gt 001 and curren119875 ge 01
CA centromere association AT association of telomere AF acentric fragments CB chromosome break CrB chromatid break AdDel additiondeletionEcrS early chromatid separation Aneu aneuploidy Polip polyploidy and gaps
Fundacao Butantan for financial support and Carolina da PazSabino for editorial support
References
[1] G Borzacchiello and F Roperto ldquoBovine papillomavirusespapillomas and cancer in cattlerdquoVeterinary Research vol 39 no5 article 45 2008
[2] M S Campo ldquoBovine papillomavirus and cancerrdquo VeterinaryJournal vol 154 no 3 pp 175ndash188 1997
[3] 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
[4] S Duensing L Y Lee A Duensing et al ldquoThe human papillo-mavirus 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
[5] M Kadaja H Isok-Paas T Laos E Ustav and M UstavldquoMechanism of genomic instability in cells infected with thehigh-risk human papillomavirusesrdquo PLoS Pathogens vol 5 no4 2009
[6] N A Hamid C Brown and K Gaston ldquoThe regulation of cellproliferation by the papillomavirus early proteinsrdquo Cellular andMolecular Life Sciences vol 66 no 10 pp 1700ndash1717 2009
[7] V Kashyap and B C Das ldquoDNA aneuploidy and infectionof human papillomavirus type 16 in preneoplastic lesions ofthe uterine cervix correlation with progression to malignancyrdquoCancer Letters vol 123 no 1 pp 47ndash52 1998
[8] D Patel A Incassati N Wang and D J McCance ldquoHumanpapillomavirus type 16 E6 and E7 cause polyploidy in humankeratinocytes and up-regulation of G2-M-phase proteinsrdquoCan-cer Research vol 64 no 4 pp 1299ndash1306 2004
[9] G Boulet C Horvath D V Broeck S Sahebali and J BogersldquoHuman papillomavirus E6 and E7 oncogenesrdquo InternationalJournal of Biochemistry andCell Biology vol 39 no 11 pp 2006ndash2011 2007
[10] V OrsquoBrien G J Grindlay and M S Campo ldquoCell transfor-mation by the E5E8 protein of bovine papillomavirus type4 p27Kip1 elevated through increased protein synthesis issequestered by cyclin D1-CDK4 complexesrdquo Journal of Biolog-ical Chemistry vol 276 no 36 pp 33861ndash33868 2001
[11] A A McBride J G Oliveira and M G McPhillips ldquoPartition-ing viral genomes in mitosis same idea different targetsrdquo CellCycle vol 5 no 14 pp 1499ndash1502 2006
[12] J G Oliveira L A Colf and A A McBride ldquoVariationsin the association of papillomavirus E2 proteins with mitoticchromosomesrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 103 no 4 pp 1047ndash10522006
[13] S Duensing and K Munger ldquoHuman papillomaviruses andcentrosomeduplication errorsmodeling the origins of genomicinstabilityrdquo Oncogene vol 21 no 40 pp 6241ndash6248 2002
8 ISRN Oncology
[14] R C Recouso R C Stocco dos Santos R Freitas et al ldquoClasto-genic effect of bracken fern (Pteridium aquilinum aracnoideum)diet in peripheral lymphocytes of human consumers prelimi-nary datardquo Veterinary and Comparative Oncology vol 1 no 1pp 22ndash29 2003
[15] R C StoccoDos Santos C J Lindsey O P Ferraz et al ldquoBovinepapillomavirus transmission and chromosomal aberrations anexperimental modelrdquo Journal of General Virology vol 79 no 9pp 2127ndash2135 1998
[16] A C De Freitas C De Carvalho O Brunner et al ldquoViralDNA sequences in peripheral blood and vertical transmissionof the virus a discussion about BPV-1rdquo Brazilian Journal ofMicrobiology vol 34 no 1 pp 76ndash78 2003
[17] C J Lindsey M E Almeida C F Vicari et al ldquoBovine papillo-mavirus DNA inmilk blood urine semen and spermatozoa ofbovine papillomavirus-infected animalsrdquo Genetics and Molecu-lar Research vol 8 no 1 pp 310ndash318 2009
[18] S Roperto R Brun F Paolini et al ldquoDetection of bovinepapillomavirus type 2 in the peripheral blood of cattle withurinary bladder tumours possible biological rolerdquo Journal ofGeneral Virology vol 89 no 12 pp 3027ndash3033 2008
[19] 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
[20] J W Moura R C Stocco Dos Santos M L Z Dagli J LDrsquoAngelino E H Birgel and W Becak ldquoChromosome aberra-tions in cattle raised on bracken fern pasturerdquo Experientia vol44 no 9 pp 785ndash788 1988
[21] M B Lioi R Barbieri G Borzacchiello et al ldquoChromosomeaberrations in cattle with chronic enzootic haematuriardquo Journalof Comparative Pathology vol 131 no 2-3 pp 233ndash236 2004
[22] C De Carvalho A C De Freitas O Brunner et al ldquoBovinepapillomavirus type 2 in reproductive tract and gametes ofslaughtered bovine femalesrdquo Brazilian Journal of Microbiologyvol 34 no 1 pp 82ndash84 2003
[23] A Yaguiu C Carvalho A C Freitas et al ldquoPapilomatosis in cat-tle in situ detection of bovine papillomavirus DNA sequencesin reproductive tissuesrdquo Brazilian Journal of MorphologicalSciences vol 23 no 3-4 pp 525ndash529 2006
[24] 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
[25] M S Campo W F H Jarrett W OrsquoNeil and R J BarronldquoLatent papillomavirus infection in cattlerdquo Research in Veteri-nary Science vol 56 no 2 pp 151ndash157 1994
[26] V Peretti F Ciotola S Albarella et al ldquoChromosome fragilityin cattle with chronic enzootic haematuriardquo Mutagenesis vol22 no 5 pp 317ndash320 2007
[27] T Ogawa Y Tomita M Okada et al ldquoBroad-spectrum detec-tion of papillomaviruses in bovine teat papillomas and healthyteat skinrdquo Journal of General Virology vol 85 no 8 pp 2191ndash2197 2004
[28] A M Leal O P Ferraz C Carvalho et al ldquoQuercetin inducesstructural chromosome aberrations and uncommon rearrange-ments in bovine cells transformed by the E7 protein of bovinepapillomavirus type 4rdquo Veterinary and Comparative Oncologyvol 1 no 1 pp 15ndash21 2003
[29] A Duensing A Chin L Wang S-F Kuan and S DuensingldquoAnalysis of centrosome overduplication in correlation to cell
division errors in high-risk human papillomavirus (HPV)-asso-ciated anal neoplasmsrdquo Virology vol 372 no 1 pp 157ndash1642008
[30] Z Liu Y Liu Y Hong L Rapp E J Androphy and J J ChenldquoBovine papillomavirus type 1 E6-induced sensitization to apo-ptosis is distinct from its transforming activityrdquo Virology vol295 no 2 pp 230ndash237 2002
[31] 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
Figure 3 Primary cell cultures (a) normal skin (b) cutaneous papilloma (c) esophagus papilloma (d) papilloma of urinary bladder fromanimal affected with enzootic hematuria (10x) The arrow indicates a cell with a different morphology
affected animals it could be discussed that the chromosomeaberrations were not caused by virus action However thesame types of aberrations were detected in cells derived fromlesions positive for virus presence Furthermore there wasa significant difference between the chromosome aberrationlevels comparing affected and not affected animals
Inmatter of fact considering the similarity of the detectedchromosome aberrations the virus action on host chromatinwas verified as effective either in blood cells or in cells derivedfrom lesions We could argue that the virus load in theperipheral blood of the affected animals was too low to bedetected in conventional PCR The same argument could beused for the not affected animals leading to the need tocompare affected to nonaffected animals and emphasizing thedifferences observed in the levels of chromosomal aberra-tions
The virus action was verified rising numerical and struc-tural chromosome aberrations This fact indicates that thevirus acts in different ways in its interaction with host
chromatin It was previously described the virus oncoproteinaction on telomeres leading to centric fusion [28] Besidesthe virus acts on mitotic spindle changing the chromosomeset and rising aneuploidy [29]
Although BPV is described as nonintegrated in host cellits action produces different types of chromosome alterationssuggesting a large interaction with the chromatin and alsowith DNA repair mechanisms [30]
We have already described the presence of virussequences in primary culture cells in different passages [31]but now we demonstrate that these sequences are activeleading to chromosome alterations
4 Conclusions
We compare for the first time the action of bovine papillo-mavirus on host cell chromatin in cultured lymphocytes andprimary culture cells describing the increase of chromosomalaberrations in both cell types The primary cells cultures
6 ISRN Oncology
(a) (b) (c)
(d) (e) (f)
(g) (h)
Figure 4 Chromosome aberrations in primary culture cells ((a) (c) (e) and (g)) and peripheral lymphocytes ((b) (d) (f) and (h)) acentricfragments ((a) (b)) chromosome break (c) chromatid break (d) chromosome rearrangement with additiondeletion ((e) (f)) telomericassociation (g) centromeric association ((e) (h)) early chromatid separation (thin arrow in (a) (e))
not only presented BPV DNA sequences but also keep thesesequences throughout different passages These data plusrecent reports [18] emphasize possible active BPV infectionin blood as well as in in vitro cultured cells
Conflict of Interests
All authors declare that there is no conflict of interestsregarding the research authorship andor publication of thispaper The authors inform that all materials and trademarks
mentioned were used just as part of experimental protocoland there are no financial gains or favoritism of business
Acknowledgments
The authors would like to thank Ministerio de CienciaTecnologia e InovacaoConselho Nacional de DesenvolvimentoCientıfico e Tecnologico (CNPq Proc 5548162006-7 and4025392011-7) FAPESP (Proc 200602439-6) Coordenacaode Aperfeicoamento Pessoal de Nıvel Superior (CAPES) and
ISRN Oncology 7
Table 2 Frequencies of different aberrations detected in blood and primary culture cells of animals affected by cutaneous papillomasesophagus papilloma and enzootic hematuria
BloodGroup CA AT AF CB CrBC 088 plusmn 124 248 plusmn 146 233 plusmn 081 175 plusmn 248 160 plusmn 022
119875 gt 005 lowastlowast119875 gt 001 and curren119875 ge 01
CA centromere association AT association of telomere AF acentric fragments CB chromosome break CrB chromatid break AdDel additiondeletionEcrS early chromatid separation Aneu aneuploidy Polip polyploidy and gaps
Fundacao Butantan for financial support and Carolina da PazSabino for editorial support
References
[1] G Borzacchiello and F Roperto ldquoBovine papillomavirusespapillomas and cancer in cattlerdquoVeterinary Research vol 39 no5 article 45 2008
[2] M S Campo ldquoBovine papillomavirus and cancerrdquo VeterinaryJournal vol 154 no 3 pp 175ndash188 1997
[3] 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
[4] S Duensing L Y Lee A Duensing et al ldquoThe human papillo-mavirus 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
[5] M Kadaja H Isok-Paas T Laos E Ustav and M UstavldquoMechanism of genomic instability in cells infected with thehigh-risk human papillomavirusesrdquo PLoS Pathogens vol 5 no4 2009
[6] N A Hamid C Brown and K Gaston ldquoThe regulation of cellproliferation by the papillomavirus early proteinsrdquo Cellular andMolecular Life Sciences vol 66 no 10 pp 1700ndash1717 2009
[7] V Kashyap and B C Das ldquoDNA aneuploidy and infectionof human papillomavirus type 16 in preneoplastic lesions ofthe uterine cervix correlation with progression to malignancyrdquoCancer Letters vol 123 no 1 pp 47ndash52 1998
[8] D Patel A Incassati N Wang and D J McCance ldquoHumanpapillomavirus type 16 E6 and E7 cause polyploidy in humankeratinocytes and up-regulation of G2-M-phase proteinsrdquoCan-cer Research vol 64 no 4 pp 1299ndash1306 2004
[9] G Boulet C Horvath D V Broeck S Sahebali and J BogersldquoHuman papillomavirus E6 and E7 oncogenesrdquo InternationalJournal of Biochemistry andCell Biology vol 39 no 11 pp 2006ndash2011 2007
[10] V OrsquoBrien G J Grindlay and M S Campo ldquoCell transfor-mation by the E5E8 protein of bovine papillomavirus type4 p27Kip1 elevated through increased protein synthesis issequestered by cyclin D1-CDK4 complexesrdquo Journal of Biolog-ical Chemistry vol 276 no 36 pp 33861ndash33868 2001
[11] A A McBride J G Oliveira and M G McPhillips ldquoPartition-ing viral genomes in mitosis same idea different targetsrdquo CellCycle vol 5 no 14 pp 1499ndash1502 2006
[12] J G Oliveira L A Colf and A A McBride ldquoVariationsin the association of papillomavirus E2 proteins with mitoticchromosomesrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 103 no 4 pp 1047ndash10522006
[13] S Duensing and K Munger ldquoHuman papillomaviruses andcentrosomeduplication errorsmodeling the origins of genomicinstabilityrdquo Oncogene vol 21 no 40 pp 6241ndash6248 2002
8 ISRN Oncology
[14] R C Recouso R C Stocco dos Santos R Freitas et al ldquoClasto-genic effect of bracken fern (Pteridium aquilinum aracnoideum)diet in peripheral lymphocytes of human consumers prelimi-nary datardquo Veterinary and Comparative Oncology vol 1 no 1pp 22ndash29 2003
[15] R C StoccoDos Santos C J Lindsey O P Ferraz et al ldquoBovinepapillomavirus transmission and chromosomal aberrations anexperimental modelrdquo Journal of General Virology vol 79 no 9pp 2127ndash2135 1998
[16] A C De Freitas C De Carvalho O Brunner et al ldquoViralDNA sequences in peripheral blood and vertical transmissionof the virus a discussion about BPV-1rdquo Brazilian Journal ofMicrobiology vol 34 no 1 pp 76ndash78 2003
[17] C J Lindsey M E Almeida C F Vicari et al ldquoBovine papillo-mavirus DNA inmilk blood urine semen and spermatozoa ofbovine papillomavirus-infected animalsrdquo Genetics and Molecu-lar Research vol 8 no 1 pp 310ndash318 2009
[18] S Roperto R Brun F Paolini et al ldquoDetection of bovinepapillomavirus type 2 in the peripheral blood of cattle withurinary bladder tumours possible biological rolerdquo Journal ofGeneral Virology vol 89 no 12 pp 3027ndash3033 2008
[19] 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
[20] J W Moura R C Stocco Dos Santos M L Z Dagli J LDrsquoAngelino E H Birgel and W Becak ldquoChromosome aberra-tions in cattle raised on bracken fern pasturerdquo Experientia vol44 no 9 pp 785ndash788 1988
[21] M B Lioi R Barbieri G Borzacchiello et al ldquoChromosomeaberrations in cattle with chronic enzootic haematuriardquo Journalof Comparative Pathology vol 131 no 2-3 pp 233ndash236 2004
[22] C De Carvalho A C De Freitas O Brunner et al ldquoBovinepapillomavirus type 2 in reproductive tract and gametes ofslaughtered bovine femalesrdquo Brazilian Journal of Microbiologyvol 34 no 1 pp 82ndash84 2003
[23] A Yaguiu C Carvalho A C Freitas et al ldquoPapilomatosis in cat-tle in situ detection of bovine papillomavirus DNA sequencesin reproductive tissuesrdquo Brazilian Journal of MorphologicalSciences vol 23 no 3-4 pp 525ndash529 2006
[24] 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
[25] M S Campo W F H Jarrett W OrsquoNeil and R J BarronldquoLatent papillomavirus infection in cattlerdquo Research in Veteri-nary Science vol 56 no 2 pp 151ndash157 1994
[26] V Peretti F Ciotola S Albarella et al ldquoChromosome fragilityin cattle with chronic enzootic haematuriardquo Mutagenesis vol22 no 5 pp 317ndash320 2007
[27] T Ogawa Y Tomita M Okada et al ldquoBroad-spectrum detec-tion of papillomaviruses in bovine teat papillomas and healthyteat skinrdquo Journal of General Virology vol 85 no 8 pp 2191ndash2197 2004
[28] A M Leal O P Ferraz C Carvalho et al ldquoQuercetin inducesstructural chromosome aberrations and uncommon rearrange-ments in bovine cells transformed by the E7 protein of bovinepapillomavirus type 4rdquo Veterinary and Comparative Oncologyvol 1 no 1 pp 15ndash21 2003
[29] A Duensing A Chin L Wang S-F Kuan and S DuensingldquoAnalysis of centrosome overduplication in correlation to cell
division errors in high-risk human papillomavirus (HPV)-asso-ciated anal neoplasmsrdquo Virology vol 372 no 1 pp 157ndash1642008
[30] Z Liu Y Liu Y Hong L Rapp E J Androphy and J J ChenldquoBovine papillomavirus type 1 E6-induced sensitization to apo-ptosis is distinct from its transforming activityrdquo Virology vol295 no 2 pp 230ndash237 2002
[31] 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
Figure 4 Chromosome aberrations in primary culture cells ((a) (c) (e) and (g)) and peripheral lymphocytes ((b) (d) (f) and (h)) acentricfragments ((a) (b)) chromosome break (c) chromatid break (d) chromosome rearrangement with additiondeletion ((e) (f)) telomericassociation (g) centromeric association ((e) (h)) early chromatid separation (thin arrow in (a) (e))
not only presented BPV DNA sequences but also keep thesesequences throughout different passages These data plusrecent reports [18] emphasize possible active BPV infectionin blood as well as in in vitro cultured cells
Conflict of Interests
All authors declare that there is no conflict of interestsregarding the research authorship andor publication of thispaper The authors inform that all materials and trademarks
mentioned were used just as part of experimental protocoland there are no financial gains or favoritism of business
Acknowledgments
The authors would like to thank Ministerio de CienciaTecnologia e InovacaoConselho Nacional de DesenvolvimentoCientıfico e Tecnologico (CNPq Proc 5548162006-7 and4025392011-7) FAPESP (Proc 200602439-6) Coordenacaode Aperfeicoamento Pessoal de Nıvel Superior (CAPES) and
ISRN Oncology 7
Table 2 Frequencies of different aberrations detected in blood and primary culture cells of animals affected by cutaneous papillomasesophagus papilloma and enzootic hematuria
BloodGroup CA AT AF CB CrBC 088 plusmn 124 248 plusmn 146 233 plusmn 081 175 plusmn 248 160 plusmn 022
119875 gt 005 lowastlowast119875 gt 001 and curren119875 ge 01
CA centromere association AT association of telomere AF acentric fragments CB chromosome break CrB chromatid break AdDel additiondeletionEcrS early chromatid separation Aneu aneuploidy Polip polyploidy and gaps
Fundacao Butantan for financial support and Carolina da PazSabino for editorial support
References
[1] G Borzacchiello and F Roperto ldquoBovine papillomavirusespapillomas and cancer in cattlerdquoVeterinary Research vol 39 no5 article 45 2008
[2] M S Campo ldquoBovine papillomavirus and cancerrdquo VeterinaryJournal vol 154 no 3 pp 175ndash188 1997
[3] 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
[4] S Duensing L Y Lee A Duensing et al ldquoThe human papillo-mavirus 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
[5] M Kadaja H Isok-Paas T Laos E Ustav and M UstavldquoMechanism of genomic instability in cells infected with thehigh-risk human papillomavirusesrdquo PLoS Pathogens vol 5 no4 2009
[6] N A Hamid C Brown and K Gaston ldquoThe regulation of cellproliferation by the papillomavirus early proteinsrdquo Cellular andMolecular Life Sciences vol 66 no 10 pp 1700ndash1717 2009
[7] V Kashyap and B C Das ldquoDNA aneuploidy and infectionof human papillomavirus type 16 in preneoplastic lesions ofthe uterine cervix correlation with progression to malignancyrdquoCancer Letters vol 123 no 1 pp 47ndash52 1998
[8] D Patel A Incassati N Wang and D J McCance ldquoHumanpapillomavirus type 16 E6 and E7 cause polyploidy in humankeratinocytes and up-regulation of G2-M-phase proteinsrdquoCan-cer Research vol 64 no 4 pp 1299ndash1306 2004
[9] G Boulet C Horvath D V Broeck S Sahebali and J BogersldquoHuman papillomavirus E6 and E7 oncogenesrdquo InternationalJournal of Biochemistry andCell Biology vol 39 no 11 pp 2006ndash2011 2007
[10] V OrsquoBrien G J Grindlay and M S Campo ldquoCell transfor-mation by the E5E8 protein of bovine papillomavirus type4 p27Kip1 elevated through increased protein synthesis issequestered by cyclin D1-CDK4 complexesrdquo Journal of Biolog-ical Chemistry vol 276 no 36 pp 33861ndash33868 2001
[11] A A McBride J G Oliveira and M G McPhillips ldquoPartition-ing viral genomes in mitosis same idea different targetsrdquo CellCycle vol 5 no 14 pp 1499ndash1502 2006
[12] J G Oliveira L A Colf and A A McBride ldquoVariationsin the association of papillomavirus E2 proteins with mitoticchromosomesrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 103 no 4 pp 1047ndash10522006
[13] S Duensing and K Munger ldquoHuman papillomaviruses andcentrosomeduplication errorsmodeling the origins of genomicinstabilityrdquo Oncogene vol 21 no 40 pp 6241ndash6248 2002
8 ISRN Oncology
[14] R C Recouso R C Stocco dos Santos R Freitas et al ldquoClasto-genic effect of bracken fern (Pteridium aquilinum aracnoideum)diet in peripheral lymphocytes of human consumers prelimi-nary datardquo Veterinary and Comparative Oncology vol 1 no 1pp 22ndash29 2003
[15] R C StoccoDos Santos C J Lindsey O P Ferraz et al ldquoBovinepapillomavirus transmission and chromosomal aberrations anexperimental modelrdquo Journal of General Virology vol 79 no 9pp 2127ndash2135 1998
[16] A C De Freitas C De Carvalho O Brunner et al ldquoViralDNA sequences in peripheral blood and vertical transmissionof the virus a discussion about BPV-1rdquo Brazilian Journal ofMicrobiology vol 34 no 1 pp 76ndash78 2003
[17] C J Lindsey M E Almeida C F Vicari et al ldquoBovine papillo-mavirus DNA inmilk blood urine semen and spermatozoa ofbovine papillomavirus-infected animalsrdquo Genetics and Molecu-lar Research vol 8 no 1 pp 310ndash318 2009
[18] S Roperto R Brun F Paolini et al ldquoDetection of bovinepapillomavirus type 2 in the peripheral blood of cattle withurinary bladder tumours possible biological rolerdquo Journal ofGeneral Virology vol 89 no 12 pp 3027ndash3033 2008
[19] 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
[20] J W Moura R C Stocco Dos Santos M L Z Dagli J LDrsquoAngelino E H Birgel and W Becak ldquoChromosome aberra-tions in cattle raised on bracken fern pasturerdquo Experientia vol44 no 9 pp 785ndash788 1988
[21] M B Lioi R Barbieri G Borzacchiello et al ldquoChromosomeaberrations in cattle with chronic enzootic haematuriardquo Journalof Comparative Pathology vol 131 no 2-3 pp 233ndash236 2004
[22] C De Carvalho A C De Freitas O Brunner et al ldquoBovinepapillomavirus type 2 in reproductive tract and gametes ofslaughtered bovine femalesrdquo Brazilian Journal of Microbiologyvol 34 no 1 pp 82ndash84 2003
[23] A Yaguiu C Carvalho A C Freitas et al ldquoPapilomatosis in cat-tle in situ detection of bovine papillomavirus DNA sequencesin reproductive tissuesrdquo Brazilian Journal of MorphologicalSciences vol 23 no 3-4 pp 525ndash529 2006
[24] 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
[25] M S Campo W F H Jarrett W OrsquoNeil and R J BarronldquoLatent papillomavirus infection in cattlerdquo Research in Veteri-nary Science vol 56 no 2 pp 151ndash157 1994
[26] V Peretti F Ciotola S Albarella et al ldquoChromosome fragilityin cattle with chronic enzootic haematuriardquo Mutagenesis vol22 no 5 pp 317ndash320 2007
[27] T Ogawa Y Tomita M Okada et al ldquoBroad-spectrum detec-tion of papillomaviruses in bovine teat papillomas and healthyteat skinrdquo Journal of General Virology vol 85 no 8 pp 2191ndash2197 2004
[28] A M Leal O P Ferraz C Carvalho et al ldquoQuercetin inducesstructural chromosome aberrations and uncommon rearrange-ments in bovine cells transformed by the E7 protein of bovinepapillomavirus type 4rdquo Veterinary and Comparative Oncologyvol 1 no 1 pp 15ndash21 2003
[29] A Duensing A Chin L Wang S-F Kuan and S DuensingldquoAnalysis of centrosome overduplication in correlation to cell
division errors in high-risk human papillomavirus (HPV)-asso-ciated anal neoplasmsrdquo Virology vol 372 no 1 pp 157ndash1642008
[30] Z Liu Y Liu Y Hong L Rapp E J Androphy and J J ChenldquoBovine papillomavirus type 1 E6-induced sensitization to apo-ptosis is distinct from its transforming activityrdquo Virology vol295 no 2 pp 230ndash237 2002
[31] 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
Table 2 Frequencies of different aberrations detected in blood and primary culture cells of animals affected by cutaneous papillomasesophagus papilloma and enzootic hematuria
BloodGroup CA AT AF CB CrBC 088 plusmn 124 248 plusmn 146 233 plusmn 081 175 plusmn 248 160 plusmn 022
119875 gt 005 lowastlowast119875 gt 001 and curren119875 ge 01
CA centromere association AT association of telomere AF acentric fragments CB chromosome break CrB chromatid break AdDel additiondeletionEcrS early chromatid separation Aneu aneuploidy Polip polyploidy and gaps
Fundacao Butantan for financial support and Carolina da PazSabino for editorial support
References
[1] G Borzacchiello and F Roperto ldquoBovine papillomavirusespapillomas and cancer in cattlerdquoVeterinary Research vol 39 no5 article 45 2008
[2] M S Campo ldquoBovine papillomavirus and cancerrdquo VeterinaryJournal vol 154 no 3 pp 175ndash188 1997
[3] 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
[4] S Duensing L Y Lee A Duensing et al ldquoThe human papillo-mavirus 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
[5] M Kadaja H Isok-Paas T Laos E Ustav and M UstavldquoMechanism of genomic instability in cells infected with thehigh-risk human papillomavirusesrdquo PLoS Pathogens vol 5 no4 2009
[6] N A Hamid C Brown and K Gaston ldquoThe regulation of cellproliferation by the papillomavirus early proteinsrdquo Cellular andMolecular Life Sciences vol 66 no 10 pp 1700ndash1717 2009
[7] V Kashyap and B C Das ldquoDNA aneuploidy and infectionof human papillomavirus type 16 in preneoplastic lesions ofthe uterine cervix correlation with progression to malignancyrdquoCancer Letters vol 123 no 1 pp 47ndash52 1998
[8] D Patel A Incassati N Wang and D J McCance ldquoHumanpapillomavirus type 16 E6 and E7 cause polyploidy in humankeratinocytes and up-regulation of G2-M-phase proteinsrdquoCan-cer Research vol 64 no 4 pp 1299ndash1306 2004
[9] G Boulet C Horvath D V Broeck S Sahebali and J BogersldquoHuman papillomavirus E6 and E7 oncogenesrdquo InternationalJournal of Biochemistry andCell Biology vol 39 no 11 pp 2006ndash2011 2007
[10] V OrsquoBrien G J Grindlay and M S Campo ldquoCell transfor-mation by the E5E8 protein of bovine papillomavirus type4 p27Kip1 elevated through increased protein synthesis issequestered by cyclin D1-CDK4 complexesrdquo Journal of Biolog-ical Chemistry vol 276 no 36 pp 33861ndash33868 2001
[11] A A McBride J G Oliveira and M G McPhillips ldquoPartition-ing viral genomes in mitosis same idea different targetsrdquo CellCycle vol 5 no 14 pp 1499ndash1502 2006
[12] J G Oliveira L A Colf and A A McBride ldquoVariationsin the association of papillomavirus E2 proteins with mitoticchromosomesrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 103 no 4 pp 1047ndash10522006
[13] S Duensing and K Munger ldquoHuman papillomaviruses andcentrosomeduplication errorsmodeling the origins of genomicinstabilityrdquo Oncogene vol 21 no 40 pp 6241ndash6248 2002
8 ISRN Oncology
[14] R C Recouso R C Stocco dos Santos R Freitas et al ldquoClasto-genic effect of bracken fern (Pteridium aquilinum aracnoideum)diet in peripheral lymphocytes of human consumers prelimi-nary datardquo Veterinary and Comparative Oncology vol 1 no 1pp 22ndash29 2003
[15] R C StoccoDos Santos C J Lindsey O P Ferraz et al ldquoBovinepapillomavirus transmission and chromosomal aberrations anexperimental modelrdquo Journal of General Virology vol 79 no 9pp 2127ndash2135 1998
[16] A C De Freitas C De Carvalho O Brunner et al ldquoViralDNA sequences in peripheral blood and vertical transmissionof the virus a discussion about BPV-1rdquo Brazilian Journal ofMicrobiology vol 34 no 1 pp 76ndash78 2003
[17] C J Lindsey M E Almeida C F Vicari et al ldquoBovine papillo-mavirus DNA inmilk blood urine semen and spermatozoa ofbovine papillomavirus-infected animalsrdquo Genetics and Molecu-lar Research vol 8 no 1 pp 310ndash318 2009
[18] S Roperto R Brun F Paolini et al ldquoDetection of bovinepapillomavirus type 2 in the peripheral blood of cattle withurinary bladder tumours possible biological rolerdquo Journal ofGeneral Virology vol 89 no 12 pp 3027ndash3033 2008
[19] 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
[20] J W Moura R C Stocco Dos Santos M L Z Dagli J LDrsquoAngelino E H Birgel and W Becak ldquoChromosome aberra-tions in cattle raised on bracken fern pasturerdquo Experientia vol44 no 9 pp 785ndash788 1988
[21] M B Lioi R Barbieri G Borzacchiello et al ldquoChromosomeaberrations in cattle with chronic enzootic haematuriardquo Journalof Comparative Pathology vol 131 no 2-3 pp 233ndash236 2004
[22] C De Carvalho A C De Freitas O Brunner et al ldquoBovinepapillomavirus type 2 in reproductive tract and gametes ofslaughtered bovine femalesrdquo Brazilian Journal of Microbiologyvol 34 no 1 pp 82ndash84 2003
[23] A Yaguiu C Carvalho A C Freitas et al ldquoPapilomatosis in cat-tle in situ detection of bovine papillomavirus DNA sequencesin reproductive tissuesrdquo Brazilian Journal of MorphologicalSciences vol 23 no 3-4 pp 525ndash529 2006
[24] 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
[25] M S Campo W F H Jarrett W OrsquoNeil and R J BarronldquoLatent papillomavirus infection in cattlerdquo Research in Veteri-nary Science vol 56 no 2 pp 151ndash157 1994
[26] V Peretti F Ciotola S Albarella et al ldquoChromosome fragilityin cattle with chronic enzootic haematuriardquo Mutagenesis vol22 no 5 pp 317ndash320 2007
[27] T Ogawa Y Tomita M Okada et al ldquoBroad-spectrum detec-tion of papillomaviruses in bovine teat papillomas and healthyteat skinrdquo Journal of General Virology vol 85 no 8 pp 2191ndash2197 2004
[28] A M Leal O P Ferraz C Carvalho et al ldquoQuercetin inducesstructural chromosome aberrations and uncommon rearrange-ments in bovine cells transformed by the E7 protein of bovinepapillomavirus type 4rdquo Veterinary and Comparative Oncologyvol 1 no 1 pp 15ndash21 2003
[29] A Duensing A Chin L Wang S-F Kuan and S DuensingldquoAnalysis of centrosome overduplication in correlation to cell
division errors in high-risk human papillomavirus (HPV)-asso-ciated anal neoplasmsrdquo Virology vol 372 no 1 pp 157ndash1642008
[30] Z Liu Y Liu Y Hong L Rapp E J Androphy and J J ChenldquoBovine papillomavirus type 1 E6-induced sensitization to apo-ptosis is distinct from its transforming activityrdquo Virology vol295 no 2 pp 230ndash237 2002
[31] 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
[14] R C Recouso R C Stocco dos Santos R Freitas et al ldquoClasto-genic effect of bracken fern (Pteridium aquilinum aracnoideum)diet in peripheral lymphocytes of human consumers prelimi-nary datardquo Veterinary and Comparative Oncology vol 1 no 1pp 22ndash29 2003
[15] R C StoccoDos Santos C J Lindsey O P Ferraz et al ldquoBovinepapillomavirus transmission and chromosomal aberrations anexperimental modelrdquo Journal of General Virology vol 79 no 9pp 2127ndash2135 1998
[16] A C De Freitas C De Carvalho O Brunner et al ldquoViralDNA sequences in peripheral blood and vertical transmissionof the virus a discussion about BPV-1rdquo Brazilian Journal ofMicrobiology vol 34 no 1 pp 76ndash78 2003
[17] C J Lindsey M E Almeida C F Vicari et al ldquoBovine papillo-mavirus DNA inmilk blood urine semen and spermatozoa ofbovine papillomavirus-infected animalsrdquo Genetics and Molecu-lar Research vol 8 no 1 pp 310ndash318 2009
[18] S Roperto R Brun F Paolini et al ldquoDetection of bovinepapillomavirus type 2 in the peripheral blood of cattle withurinary bladder tumours possible biological rolerdquo Journal ofGeneral Virology vol 89 no 12 pp 3027ndash3033 2008
[19] 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
[20] J W Moura R C Stocco Dos Santos M L Z Dagli J LDrsquoAngelino E H Birgel and W Becak ldquoChromosome aberra-tions in cattle raised on bracken fern pasturerdquo Experientia vol44 no 9 pp 785ndash788 1988
[21] M B Lioi R Barbieri G Borzacchiello et al ldquoChromosomeaberrations in cattle with chronic enzootic haematuriardquo Journalof Comparative Pathology vol 131 no 2-3 pp 233ndash236 2004
[22] C De Carvalho A C De Freitas O Brunner et al ldquoBovinepapillomavirus type 2 in reproductive tract and gametes ofslaughtered bovine femalesrdquo Brazilian Journal of Microbiologyvol 34 no 1 pp 82ndash84 2003
[23] A Yaguiu C Carvalho A C Freitas et al ldquoPapilomatosis in cat-tle in situ detection of bovine papillomavirus DNA sequencesin reproductive tissuesrdquo Brazilian Journal of MorphologicalSciences vol 23 no 3-4 pp 525ndash529 2006
[24] 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
[25] M S Campo W F H Jarrett W OrsquoNeil and R J BarronldquoLatent papillomavirus infection in cattlerdquo Research in Veteri-nary Science vol 56 no 2 pp 151ndash157 1994
[26] V Peretti F Ciotola S Albarella et al ldquoChromosome fragilityin cattle with chronic enzootic haematuriardquo Mutagenesis vol22 no 5 pp 317ndash320 2007
[27] T Ogawa Y Tomita M Okada et al ldquoBroad-spectrum detec-tion of papillomaviruses in bovine teat papillomas and healthyteat skinrdquo Journal of General Virology vol 85 no 8 pp 2191ndash2197 2004
[28] A M Leal O P Ferraz C Carvalho et al ldquoQuercetin inducesstructural chromosome aberrations and uncommon rearrange-ments in bovine cells transformed by the E7 protein of bovinepapillomavirus type 4rdquo Veterinary and Comparative Oncologyvol 1 no 1 pp 15ndash21 2003
[29] A Duensing A Chin L Wang S-F Kuan and S DuensingldquoAnalysis of centrosome overduplication in correlation to cell
division errors in high-risk human papillomavirus (HPV)-asso-ciated anal neoplasmsrdquo Virology vol 372 no 1 pp 157ndash1642008
[30] Z Liu Y Liu Y Hong L Rapp E J Androphy and J J ChenldquoBovine papillomavirus type 1 E6-induced sensitization to apo-ptosis is distinct from its transforming activityrdquo Virology vol295 no 2 pp 230ndash237 2002
[31] 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
