Protective role of Puralpha to cisplatin

Protective role of Purα to cisplatin

Rafal Kaminski, Armine Darbinyan, Nana Merabova, Satish L. Deshmane, Martyn K. White,and Kamel Khalili*Department of Neuroscience; Center for Neurovirology; Temple University School of Medicine;Philadelphia, Pennsylvania USA

AbstractBackground—The nucleic acid-binding protein Purα is involved at stalled DNA replication forks,in double-strand break (DSB) DNA repair and the cellular response to DNA replication stress.Purα also regulates homologous recombination-directed DNA repair (HRR).

Results—Cells lacking Purα showed enhanced sensitivity to cisplatin as evaluated by assays forcell viability and cell clonogenicity. This was seen both in Purα-negative MEFs and in humanglioblastoma cells treated with siRNA directed against Purα. MEFs lacking Purα also showedenhanced H2AX phosphorylation in response to cisplatin. Repair of a reporter plasmid that had beentreated with cisplatin was decreased in a reactivation assay using Purα-negative MEFs and thecapacity of nuclear extracts from Purα-negative MEFs to perform non-homologous end-joining invitro was also impaired.

Methods—We investigated the effects of the DNA damage-inducing cancer chemotherapeuticagent cisplatin on mouse embryo fibroblasts (MEFs) from PURA-/- knockout mice that lack Purα.

Conclusions—Purα has a role in the cellular response to cisplatin-induced DNA damage and mayprovide new therapeutic modalities for cisplatin-resistant tumors.

KeywordsPur-alpha; cisplatin; DNA damage; DNA repair; chemotherapy; single stranded DNA binding protein

IntroductionCisplatin, also called cisplatinum and cisdiamminedichlorido-platinum(II), is a platinum-basedcancer chemotherapeutic drug, which is often used to treat various types of cancers, includingsome carcinomas, sarcomas, lymphomas and some germ cell tumors, (e.g., seminomas andgerminomas). Cisplatin was the first member of a platinum-based class of therapeutic agents,which now also includes carboplatin and oxaliplatin.1 It is a potent antitumor agent and exertsits effect via its interaction with DNA to produce cross-linked DNA adducts that activatecheckpoint signaling pathways and thereby induce apoptosis.2,3 While most types of cancerare susceptible to cisplatin, some malignancies are not susceptible. Further, tumors that areinitially responsive may acquire resistance to cisplatin due to the occurrence of mutations orepigenetic events. A number of events have been reported to contribute to cisplatin resistanceincluding reduced cellular uptake, increased efflux from the cell, the induction of intracellulardetoxification by glutathione, defects in apoptosis induction, changes in intracellular signaltransduction pathways and alterations in DNA repair (reviewed in refs. 4 and 5). Theeffectiveness of cisplatin against testicular carcinomas has been attributed to the reduced

*Correspondence to: Kamel Khalili; 1900 North 12th Street; 015-96, Room 203; Philadelphia, Pennsylvania 19122 USA; Tel.:215.204.0678; Fax: 215.204.0679; Email: E-mail: [email protected].

NIH Public AccessAuthor ManuscriptCancer Biol Ther. Author manuscript; available in PMC 2009 December 1.

Published in final edited form as:Cancer Biol Ther. 2008 December ; 7(12): 1926–1935.

NIH

-PA Author Manuscript

NIH


NIH


capacity of testis tumor cells to repair cisplatin-damaged DNA.6 A number of changes in DNArepair have been reported to be associated with platinum resistance including enzymes involvedin nucleotide excision repair (e.g., xeroderma pigmentosum, complementation group D protein,XPD),7 base excision repair (e.g., DNA polymerase β),8 the homologous recombination repairsystem (e.g., the Rad51-paralog protein, X-ray repair-complementing protein defective inchinese hamster-3, XRCC3)9 and topoisomerase I.10

Our recent studies on DNA repair have focused on the role of the protein Purα.11,12 Purα isa cellular regulatory protein that was first isolated from mouse brain using an assay based onits ability to bind to a DNA sequence that is found in the mouse myelin basic protein genepromoter.13,14 Similarly, human Purα binds to a DNA sequence that is found in the humanc-Myc gene promoter and was cloned from a HeLa cell cDNA library and sequenced.15,16Mouse Purα17 differs from human Purα at only two amino acid residues16 and the DNA-binding domain of Purα is especially strongly conserved throughout evolution. Purα is amember of the Pur family of proteins together with Purβ16 and Purγ18 and it is expressed invirtually every animal tissue.19 Purα is a multifunctional protein, which binds both DNA andRNA and is thought to function in the initiation of DNA replication, control of transcriptionand mRNA translation.19,20 Recently, Purα was shown to be involved in transporting andtargeting mRNAs in neuronal cells.21-23

Purα may also be involved in cell cycle regulation and in the process of oncogenictransformation since it can bind to several regulatory proteins including the retinoblastomatumor suppressor protein,24 E2F-1,25,26 Sp1,27 and YB-1.28 Levels of Purα vary throughthe cell cycle, declining at S-phase and peaking during mitosis.29 Microinjection of Purα intomouse fibroblasts causes cell cycle arrest at both the G1/S or G2/M checkpoints.30 Expressionof Purα in Ras-transformed mouse fibroblasts inhibited their growth in soft agar.31 Ectopicoverexpression of Purα was found to suppress growth of transformed and tumor cells includingglioblastomas.32 Some data also points to a role for Purα downregulation in leukemogenesis.33,34

The creation of knockout mice with targeted inactivation of the PURA gene, which encodesPurα, revealed an essential role for Purα in postnatal brain development.35 Mice withdisruption of both PURA alleles (PURA-/-) develop neurological problems at two weeks ofage and die by four weeks. The creation of these knockout mice has made available mouseembryo fibroblast primary cultures (MEFs) derived from the PURA-/- mice and PURA+/+

controls that can be used as an experimental system to examine the cellular functions of Purα.

Recently, we have found that Purα can affect cellular DNA repair, specifically that Purα hasa role as a caretaker protein that is involved in the repair of double-strand breaks (DSBs), whichare produced after DNA damage.11 We have also found that Purα regulates homologousrecombination-directed DNA repair (HRR) and the expression of the HRR protein Rad51.12In light of these findings implicating Purα in DNA repair and the earlier reports of theinvolvement of DNA repair processes in cisplatin resistance that are discussed above, weinvestigated whether Purα may have a role in the cellular response to DNA damage inducedby cisplatin.

ResultsPurα-negative MEFs (Purα-MEFs) have an increased sensitivity to cisplatin in cell viabilityand clonogenicity assays

The effect of cisplatin on Purα positive and negative MEFs was examined (Fig. 1). The lackof Purα protein expression in the Purα-negative MEFs was confirmed by Western blot (Fig.1A). The sensitivity of Purα-negative MEFs to cisplatin was greater at all concentrations tested,

Kaminski et al. Page 2

Cancer Biol Ther. Author manuscript; available in PMC 2009 December 1.

NIH


NIH


NIH


up to 2 μM. Similarly a time course for the decline of cell viability with 0.5 μM cisplatin showeda faster rate of cell death for the Purα-negative MEFs compared to the Purα-positive MEFs(Fig. 1B). With respect to clonogenicity, the sensitivity of Purα-negative MEFs to cisplatinwas greater at all concentrations tested, up to 2 μM (Fig. 1C).

Purα-negative MEFs show faster cell cycle transition and more γH2AX as measured by FACSanalysis and immunocytochemistry

The effect of cisplatin on Purα positive and negative MEFs was examined using eitherasynchronous cultures or cultures that had been synchronized by serum starvation followed byrelease from starvation (Fig. 2). As we have reported before,32,44 Purα is growth inhibitoryand slows progression through the cell cycle. Thus, in both the presence and absence ofcisplatin, Purα-negative cells exit G1/G0 more quickly than Purα-positive cells (Fig. 2A and Erespectively). Similarly, untreated Purα-negative cells enter S-phase more rapidly than Purα-positive cells (28 and 24 hours respectively; Fig. 2B). Both Purα-negative and Purα-positivecells exit S-phase by 27 h (Fig. 2B). Interestingly, cisplatin-treated cells do not exit S-phasebut accumulate in this phase, presumably due to unresolved DNA damage (Fig. 2F). Again,the Purα-negative cells accumulate more rapidly than the Purα-positive cells. Likewise, whileuntreated cells begin to reach G2/M by 24-27 h (Fig. 2C), cisplatin-treated cells do not reachG2/M (Fig. 2G). No γH2AX accumulation was detected in untreated cells (Fig. 2D). However,γH2AX accumulation was detected in the Purα-negative but not the Purα-positive cells at thelater time points (24-27 h; Fig. 2H). In a separate experiment, increased accumulation ofγH2AX in Purα-negative cells relative to Purα-positive cells was also demonstrated byimmunocytochemistry (Fig. 2I). Cells arrested in G0/G1 were released from serum starvationand treated with cisplatin. Foci of phospho-histone2AX (Ser139; γH2AX) were observed byfluorescence microscopy at 0.5 h post-treatment. Extensive γH2AX foci formation wasobserved in the nuclei of cells that lack Purα (right-hand photograph, Fig. 2I). These data alsoindicate that Purα-negative cells are more sensitive than Purα-positive cells for the inductionof γH2AX by cisplatin.

Purα-negative cells are impaired in their ability to reactivate transfected platinated reporterplasmid

This assay measures the ability of cells to repair DNA damage that had been inflicted to areporter plasmid by in vitro treatment of the plasmid with cisplatin (platination). Purα positiveand negative MEFs were transfected with platinated and untreated control plasmid andluciferase measured at various time points posttransfection. As shown in Figure 3A, repair ofthe platinated plasmid was significantly more rapid in the Purα-positive MEFs than in thePurα-negative MEFs. In another experiment, the transfections were performed with or withoutan expression plasmid for T7-tagged Purα (Fig. 3B). Again, repair of the platinated plasmidwas significantly more rapid in the Purα-positive MEFs than in the Purα-negative MEFs. Inthe cases where T7-tagged Purα was cotransfected, repair of the platinated plasmid wassignificantly improved in the Purα-negative MEFs but only at the later time points (21-27hours). The cell extracts used in Figure 3B were analyzed by Western blot with antibody toPurα (Fig. 3C). The presence and absence of Purα was confirmed in the Purα positive andnegative MEFs respectively as indicated. The cells that were transfected with T7-Purα showedan additional band, which ran at a slightly higher molecular weight and appeared at the latertime points (21-27 hours), thus correlating with the improved rates of platinated plasmid repair.

Purα-MEFs show enhanced sensitivity to H2O2 in the comet assayThe comet assay is indicative of fragmented DNA “tailing” from a cell subject to in situ alkalineagarose gel electrophoresis and is thus a measure of cellular DNA fragmentation and its repair.Purα positive and negative MEFs were treated with and without cisplatin in the presence or



NIH


NIH


NIH


absence of hydrogen peroxide. As shown in Figure 4, cisplatin alone caused no increase inOlive Tail Moment (OTM) indicating no increase in DNA fragmentation, presumably due tothe capacity of cisplatin to induce interstrand DNA crosslinking.2,3 Treatment of cells withhydrogen peroxide induced significant DNA fragmentation (time zero), which was more severein the Purα-negative cells (left-hand panel) than in the Purα-positive cells. Interestingly,combined treatment of Purα-negative cells with H2O2 and cisplatin resulted in a decreasedOTM at time zero, which is consistent with cisplatin-induced formation of cross-links thatretard DNA migration. On the other hand, no significant change in OTM was detected underthe same conditions for the Purα-positive cells at time zero, suggesting that Purα is protectiveagainst the formation of cisplatin-induced DNA-DNA cross-links.

After 20 hours, DNA fragmentation was near background in the Purα-positive cells, while therewas still significant DNA fragmentation in the Purα-negative cells. Thus initial DNA damageis more extensive in Purα-negative cells and is also repaired more slowly.

Purα-negative cells exhibit impaired nonhomologous end-joining repair activityThe activity of nuclear extracts from cells to concatemerate linear plasmid DNA withunmatching termini is a measure of nonhomologous end-joining (NHEJ) double-strand breakDNA repair activity.41 Figure 5A shows a representative NHEJ assay with the monomericlinear plasmid DNA indicated by the arrowhead and the multimeric DNA concatamersindicated by the bracket. The data indicate that the nuclear extracts from Purα-negative cellshave substantially lower NHEJ activity than the nuclear extracts from Purα-positive cells. Lane1 shows the negative control with no nuclear extract. Expression of Purα in these nuclearextracts was measured by Western blot with PCNA as a loading control (Fig. 5B).

SiRNA directed against Purα sensitizes human U87MG glioblastoma cells and HeLa cervicalcarcinoma cells to the cytotoxic effects of cisplatin

Transfection of U87MG cells with siRNA directed against Purα (Purα-siRNA) caused asignificant reduction of the level of expression of cellular Purα protein when compared to cellstransfected with a control non-targeting siRNA (NT-siRNA), (Fig. 6A). This resulted in asignificant decrease in viability of the cells in response to different concentrations of cisplatinas measured by trypan blue exclusion (Fig. 6B). Similarly there was a significant decrease inthe clonogenicity of the cells in response to different concentrations of cisplatin as measuredby assay of colony forming units (CFU; Fig. 6C). Similar results were obtained for HeLa cells(Fig. 6D-F). Note that the Western blot in Figure 6A was performed with the same U87 MGcells as the data shown in Figure 6B and C and the Western blot in Figure 6D was performedwith the same HeLa cells as the data shown in Figure 6E and F.

siRNA directed against Purα sensitizes some human brain and prostate tumor cell types tothe cytotoxic effects of cisplatin: T98G and LN229 glioblastoma cells, Daoy medulloblastomacells, C4-2, C4-2B, LNCaP and DU145 prostate carcinoma cells

In order to examine how different types of human brain and prostate tumor cell types may beaffected in their sensitivity to cisplatin by Purα knockdown, we selected these three humanbrain tumor cell lines and four human prostate tumor cell lines. As shown inset in Figure 7A-G, all cell types showed significant knockdown in Purα protein level upon transfection withPurα-siRNA (lanes 3) when compared to NT-siRNA (lanes 2) or untransfected control (lane1). Viability was measured by assaying the amount of protein remaining in the cell monolayerfollowing cisplatin treatment and washing with PBS and was expressed on the Y-axes relativeto untreated control (100%). For the human brain tumors (Figs. 7A-C), cisplatin was toxic forall cells and siRNA for Purα had a protective trend. The extent of this protective trend wasvariable between the three lines. For example, T98G was relatively resistant to cisplatin, with50% killing at about 2 μM cisplatin, and was significantly sensitized to cisplatin by siRNA for



NIH


NIH


NIH


Purα (Fig. 7A). On the other hand, Daoy was very sensitive to cisplatin, with 50% killing atabout 0.1 μM cisplatin, and siRNA for Purα had little effect (Fig. 7C). LN229 has anintermediate phenotype (Fig. 7B). For the human prostate tumor cell lines (Fig. 7D and G), asimilar situation was found with respect to the effect of Purα knockdown on sensitivity tocisplatin. All four of the human prostate carcinoma cell lines showed significant knockdownin Purα protein levels upon transfection with Purα-siRNA (insets, lanes 3) when compared toNT-siRNA (insets, lanes 2) or untransfected control (insets, lane 1). While cisplatin was toxicfor all of these prostate cells and siRNA for Purα tended to be protective, as was the case forthe brain tumors, the extent of protection was variable between lines. LNCaP and DU145 wererelatively resistant to cisplatin and were significantly sensitized to cisplatin by siRNA forPurα (Fig. 7F and G respectively). On the other hand, C4-2 and C4-2B were more sensitive tocisplatin and siRNA for Purα had only a slight protective effect (Fig. 7D and E respectively).

DiscussionPurα is a highly conserved protein that has multiple functions that are executed through itsability to bind to a variety of proteins and to nucleic acids, both DNA and RNA. With regardto tumori-genesis, many studies have indicated that Purα has the properties of a tumorsuppressor protein through its interactions with pRb, E2F1, etc. However, we have foundevidence that Purα may be involved in DNA repair11,12 and thus we reasoned that Purα mayplay a role in the cellular response to chemotherapeutic agents that act by damaging DNA, e.g.,cisplatin. Since we had created homozygous Purα-knockout mice,35 we were able to prepareMEFs from these mice and create a pair of stable cell lines, one of which was Purα-negativeand the other of which had Purα re-introduced by ectopic expression. This allowed us to directlyexamine the effect of Purα on cisplatin sensitivity. Our data, generated from these cells, clearlyshow that lack of Purα is associated with an increased sensitivity to cisplatin. Thus Purα-negative cells lose viability more rapidly than Purα-positive cells at a fixed concentration ofcisplatin and Purα-negative cells show a lower cisplatin concentration-dependence for loss ofviability and clonogenicity than Purα-positive cells for a fixed time of treatment.

The data suggest that Purα increases the susceptibility to the incurrence of DNA damage aswell as slowing down subsequent DNA repair. Thus, the induction of H2AX phosphorylation,which is indicative of double-strand DNA breaks, is greater in the Purα-negative cells than thePurα-positive cells. Further, the data from the comet assay, which directly measures cellularDNA fragmentation, shows that hydrogen peroxide inflicts more DNA damage in the Purα-negative cells than in the Purα-positive cells when measured at time zero following treatment.This indicates that Purα has a protective effect against DNA damage and perhaps Purα boundto DNA in vivo shields the DNA from genotoxic chemicals. The data indicate that Purα alsohas a role in the subsequent repair of DNA damage. Thus when reporter plasmid, which hadbeen damaged in vitro by treatment with cisplatin, was introduced into cells by transfection,the DNA was repaired more rapidly by the Purα-positive cells compared to the Purα-negativecells.

What is the role of Purα in DNA repair? We previously reported that Purα has a role inregulating homologous recombination-directed (HRR) double-strand DNA repair through itseffects on expression of the HRR protein, Rad51.12 We have now found that Purα has a rolein non-homologous end-joining (NHEJ) repair. Thus nuclear extracts prepared from Purα-negative cells were markedly impaired in their ability to catamerate plasmid in an in vitro NHEJassay than nuclear extracts prepared from Purα-positive cells. Perhaps, Purα with its ability tobind both DNA and protein is involved in the recruitment of DNA repair proteins to lesions inDNA.



NIH


NIH


NIH


Since Purα is protective against cisplatin induction of DNA damage and promotes DNA repair,we reasoned that selective knockdown of Purα by an RNA interference approach in humantumor cells might sensitize them to the chemotherapeutic effects of cisplatin. As was discussedin detail in the Introduction, cisplatin-resistance in human tumors can occur by manymechanisms that may be both genetic and epigenetic. One such mechanism is enhanced DNArepair mechanisms. We examined three human brain tumor cell lines and four prostatecarcinoma cell lines. Indeed, we found that an siRNA directed against Purα couldchemosensitize the tumor cells to cisplatin but that this effect was variable between the celllines. Interestingly, the degree of sensitivity to cisplatin was inversely correlated to thechemoprotection afforded by Purα siRNA, i.e., cell lines that were more sensitive to cisplatin,e.g., Daoy, C4-2, did not respond much to Purα siRNA, whereas the cell lines that were moreresistant to cisplatin showed significant chemosensitization after Purα siRNA treatment, e.g.,T98G, DU145. These data indicate that in this small set of tumor samples, knockdown ofPurα expression led to an increase in the responsiveness to cisplatin for those tumors that wererelatively cisplatin-resistant. Thus it is possible that downregulation of Purα in cisplatin-resistant tumors may provide a new therapeutic modality.

Material and MethodsCell cultures and transfection

To provide a well-controlled system to examine the effects of Purα, Purα+ and Purα- cell lineswere derived in parallel from the same primary cell cultures as follows. Primary mouse embryofibroblasts (MEFs) were initially derived from homozygous PURA-/- mouse embryos35prepared from 13-day pregnant mice using standard techniques. PURA-/-(+Purα) MEFs andPURA-/-(-Purα) MEFs, which were used in these experiments and are abbreviated to Purα+ andPurα- respectively, were established by stably transfecting PURA-/- MEFs with pTRE-Purαcontaining full length Purα cDNA, which is constitutively expressed, or pcDNA3 empty vectorrespectively.12 MEFs were cultured in DMEM medium supplemented with 10% FBS andantibiotics. Cells were maintained in a humid incubator at 37°C with 7% CO2. U87 MG, LN229and T98G human glioblastoma, HeLa human cervical carcinoma, LNCaP, C4-2 and C4-2Bhuman prostate adenocarcinoma and Daoy human medulloblastoma cell lines were obtainedfrom the ATCC. Cells were transfected with plasmids using Lipofectamine 2000 transfectionreagent (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. Cells weretransfected with siRNA using Oligofectamine (InVitrogen) according to the manufacturer'sprotocol.

Treatment of cells with cisplatinCells were treated with cisplatin (Bedford Laboratories, Bedford, OH, as a 1 mg/ml aqueousstock solution) as follows. To generate a dose-response curve, Purα+ and Purα- cells were platedand treated with different concentrations of cisplatin (0, 0.5, 1 and 2 μM) for 3 hr at 37°C.Viability was measured after 5 days. To investigate the time course of the effect of cisplatin,Purα+ and Purα- cells were plated in 100 mm dishes in equal numbers in triplicate in two sets.One set of each cell line was treated with 0.5 μM cisplatin in growth medium for 3 hours,washed with saline and cell viability evaluated. The percentage of cells in the treated set relativeto the untreated set was determined for each cell type at 1, 2, 3, 4 and 5 days following treatment.

Cell viability assayTrypan blue exclusion assay was performed to assess cell viability. This viability assaymeasures the percentage of a cell suspension that is able to exclude Trypan blue dye. In brief,cell suspension was diluted 1:1 with 0.4% Trypan blue and cells were counted with ahemocytometer.



NIH


NIH


NIH


Clonogenic assayPurα+ and Purα- cells were plated and treated with or without 0.5, 1 and 2 μg/ml cisplatin asdescribed above. Cells were grown for 10-14 days, fixed and stained with methylene blue.Colonies with more than 50 cells were counted and the percentage of colonies formed in treatedplates relative to the untreated plates was calculated for each cell type.

Flow cytometric analysis for γH2AXCells were harvested, washed with PBS and fixed in suspension in 1% methanol-freeformaldehyde in PBS on ice for 20 min. Cells were then resuspended in 73% ethanol for 16-20h at -20°C, washed with PBS and gently resuspended in 0.2% Triton X-100 in PBS/1% BSAfor 30 min. Following low speed centrifugation, cells were incubated with anti-phospho-H2AX(Ser139)-FITC-conjugated antibody (mouse monoclonal, Upstate Biotechnologies,#16-202A) in 1% BSA in PBS overnight at 4°C. This antibody is also known as anti-γH2AXand only recognizes H2AX that is phosphorylated on the serine at position 139. Cells werethen washed with PBS and stained with propidium iodide in PBS/RNase A. Flow cytometrywas performed with a COULTER® EPICS® FACScan flow cytometer to determine the cellcycle distribution and measure the green fluorescence.

Reactivation of platinated plasmidThe ability of cells to repair DNA was determined by transfection with a reporter plasmid withDNA that had been damaged in vitro with cisplatin and measuring reconstitution of reporterfunction. The pCMV-luciferase reporter plasmid was incubated in TE pH 7.4 buffer with orwithout 5 μM cisplatin at 37°C for 3 h. The yield of this platination procedure is 1.5 +/- 1.4pg/μg of DNA which is equivalent to 1 adduct per 1 kb of platinated DNA.36 Purα+ andPurα- cells were transfected with platinated and unplatinated plasmids together with pRL-TKinternal control reporter plasmid, which expresses Renilla reniformis luciferase from theHerpes simplex virus thymidine kinase promoter and serves as a control for transfectionefficiency. Protein extracts were prepared at various time points after transfection and aluciferase assay was performed using the Promega Dual Luciferase Assay Kit (Promega,Madison, WI USA) according to the manufacturer's instructions. Quantitation was with aluminometer (Femptomaster FB12, Zylux Corporation). The ratio of Photinus pyralis fireflyluciferase activity measured for the platinated plasmid to that for the control plasmid wascalculated for each time point and normalized to Renilla luciferase activity. In one set ofexperiments, the transfection step also included pEBV-Purα, which expresses Purα with a T7epitope tag, or control vector pcDNA3.

Immunocytochemistry for γH2AXCells were plated for immunocytochemistry onto poly-D-lysine coated glass chamber slides,treated with and without cisplatin for 0.5, 2 and 5 h, and then were fixed for 15 minutes in 3%paraformaldehyde at room temperature. After a wash in phosphate buffered saline (PBS), cellswere permeabilized for 15 min at room temperature in PBS containing 0.3% Triton X-100.Slides were washed three times in PBS for 5 min and subsequently blocked with PBS containing10% normal goat serum for 30 min. Subsequently, 2 μg/ml anti-γH2AX FITC-conjugatedmonoclonal antibody (mouse monoclonal, Upstate Biotechnologies, #16-202A) was appliedto the slides for 60 min at room temperature. After extensive washing, slides were mountedwith coverslips using antifading solution containing DAPI. Slides were washed 3X with PBS,mounted and examined by fluorescence microscopy.

Comet assayComet nuclei formation was analyzed by alkaline comet assay as we have previously described.37 Two sets of serum-starved (72 hr) Purα+ and Purα- cells were treated with or without



NIH


NIH


NIH


cisplatin (100 μM for 3 hr at 37°C) and/or H2O2 (20 μM for 10 min at 37°C) and one set wasanalyzed by comet assay. The second set was incubated in complete medium for 20 h and thenanalyzed by comet assay. Single cell gel electrophoresis was performed at the third dayfollowing induction. The comet assay was performed under alkaline conditions.38 About105 cells in 250 μl of PBS were mixed with 750 μl of 1.33% low-melting-point agarose, typeVII in PBS (Sigma A-4018). One hundred microliters of cell suspension were spread on afrosted microscope slide that had been precoated with 1% N-agarose, type I-A in H2O (SigmaA-0169). Slides were placed in cold lysis solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris[pH 10] and 1% Triton X-100, added freshly before use) for 1 h at 4°C. Slides were incubatedfor 40 min in alkaline unwinding buffer (300 mM NaOH and 1 mM EDTA, pH > 13) in thedark at 4°C. Electrophoresis was conducted for 30 min at 25 V (0.72 V/cm) and 300 mA. Slideswere washed with distilled H2O 3 × 5 min, air dried and stained for analysis with propidiumiodide (PI) (2.5 μg/ml) in sodium citrate pH 8.2 and covered with cover slips. Images of atleast 100 cells per sample (50 cells/slide) were evaluated using a fluorescence microscope andComet 5.0 image analysis software (Kinetic Imaging, Liverpool, UK). Necrotic and apoptoticcells, identified by their microscopic appearance (comets with no heads and nearly all DNAin the tail), were excluded from the analysis.39 The mean of the olive tail moment (OTM) (theproduct of the tail length and the fraction of the total DNA in the tail) was calculated as ameasure of DNA damage.40

Nonhomologous end joining (NHEJ) assay for double strand DNA break repairThe NHEJ assay was performed by a modification of the method of Baumann and West41 aswe have previously described.42,43 Cells were untreated or treated with 1 μg/ml (3.3 μM)cisplatin for 20 hours. Nuclear extracts were prepared as follows. Cells were harvested, washedthree times with ice-cold PBS and resuspended in ice-cold hypotonic lysis buffer (10 mMHEPES pH 7.9, 1.5 mM MgCl2, 10 mM KCl and protease inhibitors). After 10 minutes on ice,lysates were centrifuged for 5 min and the cell pellets resuspended in lysis buffer and placedon ice for 10 min. After centrifuging, cell pellets were resuspended in ice-cold hypertonicnuclear lysis buffer (20 mM HEPES pH 7.9, 1.5 mM MgCl2, 500 mM NaCl, 25% glycerol(vol/vol), 0.2 mM EDTA and protease inhibitors). After 1 hr on ice and 3 rapid cycles offreezing-thawing (liquid nitrogen and 37°C water bath), the nuclear lysates were cleared bycentrifugation. Supernatants were dialyzed for 10 hr against buffer containing 25 mM Tris HCl(pH 7.5), l mM EDTA, 10% glycerol (vol/vol) and protease inhibitors.

The NHEJ assays were set up as follows. DNA substrate for the reaction was made by cuttingthe vector plasmid pBlueScriptIISK (Stratagene, La Jolla, CA) with the restriction enzymesBamHI and EcoRV. The resulting 3 Kb linear DNA fragment was purified on an agarose gel.The end-joining reactions were carried out in 25 mM Tris OAc (pH 7.5), 100 mM KOAc, 10mM MgOAc, 1 mM DTT, 2 mM ATP and 200 mM dNTPs. Nuclear extracts (50 μg) wereincubated for 5 min at 37°C before the addition of the DNA substrate (400 ng). After incubationfor 1 hr at 37°C, DNA products were deproteinized by treatment with 100 μg/ml proteinase Kand analyzed by electrophoresis through a 0.7% agarose gel.

Treatment of human tumor cell lines with siRNA targeting PurαA variety of human tumor cell lines (listed in the cell culture section) were treated with eitherPurα siRNA or non-targeting siRNA using Oligofectamine according to the manufacturer'sinstructions (Invitrogen). Smartpool Purα siRNA and non-targeting siRNA were obtained fromDharmacon (Lafayette, CO) and were used at a final concentration of 50 nM. The viability andclonogenicity of cultures were then measured as described above. Western blot analysis ofprotein extracts from untransfected cells or cells transfected with Purα-specific siRNA usinganti-Purα was used to ascertain Purα knock-down with anti-α-tubulin antibody used as aloading control.



NIH


NIH


NIH


AntibodiesThe following antibodies were used: FITC-conjugated anti-phospho-histone H2AX (Ser139)(Upstate Biotechnology, Lake Placid, NY), anti-phospho-histone H2AX (Ser139) (UpstateBiotechnology). Rabbit polyclonal anti-Purα was used as previously described (Kaminski etal., 2008) except for Figure 1 where mouse monoclonal antibody (clone 10B12) kindlyprovided by Dr. Ed Johnson (Eastern Virginia Medical School, Norfolk, VA).

AcknowledgementsWe thank past and present members of the Center for Neurovirology for their insightful discussion and sharing ofideas and reagents. We also wish to thank C. Schriver for editorial assistance. This work was supported by grantsawarded by the NIH to M.K.W. and K.K.

References1. Kelland L. The resurgence of platinum-based cancer chemotherapy. Nat Rev Cancer 2007;7:573–84.

[PubMed: 17625587]2. Siddik ZH. Cisplatin: Mode of cytotoxic action and molecular basis of resistance. Oncogene

2003;22:7265–79. [PubMed: 14576837]3. Wang D, Lippard SJ. Cellular processing of platinum anticancer drugs. Nat Rev Drug Discov

2005;4:307–20. [PubMed: 15789122]4. Helleday T, Petermann E, Lundin C, Hodgson B, Sharma RA. DNA repair pathways as targets for

cancer therapy. Nat Rev Cancer 2008;8:193–204. [PubMed: 18256616]5. Stewart DJ. Mechanisms of resistance to cisplatin and carboplatin. Crit Rev Oncol Hematol

2007;63:12–31. [PubMed: 17336087]6. Köberle B, Grimaldi KA, Sunters A, Hartley JA, Kelland LR, Masters JR. DNA repair capacity and

cisplatin sensitivity of human testis tumour cells. Int J Cancer 1997;70:551–5. [PubMed: 9052754]7. Rosell R, Taron M, Barnadas A, Scagliotti G, Sarries C, Roig B. Nucleotide excision repair pathways

involved in Cisplatin resistance in non-small-cell lung cancer. Cancer 2003;10:297–305.8. Bergoglio V, Canitrot Y, Hogarth L, Minto L, Howell SB, Cazaux C, Hoffmann JS. Enhanced

expression and activity of DNA polymerase beta in human ovarian tumor cells: Impact on sensitivitytowards antitumor agents. Oncogene 2001;20:6181–7. [PubMed: 11593426]

9. Xu ZY, Loignon M, Han FY, Panasci L, Aloyz R. Xrcc3 induces cisplatin resistance by stimulationof Rad51-related recombinational repair, S-phase checkpoint activation and reduced apoptosis. JPharmacol Exp Ther 2005;314:495–505. [PubMed: 15843498]

10. Ali-Osman F, Berger MS, Rajagopal S, Spence A, Livingston RB. Topoisomerase II inhibition andaltered kinetics of formation and repair of nitrosourea and cisplatin-induced DNA interstrand cross-links and cytotoxicity in human glioblastoma cells. Cancer Res 1993;53:5663–8. [PubMed: 8242621]

11. Wang H, Wang M, Reiss K, Darbinian-Sarkissian N, Johnson EM, Iliakis G, Amini S, Khalili K,Rappaport J. Evidence for the involvement of Puralpha in response to DNA replication stress. CancerBiol Ther 2007;6:596–602. [PubMed: 17374989]

12. Wang H, White MK, Kaminski R, Darbinian N, Johnson EM, Amini S, Khalili K, Rappaport J. Roleof Purα in the modulation of homologous recombination-directed DNA repair by HIV-1 Tat.Anticancer Res 2008;28:1441–8. [PubMed: 18630497]

13. Haas S, Gordon J, Khalili K. A developmentally regulated DNA-binding protein from mouse brainstimulates myelin basic protein gene expression. Mol Cell Biol 1993;13:3103–12. [PubMed:7682655]

14. Haas S, Thatikunta P, Steplewski A, Johnson EM, Khalili K, Amini S. A 39-kD DNA-binding proteinfrom mouse brain stimulates transcription of myelin basic protein gene in oligodendrocytic cells. JCell Biol 1995;130:1171–9. [PubMed: 7657701]

15. Bergemann AD, Johnson EM. The HeLa Pur factor binds single-stranded DNA at a specific elementconserved in gene flanking regions and origins of DNA replication. Mol Cell Biol 1992;12:1257–65. [PubMed: 1545807]



NIH


NIH


NIH


16. Bergemann AD, Ma ZW, Johnson EM. Sequence of cDNA comprising the human pur gene andsequence-specific single-stranded DNA-binding properties of the encoded protein. Mol Cel Biol1992;12:5673–82.

17. Ma ZW, Bergemann AD, Johnson EM. Conservation in human and mouse Puralpha of a motifcommon to several proteins involved in initiation of DNA replication. Gene 1994;149:311–4.[PubMed: 7959008]

18. Liu H, Johnson EM. Distinct proteins encoded by alternative transcripts of the PURG gene, locatedcontrapodal to WRN on chromosome 8, determined by differential termination/polyadenylation.Nucleic Acids Res 2002;30:2417–26. [PubMed: 12034829]

19. Johnson EM. The Pur protein family: Clues to function from recent studies on cancer and AIDS.Anticancer Res 2003;23:2093–100. [PubMed: 12894583]

20. Gallia GL, Johnson EM, Khalili K. Puralpha: A multifunctional single-stranded DNA- and RNA-binding protein. Nucleic Acids Res 2000;28:3197–205. [PubMed: 10954586]

21. Kanai Y, Dohmae N, Hirokawa N. Kinesin transports RNA: Isolation and characterization of an RNA-transporting granule. Neuron 2004;43:513–25. [PubMed: 15312650]

22. Johnson EM, Kinoshita Y, Weinreb DB, Wortman MJ, Simon R, Khalili K, Winckler B, Gordon J.Role of Puralpha in targeting mRNA to sites of translation in hippocampal neuronal dendrites. JNeurosci Res 2006;83:929–43. [PubMed: 16511857]

23. Kaminski R, Darbinian N, Sawaya BE, Slonina D, Amini S, Johnson EM, Rappaport J, Khalili K,Darbinyan A. Puralpha as a cellular co-factor of Rev/RRE-mediated expression of HIV-1 intron-containing mRNA. J Cell Biochem 2008;103:1231–45. [PubMed: 17722108]

24. Johnson EM, Chen PL, Krachmarov CP, Barr SM, Kanovsky M, Ma ZW, Lee WH. Association ofhuman Puralpha with the retinoblastoma protein, Rb, regulates binding to the single-stranded DNAPur alpha recognition element. J Biol Chem 1995;270:24352–60. [PubMed: 7592647]

25. Darbinian N, Gallia GL, Kundu M, Shcherbik N, Tretiakova A, Giordano A, Khalili K. Associationof Puralpha and E2F-1 suppresses transcriptional activity of E2F-1. Oncogene 1999;18:6398–402.[PubMed: 10597240]

26. Darbinian N, White MK, Khalili K. Regulation of the Pur-alpha promoter by E2F-1. J Cell Biochem2006;99:1052–63. [PubMed: 16741925]

27. Tretiakova A, Steplewski A, Johnson EM, Khalili K, Amini S. Regulation of myelin basic proteingene transcription by Sp1 and Puralpha: Evidence for association of Sp1 and Puralpha in brain. JCell Physiol 1999;181:160–8. [PubMed: 10457364]

28. Safak M, Gallia GL, Khalili K. Reciprocal interaction between two cellular proteins, Puralpha andYB-1, modulates transcriptional activity of JCVCY in glial cells. Mol Cell Biol 1999;19:2712–23.[PubMed: 10082537]

29. Itoh H, Wortman MJ, Kanovsky M, Uson RR, Gordon RE, Alfano N, Johnson EM. Alterations inPur(alpha) levels and intracellular localization in the CV-1 cell cycle. Cell Growth Differ1998;9:651–65. [PubMed: 9716182]

30. Stacey DW, Hitomi M, Kanovsky M, Gan L, Johnson EM. Cell cycle arrest and morphologicalalterations following microinjection of NIH3T3 cells with Puralpha. Oncogene 1999;18:4254–61.[PubMed: 10435638]

31. Barr SM, Johnson EM. Ras-induced colony formation and anchorage-independent growth inhibitedby elevated expression of Puralpha in NIH3T3 cells. J Cell Biochem 2001;81:621–38. [PubMed:11329617]

32. Darbinian N, Gallia GL, King J, Del Valle L, Johnson EM, Khalili K. Growth inhibition ofglioblastoma cells by human Pur(alpha). J Cell Physiol 2001;189:334–40. [PubMed: 11748591]

33. Bruchova H, Borovanova T, Klamova H, Brdicka R. Gene expression profiling in chronic myeloidleukemia patients treated with hydroxyurea. Leuk Lymphoma 2002;43:1289–95. [PubMed:12152998]

34. Lezon-Geyda K, Najfeld V, Johnson EM. Deletions of PURA, at 5q31 and PURB, at 7p13, inmyelodysplastic syndrome and progression to acute myelogenous leukemia. Leukemia 2001;15:954–62. [PubMed: 11417483]

35. Khalili K, Del Valle L, Muralidharan V, Gault WJ, Darbinian N, Otte J, Meier E, Johnson EM, DanielDC, Kinoshita Y, Amini S, Gordon J. Puralpha is essential for postnatal brain development and



NIH


NIH


NIH


developmentally coupled cellular proliferation as revealed by genetic inactivation in the mouse. MolCell Biol 2003;23:6857–75. [PubMed: 12972605]

36. Cenni B, Kim HK, Bubley GJ, Aebi S, Fink D, Teicher BA, Howell SB, Christen RD. Loss of DNAmismatch repair facilitates reactivation of a reporter plasmid damaged by cisplatin. Br J Cancer1999;80:699–704. [PubMed: 10360646]

37. Merabova N, Kaniowska D, Kaminski R, Deshmane SL, White MK, Amini S, Darbinyan A, KhaliliK. JC virus agnoprotein inhibits in vitro differentiation of oligodendrocytes and promotes apoptosis.J Virol 2008;82:1558–69. [PubMed: 17989177]

38. Singh NP, McCoy MT, Tice RR, Schneider EL. A simple technique for quantitation of low levels ofDNA damage in individual cells. Exp Cell Res 1988;175:184–91. [PubMed: 3345800]

39. Speit GH, Hartmann A. The comet assay (single-cell gel test). A sensitive genotoxicity test for thedetection of DNA damage and repair. Methods Mol Biol 1999;113:203–12. [PubMed: 10443422]

40. Olive PL, Banath JP, Durand RE. Heterogeneity in radiation-induced DNA damage and repair intumor and normal cells measured using the “comet” assay. Radiat Res 1990;122:86–94. [PubMed:2320728]

41. Baumann P, West SC. DNA end-joining catalyzed by human cell-free extracts. Proc Natl Acad SciUSA 1998;95:14066–70. [PubMed: 9826654]

42. Darbinyan A, Siddiqui KM, Slonina D, Darbinian N, Amini S, White MK, Khalili K. Role of JC virusagnoprotein in DNA repair. J Virol 2004;78:8593–600. [PubMed: 15280468]

43. Darbinyan A, White MK, Akan S, Radhakrishnan S, Del Valle L, Amini S, Khalili K. Alterations ofDNA damage repair pathways resulting from JCV infection. Virology 2007;364:73–86. [PubMed:17368705]

44. Darbinian N, White MK, Gallia GL, Amini S, Rappaport J, Khalili K. Interaction between the Puraand E2F-1 transcription factors. Anticancer Res 2004;24:2585–94. [PubMed: 15517862]



NIH


NIH


NIH


Figure 1.Cell viability and clonogenicity of Purα-positive and -negative cells in response to cisplatintreatment. (A) Purα positive and negative MEFs were analyzed by Western blot to confirmtheir Purα status. (B) The dose-dependence of the effect of cisplatin (3 h) on the viability ofthe Purα positive and negative MEFs was measured by trypan blue exclusion assay. (C) Thetime-dependence of the effect of cisplatin (0.5 μM) on the viability of the Purα positive andnegative MEFs was measured by trypan blue exclusion assay. (D) The dose-dependence of theeffect of cisplatin (3 h) on the clonogenicity of the Purα positive and negative MEFs wasmeasured.



NIH


NIH


NIH


Figure 2.Effect of cisplatin treatment on cell cycle parameters and γH2AX expression in synchronizedPurα-positive and -negative cells measured by FACS analysis and immunocytochemistry.Untreated Purα positive and negative MEFs were synchronized by culture in serum-freemedium, treated with and without cisplatin, transferred to complete medium and analyzed byFACS for cell cycle phase and H2AX expression. (A) Untreated cells in G0/G1. (B) Untreatedcells in S phase. (C) Untreated cells in G2/M. (D) Expression of γH2AX by untreated cells.(E) Cisplatin-treated cells in G0/G1. (F) Cisplatin-treated cells in S phase. (G) Cisplatin-treatedcells in G2/M. (H) Expression of γH2AX by cisplatin-treated cells. (I) In a separate experiment,untreated Purα positive and negative MEFs were arrested in G1/G0 by serum starvation,



NIH


NIH


NIH


released from starvation for 18 h and were treated with and without cisplatin for 1 h. At 0.5 hafter treatment, cells were labeled with antibody to γH2AX and examined by fluorescencemicroscopy as described in Materials and Methods.



NIH


NIH


NIH


Figure 3.Reactivation of transfected platinated reporter plasmid in Purα-positive and -negative cells.Luciferase reporter plasmid was treated in vitro with and without cisplatin as described inMaterials and Methods and then introduced into Purα-positive and -negative cells bytransfection. (A) Time course of restoration of luciferase activity. (B) In this experiment,Purα-positive and -negative cells were seeded into 6 well plates (1 × 105 cells/well) transfectedwith platinated and control plasmids (30 ng/well) in combination with an expression plasmidfor T7-tagged Purα (pEBV-Purα). For each transfection, the total amount of DNA used wasequalized to 1 μg/well by the addition of empty vector plasmid (pcDNA3.1). Again, the time



NIH


NIH


NIH


course of restoration of luciferase activity was measured. (C) The time course for the expressionof Purα in the transfected cultures from (B).



NIH


NIH


NIH


Figure 4.Comet nuclei formation in Purα-positive and -negative cells. Serum-starved Purα+ and Purα-

cells were treated with or without cisplatin and/or H2O2 and analyzed by alkaline comet assayas described in Materials and Methods. The results are representative of three separateexperiments.



NIH


NIH


NIH


Figure 5.Nonhomologous end-joining (NHEJ) activity in nuclear extracts prepared from Purα-positiveand -negative cells. (A) Purα+ and Purα- cells were treated with or without cisplatin and nuclearextracts were prepared and used in NHEJ reactions in vitro. The arrowhead indicates the linearmonomer plasmid DNA substrate. The bracket indicates the multimeric products of NHEJ. (B)The expression and lack of expression of Purα was confirmed by Western blot for the Purα+

and Purα- cells respectively. PCNA was used a control for protein loading of the nuclearextracts on the gel.



NIH


NIH


NIH


Figure 6.Effect of siRNA-Purα on the cell viability and clonogenicity of U87MG and HeLa cells inresponse to cisplatin treatment. U87MG human glioblastoma cells and HeLa cervicalcarcinoma cells were transfected with siRNA to Purα (siRNA-Purα) or a control non-targetingsiRNA (NT-siRNA) and treated with or without cisplatin as described in Materials andMethods. (A) Knockdown of the Purα protein expression level in U87MG cells by siRNA-Purα was demonstrated by Western blot. (B) Cell viability of U87MG cells at different cisplatinconcentrations as measured by trypan blue exclusion. (C) Clonogenicity of U87MG cells atdifferent cisplatin concentrations. (D) Knockdown of the Purα protein expression level in HeLacells by siRNA-Purα was demonstrated by Western blot. (E) Cell viability of HeLa cells atdifferent cisplatin concentrations as measured by trypan blue exclusion. (F) Clonogenicity ofHeLa cells at different cisplatin concentrations.



NIH


NIH


NIH


Figure 7.(See previous page). Effect of siRNA-Purα on the cell viability of different human brain tumorand prostate tumor cell types in response to cisplatin treatment. (A) T98G and (B) LN229human glioblastoma cells, (C) Daoy human medulloblastoma cells, (D) C4-2, (E) C4-2B, (F)LNCaP and (G) DU145 human prostate cancer cells were untransfected (-) or transfected withsiRNA to Purα (siRNA-Purα) or a control non-targeting siRNA (NT-siRNA) and treated withor without cisplatin as described in Materials and Methods. Knockdown of the Purα proteinexpression level in each cell line by siRNA-Purα was demonstrated by Western blot and isshown inset in each Panel. For each cell type, the total amount of cell protein remaining in the



NIH


NIH


NIH


washed cell monolayer after five days treatment with different cisplatin concentrations wasmeasured by Bradford assay and compared to untreated cells (100%).



NIH


NIH


NIH


Date post:	22-Nov-2023
Category:	Documents
Upload:	independent
View:	0 times
Download:	0 times

Protective role of Puralpha to cisplatin

Documents