Viiruse ja peremeesraku interaktsioonid inimese papilloomiviiruse elutsüklis

Väitekirja elektrooniline versioon ei sisalda publikatsioone.Papilloomiviirused on väga laialdase levikuga ning pea kõik inimesed nakatuvad mingil eluetapil selle viirusega. Üldiselt kulgeb HPV infektsioon üsna kergelt, põhjustades nahal või limaskestadel healoomulisi vohandeid, soolatüükaid (β-HPV) või kondüloome (α-HPV). Kliiniliselt oluliseks peetakse aga papilloomiviirusi seetõttu, et selle viiruse infektsiooniga on seotud enamik emakakaela vähi juhtumitest. Arvatakse, et emakakaela vähi tekke üks olulisemaid etappe on raku nakatumine kõrgesse riskigruppi kuuluva papilloomiviiruse subtüübiga. Kui soolatüükaid ja kondüloome põhjustavad madala riskiga inimese papilloomiviiruste subtüüpide infektsioon aja möödudes kaob, siis emakakaela vähki põhjustavate viiruste infektsioon muutub kergesti pikaajaliseks. Sellisesse faasi jõudnud nakkuse ravivõimalused on aga väga piiratud, mistõttu on HPV infektsiooni varajane avastamine eduka ravi seisukohalt ülioluline. Papilloomiviiruse infektsioon kulgeb tavaliselt läbi ekstrakromosomaalse rõngasmolekuli ning viiruse elutsükli edukaks lõpetamiseks ei ole integreerumine peremeesraku pärilikku materjali (DNA) tingimata vajalik. Vähirakkude geneetilisel uurimisel on aga muuhulgas HPV nakkuse tuvastamisele avastatud ka asjaolu, et väga paljudel juhtudel on papilloomiviiruse genoomi osad integreerunud peremeesraku kromosoomidesse, muutes HPV DNA üheks osaks peremeesraku pärilikust materjalist. HPV genoomi osaline integratsioon rikub ära aga mitmete viiruse jaoks oluliste valkude ekspressiooni (s.h. DNA replikatsiooniks ja virioni moodustamiseks vajalike valkude sünteesi). Seega peetakse integreerumist viiruse seisukohalt tupikteeks. Peremeesraku seisukohalt aga peetakse pahaloomuliseks kasvajaks arenenud rakkude DNAs tuvastatud suuri ja tagasipöördumatuid muutusi just viiruse integratsiooni tagajärjeks. Viiruse integratsioon võib toimuda mistahes kromosoomi piirkonda, ka nendesse piirkondadesse, mis on peremeesraku kasvu ja jagunemise kontrolli seisukohalt väga olulised. Kuna nakatunud rakk sisaldab keskmiselt 100 ekstrakromosomaalset viiruse genoomi koopiat ning massilist genoomide integratsiooni tavaliselt ei toimu, tekib olukord, kus ühes rakus eksisteerib kaks erinevat viiruse DNA vormi: üks, puudulike geenidega integreerunud vorm ja teine, täiesti funktsionaalne ekstrakromosomaalne vorm. Kuigi integreerunud viiruse DNAlt ei saa enam ekspresseerida DNA paljundamiseks vajalikke replikatsioonivalke, siis ekstrakromosomaalselt molekulilt replikatsioonivalkude sünteesiks mingeid takistusi ei ole. Selline olukord on aga peremeesraku genoomse stabiilsuse seisukohalt väga ohtlik, kuna integreerunud HPV järjestused sisaldavad viiruse DNA paljundamiseks vajalikku alguskohta (replikatsiooni origin-i), mis ei ole rakuliste regulatsiooni faktoritega kontrollitav. Sellelt järjestuselt alustatud DNA replikatsioon, põhjustab nii integreerunud HPV DNA kui ka sellega külgnevate peremeesraku genoomi osade kontrollimatut paljundamist (amplifikatsiooni). Olenevalt integratsiooni kohast, võivad integreerunud HPV järjestusega külgnevad alad sisaldada onkogeene või muid raku kasvu ja jagunemisega seotud regulatoorseid geene, mille amplifikatsioon võib põhjustada raku genoomset ebastabiilsust ning kontrollimatut kasvu ja jagunemist. Lisaks võib põhjustada HPV-dele omane litsentseerimata DNA re-replikatsioon replikatsiooni komplekside takerdumisi, mis viib ebatüüpiliste DNA replikatsiooni produktide kuhjumiseni raku tuumas. Rakulise DNA reparatsiooni radade võtmemolekulide lokalisatsioon HPV DNA replikatsiooni tsentritesse viitab rakuliste mehhanismide olulisele rollile tekkinud kaheahelaliste DNA katkete parandamisel. Kui enamik kaheahelalisi DNA katkeid parandatakse õigesti, siis antud väitekirjas kirjeldatud viiruse integratsiooni koha translokatsioon uude kromosoomi piirkonda viitab ebaõnnestunud katsele parandada DNA amplifikatsiooni tagajärjel tekkinud DNA produkte. Seega, integreerunud HPV järjestuste amplifikatsioon ja peremeesraku vastusena aktiveeritud DNA reparatsiooni mehhanismid võivad viia peremeesraku genoomse ebastabiilsuseni ja vähkkasvaja tekkeni. Inimese papilloomiviiruste uurimine nende looduslikes peremeesrakkudes on üsna keerukas just keratinotsüütide keeruka elutsükli tõttu, mis on aluseks nahkkoe kihistunud struktuuri moodustumisel. Kui diferentseerumata basaalsed keratinotsüüdid on pidevas jagunemises ja vastutavad basaalrakkude populatsiooni uuenemise eest, siis diferentsieerumisradadele suunatud rakud läbivad kontrollitud rakusurmaga lõppeva elutsükli. Mitmes eri diferentseerumisastmes olevate keratinotsüütide populatsioon tekitab nahkkoe kihistunud struktuuri, mille peamiseks ülesandeks on tagada tugev (ja läbimatu) barjäär sise- ja väliskeskkonna vahel. Kuna viirus nakatab diferentseerumata basaalseid keratinotsüüte ja pakib oma viiruspartiklid alles terminaalselt diferentseerunud rakkudes, on papilloomiviirused kohanenud kõikide peremeesraku eluetappidega. Lühidalt, papilloomiviirus nakatab basaalseid keratinotsüüte mikrovigastuste kaudu ning peale viiruse geneetilise materjali jõudmist raku tuuma, toimub esmalt aktiivne viirusgenoomi paljundamine. Peale optimaalse genoomi koopiaarvu saavutamist, lülitub viirus ümber stabiilsele genoomi säilimisele ning jääb ootama peremeesraku diferentseerumist. Sel ajal toimub HPV- ja peremeesraku geneetilise materjali sünkroonne paljundamine ning viiruse genoomide peaaegu ühtlane jaotumine tütarrakkude vahel. Terminaalselt diferentseerunud rakkudes toimub taaskord aktiivne genoomi paljundamine (amplifikatsioon) ning viimase etapina viiruspartiklite moodustumine. Laboritingimustes on keratinotsüütide kasvatamine ja elutsükli mimikeerimine tehniliselt keerukas, ajakulukas ja küllaltki kallis. Lisaks ei ole otstarbekas niigi kapriisset rakukultuuri kasutada laiamahuliseks kemikaalide skriinimiseks, et leida HPV elutsüklit pärssivaid komponente. Meie laboris välja töötatud sääreluu kasvaja rakuliiinil U2OS põhinev HPV uurimismudel osutus edukaks mitmete HPV subtüüpide (β-HPV5, β-HPV8, α-HPV6b, α-HPV11, α-HPV16, α-HPV18) transientse, stabiilse kui ka amplifikatsioonilise DNA replikatsiooni jälgimisel. Kuna U2OS rakud ei ole looduslikud papilloomiviiruse peremeesrakud, pidasime vajalikuks kaardistada viiruse geenide avaldumist, et tõsta U2OS rakusüsteemi usaldusväärsust HPV uuringutes. Antud töös kirjeldati HPV11 geenide ekspressiooni, kuid sarnane analüüs on läbi viidud ka HPV5 ning HPV18 subtüübiga. Kõiki saadud tulemusi on võrreldud viiruse loomulikest peremeesrakkudest, keratinotsüütidest, saadud tulemustega ning nende sarnasuse tõttu oleme arvamusel, et U2OS süsteem on sobiv keskkond papilloomiviiruste uurimiseks ning oma lihtsuse ja odavuse poolest ideaalne ka esialgsete ravimi kandidaatide skriinimiseks. Lisaks tuvastati U2OS süsteemis 1n HPV molekulide sünteesi kõrval ka suuremaid, 2n ja rohkem, järjestikku paigutunud HPV genoomi koopiatega rõngasmolekule (oligomeerid). Sarnaseid vaheprodukte on täheldatud nii varasemates kui ka antud töö raames kogutud HPV-positiivsetest kliinilistest koeproovidest. HPV-spetsiifiliste oligomeeride tekke täpsemal uurimisel tekkis hüpotees, et nende moodustumisel võib olla oma roll rakulise rekombinatsioonilise DNA replikatsiooni laadil. Oligomeersed HPV molekulid võivad osutuda kasulikeks, et tagada kiire HPV genoomi koopia arvu tõus vegetatiivses viiruse elutsükli faasis kui ka viiruse genoomi jagunemisel kahe tütarraku vahel. Kuna viiruspartiklisse pakitakse vaid üks HPV genoom, pole täpselt teada, milliseid mehhanisme kasutatakse oligomeersete DNA vormide konverteerimisel tagasi 1n HPV genoomideks.Human papillomaviruses (HPVs) are widely distributed and infect almost all people at some point. Generally, the viral infection does not cause significant illness but may generate benign warts on skin (β-HPVs) and condylomas on genitals (α-HPVs). The clinical importance of papillomaviruses is associated with the fact that HPV genomes (or parts of them) are found in almost all cervical cancer cases. It is believed that infection with the papillomaviruses that belong to the high-risk group of α-HPVs (e.g. HPV16, HPV18) is one of the most important steps in the development of cervical cancer. When infection with low-risk α-HPVs (e.g. HPV6, HPV11) is eventually eliminated by the immune system (usually taking up to 2 years), then infection with high-risk HPVs becomes more easily persistent. The limited treatment options to cure persistent HPV infection make the early detection of HPV infection highly important. Successful progression through the HPV life cycle is mediated through extrachromosomal molecules, and integration of the viral genome into host cell chromosomes is not essential. However, in cervical cancer cells, parts of HPV genomes are often found integrated into the host genetic material. This partial integration of HPV genomes disrupts the expression of many viral genes (including genes that encode proteins for DNA replication and capsid formation), and therefore, integration into host chromosomes is considered a dead end for the virus. On the other hand, as the integration site is not determined, the uncontrollable growth of cancer cells may result from viral integration into chromosome regions responsible for the regulation of cell growth. Additionally, the large-scale genetic changes found in cervical cancer cells are probably triggered by the viral integration event. Moreover, viral DNA integration encompasses only a few viral genomes, while the majority remains extrachromosomal molecules (cells carry approximately 100 copies of HPV genomes). The co-occurrence of two forms of viral DNA in one cell is a real threat to genomic stability, as HPVs integrate into the host genetic material in a way that the viral DNA replication origin that cannot be controlled by cellular checkpoint pathways is intact. The integration event usually disrupts the genes that encode the replication proteins; however, the expression of replication proteins from extrachromosomal molecules is still possible. Multiple replication initiations from the integrated DNA replication origin by episome-derived viral replication proteins lead to the amplification of not only the integrated viral DNA but also the flanking cellular sequences. Depending on the site of the integration, the flanking cellular sequences may contain oncogenes or other regulatory elements which amplification may lead again to uncontrollable cell growth or to genomic instability. In addition, the unlicensed DNA re-replication characteristic of HPVs often cause the collision of replication forks and the accumulation of aberrant DNA replication products. The co-localization of cellular DNA damage response (DDR) pathway key molecules to HPV DNA replication centers indicate to the generation of double-stranded DNA breaks by unlicensed DNA re-replication. It seems that the activation of cellular DDR pathways has an important role in repairing double-stranded DNA breaks that occur during viral DNA replication. While most of the double-stranded breaks are repaired properly, the cross-chromosomal translocations of viral integration locus detected within this thesis may result from failed attempt to repair the DNA damage. The generation of multiple modifications in host cell genomic material may eventually lead to the genomic instability and to the formation of malignancy. Keratinocytes, the native host cells for papillomavirus, are responsible for the continuous renewal of epithelial tissue and therefore have adapted a very complex life cycle. While undifferentiated basal keratinocytes are responsible for renewal of the basal cell compartment, they also undergo terminal differentiation and form the stratified structure of the epithelia. As papillomaviruses infect the basal layer of undifferentiated keratinocytes and pack their viral particles at terminally differentiated cell residues, the viral life cycle has adjusted to all stages of the host cell life cycle. The cultivation of keratinocytes in cell culture and mimicking their differentiation program is technically demanding, time-consuming and cost-intensive. In addition, keratinocytes are not suitable for the high-throughput screening of chemical compounds to find drugs that could reduce papillomavirus viability. The need for a simpler system to study the various aspects of the HPV life cycle has long inspired our research group to find a cell line that is easy to culture and where the replication of HPV genomes could be monitored. The development of a U2OS cell-based system provided a suitable environment for HPV, as the transient, stable, and amplificational genome replication of various types of HPV (β-HPV5, β-HPV8, α-HPV6b, α-HPV11, α-HPV16, and α-HPV18) was detected. Interestingly, while HPVs function through unit-sized (1n) extrachromosomal molecules and ultimately only one genome is packed into viral particles, analysis of replication intermediates detected larger than unit-sized HPV-specific molecules in addition to 1n molecules. Subsequent analysis of replication intermediates confirmed that the larger molecules were head-to-tail concatemers (further referred to as oligomers). Similar DNA replication products have also been described in previously analyzed clinical samples and were also detected in HPV16 and HPV18-positive patient probes collected within this thesis. Further analysis of the formation of oligomeric forms of HPV molecules suggested that recombination-dependent DNA replication is involved in this process. As the human osteosarcoma cell line U2OS is not the native host for papillomavirus, the suitability of this cell line in the study of papillomaviruses was further confirmed by the complete mapping of HPV11 transcripts. Similar analysis has also been carried out with HPV5 and HPV18 by other members in our research group. The similarity of gene expression of all three HPV types with previously reported results in native host cells or in clinical samples confirmed that U2OS cells are suitable for research of human papillomaviruses as well as for preliminary studies of anti-HPV drugs

Mechanism of Genomic Instability in Cells Infected with the High-Risk Human Papillomaviruses

In HPV–related cancers, the “high-risk” human papillomaviruses (HPVs) are frequently found integrated into the cellular genome. The integrated subgenomic HPV fragments express viral oncoproteins and carry an origin of DNA replication that is capable of initiating bidirectional DNA re-replication in the presence of HPV replication proteins E1 and E2, which ultimately leads to rearrangements within the locus of the integrated viral DNA. The current study indicates that the E1- and E2-dependent DNA replication from the integrated HPV origin follows the “onion skin”–type replication mode and generates a heterogeneous population of replication intermediates. These include linear, branched, open circular, and supercoiled plasmids, as identified by two-dimensional neutral-neutral gel-electrophoresis. We used immunofluorescence analysis to show that the DNA repair/recombination centers are assembled at the sites of the integrated HPV replication. These centers recruit viral and cellular replication proteins, the MRE complex, Ku70/80, ATM, Chk2, and, to some extent, ATRIP and Chk1 (S317). In addition, the synthesis of histone γH2AX, which is a hallmark of DNA double strand breaks, is induced, and Chk2 is activated by phosphorylation in the HPV–replicating cells. These changes suggest that the integrated HPV replication intermediates are processed by the activated cellular DNA repair/recombination machinery, which results in cross-chromosomal translocations as detected by metaphase FISH. We also confirmed that the replicating HPV episomes that expressed the physiological levels of viral replication proteins could induce genomic instability in the cells with integrated HPV. We conclude that the HPV replication origin within the host chromosome is one of the key factors that triggers the development of HPV–associated cancers. It could be used as a starting point for the “onion skin”–type of DNA replication whenever the HPV plasmid exists in the same cell, which endangers the host genomic integrity during the initial integration and after the de novo infection

DNA replication initiated from the integrated HPV origin generates various low-molecular-weight DNA products.

<p>High-molecular-weight (HMW) DNA and low-molecular-weight (LMW) DNA were fractionated and purified by Hirt lysis 24 hrs post-transfection from HeLa cells (A) and from SiHa cells (B) that were both transfected as follows: mock-transfection (lanes 1 and 5); 5 µg of HPV18 E1 expression plasmid (lanes 2 and 6); 2 µg of E2 expression plasmid (lanes 3 and 7); 5 µg of HPV18 E1 and 2 µg of E2 expression plasmids (lanes 4 and 8). A 3 µg portion of HMW DNA and three times the respective amount of LMW DNA were digested with HindIII (A) or Acc65I/BshTI (B) and separated on a one-dimensional gel. The integrated HPV URR-specific signals were detected by Southern blot analysis. (C) Schematic presentation of the migration of dsDNA linear, supercoiled, and open circle molecules on 2D neutral-neutral gels. (D, E) 5 µg of HPV18 E1 and 2 µg of E2 expression plasmids were transfected into HeLa cells and LMW DNA was purified by Hirt lysis 48 hrs post-transfection. Extracted DNA was digested with DpnI and fractionated by the CsCl-ethidium bromide density gradient. The fraction of linear fragments (lin) and open circular molecules (oc) (D) and the fraction of supercoiled circular plasmids (sc) (E) were separated on a 2D gel, transferred to a nylon filter, and probed with the HPV18 genomic fragment (from nt 3917 to1575). Numbers shown on the axes represent the markers of linear (lin) and supercoiled circular (sc) DNA forms. Black arrowhead indicates the shift that was caused by mtDNA. (F) Schematic presentation of HPV16 integration locus within chromosome 13 in SiHa cells, where the cleavage sites of Acc65I, Eco91I, BshTI, and BcuI as well as their distances from HPV origin are presented. (G) Schematic presentation of the migration of replication forks and replication puffs on 2D neutral-neutral gels. (H,I) SiHa cells were co-transfected with 5 µg of HPV18 E1 and 2 µg of E2 expression plasmids. LMW DNA was extracted 24 hrs post-transfection and digested with Acc65I-BshTI (H) and Eco91I-BcuI (I). Respective HPV16 genome fragments were used as probes on 2D Southern blots. (J) SiHa cells were co-transfected with 1 µg of HPV18 E1 and 2 µg of E2 expression plasmids. Extracted LMW DNA was digested with Eco91I-BcuI and analyzed by 2D Southern blots.</p

Replication centers of the integrated HPV recruit Mre11-Nbs1-Rad50 complex and Ku70/80 heterodimer.

<p>HeLa cells were transfected and analyzed as described previously. Co-immunostaining of HPV18 E1 (Alexa Fluor 568, second column) and the following proteins are presented: Mre11, Nbs1, Rad50, and Ku70/80 proteins (Alexa Fluor 488, first column). The localizations of the E1 and the respective DNA repair proteins are also shown in the third column as a merged image and DAPI stained nuclei are presented in the fourth column.</p

Re-replication of the integrated HPV induces the instability of chromosome structure.

<p>SiHa cells were transfected with 10 µg of HPV18 E1 and 5 µg of E2 expression plasmids. Single cell subcloning was performed 72 h after transfection <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000397#ppat.1000397-Kadaja2" target="_blank">[30]</a>. SiHa cells and the subclones with novel restriction patterns were analyzed by FISH. (A) FISH analysis of the interphase nuclei (left panel) and metaphase chromosome spreads (the middle and the right panels) of SiHa cells. (B) Interphase FISH (left panel) and metaphase FISH (the middle and the right panels) analyses of the subclone with cross-chromosomal translocation. The integrated HPV16 is visible as green (Alexa Fluor 488), and the subtelomeric region of the chromosome 13 is visible as red (Texas Red). The translocation of 13q is indicated with the white arrowhead.</p

The HPV genome induces genomic instability in cells harboring the integrated HPV.

<p>Southern blot analyses of the HeLa (A, B) and SiHa cells (C, D) co-transfected with 1 µg of HPV16 genome and 1 µg of linearized pEGFPN-1 (A, C) or with 1 µg of HPV18 genome and 1 µg of linearized pEGFPN-1 (B, D). Low-molecular-weight DNA was extracted 24 h and 48 h after transfection and digested with restriction enzymes, as indicated in the figure. HPV plasmids were detected with a ³²P-labeled HPV16 or HPV18 genome probe, respectively. (E, F) The restriction analyses of untreated SiHa cells (E, lanes 1–3), HPV16-transfected SiHa cells five weeks post-transfection (E, lanes 4–6), HPV18-transfected SiHa cells five weeks post-transfection (E, lanes 13–15; F, lanes 1–3), as well as the subclones of the HPV16-transfected SiHa cells (E, lanes 7–12) and HPV18-transfected SiHa cells (E, lanes 16–18; F, lanes 4–6). Digested total DNA was analyzed by Southern blotting, where the HPV16 (E) or HPV18 (F) genomes were used as probes. Restriction enzymes are indicated in the figure.</p

DNA replication of the integrated HPV takes place at specific nuclear foci.

<p>HeLa and SiHa cells were co-transfected with 5 µg of HPV18 E1 and 2 µg of E2 expression plasmids. Cells were analyzed 20 hrs post-transfection. (A) Co-immunostaining of E1 (Alexa Fluor 568, first column) and BrdU (FITC, second column). Cells were pulse-labeled with BrdU for 2 hrs prior to IF analysis. In the third column, the localizations of E1 and BrdU in the same cells are presented as a merged image. (B) Combined immunofluorescence and FISH analysis to detect the integrated HPV DNA (Alexa Fluor 488, first column) and the HPV E1 protein (Alexa Fluor 568, second column) in SiHa and HeLa cells. Localizations of the E1 and the integrated HPV DNA are also presented in the third column as a merged image. DNA was counterstained with DAPI (fourth column).</p

Mechanism of the genomic instability of the cells harboring the replication origin of integrated HPV.

<p>If HPV plasmid is present in the cells harboring integrated HPV, the DNA re-replication from the integrated HPV origin is initiated, and ATM and ATR signaling pathways respond, respectively, to the produced DSBs and ssDNA. In most cases, the cells will either become apoptotic or the damage sites will be properly repaired by homologous recombination and non-homologous end-joining. In addition, duplications within the HPV locus have previously been detected. In the current study, the excision and generation of extrachromosomal copies of the HPV locus and cross-chromosomal translocations were also detected. All these mutations require DSBs that could be generated, such as through head-to-tail fork collision or by encounter of the re-replication fork with the Okazaki fragments of the previous fork. Supported by our observations, we speculate that the cellular defense against the integrated HPV re-replication is primary coordinated by ATM. ATR pathways do not interfere with a properly working E1-driven replication fork but, rather, are responsible for the repair of the damage that is caused by fork collision and dissociation that reveals the RPA coated ssDNA without the generation of DSBs. DNA replication/repair factors that are shown to be within the HPV replication centers in the current work, are colored in the figure.</p

Activation of the ATM-Chk2 signaling pathway.

<p>(A–C) HeLa cells were transfected as follows: 5 µg of HPV18 E1 and 2 µg of HPV18 E2 expression plasmids (lane 1); 5 µg of HPV18 E1 expression plasmid (lane 2); 2 µg of E2 expression plasmid (lane 3); and a mock-transfection (lane 4). In every transfection, the amount of plasmid was adjusted to 10 µg with a carrier plasmid (pauxoMCF). Non-transfected HeLa cells are presented in lane 5 and HeLa cells that were treated 1 h with etoposide (50 µM) prior to the analysis in lane 6. Western blot analyses were performed at a 24 hrs time point to detect HPV18 E1 (A, panel a), HPV18 E2 (A, panel b), gamma histone H2AX (phosphorylated at S139) (A, panel c), and β-actin (A, panel d). Western blot analyses of Chk2 phosphorylated at Thr68 and Ser19 were performed after the immunoprecipitation with the anti-Chk2 antibody (B, panels a, b, c). Chk1 phosphorylated at Ser317 was detected from extracts that were immunoprecipitated with the anti-Chk1 antibody (C, panels a, b). (D) HeLa cells were transfected either with 2 µg of circular HPV18 genome, 2 µg of pBabePuro and 6 µg of carrier plasmid (lane 1), or with 2 µg of pBabePuro and 8 µg of carrier plasmid (lane 2). Untransfected cells were removed with puromycin treatment (2 µg/ml) 24–48 h posttransfection. Western blot analyses with anti-Chk2 (panel a) and anti-Chk2-Ser19 (panel b) antibodies were performed after the immunoprecipitation with the anti-Chk2 antibody at a 72 h time point. Untreated HeLa cells are shown in lane 3 and etoposide-treated HeLa cells in lane 4.</p