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• W2169182876 abstract "Hepatitis B virus X protein (pX) is implicated in hepatocellular carcinoma pathogenesis by an unknown mechanism. Employing the tetracycline-regulated pX-expressing 4pX-1 cell line, derived from the murine AML12 hepatocyte cell line, we demonstrate that pX induces partial polyploidy (>4N DNA). Depletion of p53 in 4pX-1 cells increases by 5-fold the polyploid cells in response to pX expression, indicating that p53 antagonizes pX-induced polyploidy. Dual-parameter flow cytometric analyses show pX-dependent bromodeoxyuridine (BrdUrd) incorporation in 4pX-1 cells containing 4N and >4N DNA, suggesting pX induces DNA re-replication. Interestingly, pX increases expression of endogenous replication initiation factors Cdc6 and Cdtl while suppressing geminin expression, a negative regulator of rereplication. In comparison to a geminin knockdown 4pX-1 cell line used as DNA re-replication control, the Cdt1/geminin ratio is greater in 4pX-1 cells expressing pX, indicating that pX promotes DNA re-replication. In support of this conclusion, pX-expressing 4pX-1 cells, similar to the geminin knockdown 4pX-1 cells, continue to incorporate BrdUrd in the G2 phase and exhibit nuclear Cdc6 and MCM5 co-localization and the absence of geminin. In addition, pX expression activates the ATR kinase, the sensor of DNA re-replication, which in turn phosphorylates RAD17 and H2AX. Interestingly, phospho-H2AX-positive and BrdUrd -positive cells progress through mitosis, demonstrating a link between pX-induced DNA re-replication and polyploidy. Our studies high-light a novel function of pX that likely contributes to hepatocellular carcinoma pathogenesis. Hepatitis B virus X protein (pX) is implicated in hepatocellular carcinoma pathogenesis by an unknown mechanism. Employing the tetracycline-regulated pX-expressing 4pX-1 cell line, derived from the murine AML12 hepatocyte cell line, we demonstrate that pX induces partial polyploidy (>4N DNA). Depletion of p53 in 4pX-1 cells increases by 5-fold the polyploid cells in response to pX expression, indicating that p53 antagonizes pX-induced polyploidy. Dual-parameter flow cytometric analyses show pX-dependent bromodeoxyuridine (BrdUrd) incorporation in 4pX-1 cells containing 4N and >4N DNA, suggesting pX induces DNA re-replication. Interestingly, pX increases expression of endogenous replication initiation factors Cdc6 and Cdtl while suppressing geminin expression, a negative regulator of rereplication. In comparison to a geminin knockdown 4pX-1 cell line used as DNA re-replication control, the Cdt1/geminin ratio is greater in 4pX-1 cells expressing pX, indicating that pX promotes DNA re-replication. In support of this conclusion, pX-expressing 4pX-1 cells, similar to the geminin knockdown 4pX-1 cells, continue to incorporate BrdUrd in the G2 phase and exhibit nuclear Cdc6 and MCM5 co-localization and the absence of geminin. In addition, pX expression activates the ATR kinase, the sensor of DNA re-replication, which in turn phosphorylates RAD17 and H2AX. Interestingly, phospho-H2AX-positive and BrdUrd -positive cells progress through mitosis, demonstrating a link between pX-induced DNA re-replication and polyploidy. Our studies high-light a novel function of pX that likely contributes to hepatocellular carcinoma pathogenesis. Chronic hepatitis B virus (HBV) 4The abbreviations used are: HBV, hepatitis B virus; HCC, hepatocellular carcinoma; pre-RC, pre-replicative complex; pX, X protein; MAPK, mitogen-activated protein kinase; ATM, ataxia telangiectasia mutated; ATR, ATM- and Rad3-related; PBS, phosphate-buffered saline; WCE, whole cell extract; FCS, fetal calf serum; BrdUrd, bromodeoxyuridine; MCM, minichromosome maintenance; H2AX, histone H2A family, member X. 4The abbreviations used are: HBV, hepatitis B virus; HCC, hepatocellular carcinoma; pre-RC, pre-replicative complex; pX, X protein; MAPK, mitogen-activated protein kinase; ATM, ataxia telangiectasia mutated; ATR, ATM- and Rad3-related; PBS, phosphate-buffered saline; WCE, whole cell extract; FCS, fetal calf serum; BrdUrd, bromodeoxyuridine; MCM, minichromosome maintenance; H2AX, histone H2A family, member X. infection results in the development of hepatocellular carcinoma (HCC) by the fourth or fifth decade (1Beasley R.P. Hwang L.Y. Lin C.C. Chien C.S. Lancet. 1981; 2: 1129-1133Abstract PubMed Scopus (401) Google Scholar) by an unknown mechanism. In HBV-mediated HCC the rate of chromosomal aberrations is significantly increased in comparison to HCC associated with other risk factors (2Cougot D. Neuveut C. Buendia M.A. J Clin Virol. 2005; 34: S75-S78Crossref PubMed Scopus (160) Google Scholar, 3Marchio A. Meddeb M. Pineau P. Danglot G. Tiollais P. Bernheim A. Dejean A. Genes Chromosomes Cancer. 1997; 18: 59-65Crossref PubMed Scopus (260) Google Scholar, 4Zhang S.-H. Cong W.-M. Xian Z.H. Dong H. Wu M.-C. J. Cancer Res. Clin. Oncol. 2004; 130: 757-761Crossref PubMed Scopus (19) Google Scholar). However, the mechanism by which genomic changes initiate HCC development is not yet understood (5Kawai H. Suda T. Aoyagi Y. Isokawa O. Mita Y. Waguri N. Kuroiwa T. Igarashi M. Tsukada K. Mori S. Shimizu T. Suzuki Y. Abe Y. Takahashi T. Nomoto M. Asakura H. Hepatology. 2000; 31: 1246-1250Crossref PubMed Scopus (60) Google Scholar, 6Thorgeirsson S.S. Grisham J.W. Nat. Genet. 2002; 31: 339-346Crossref PubMed Scopus (1256) Google Scholar, 7Wilkens L. Flemming P. Gebel M. Bleck J. Terkamp C. Wingen L. Kreipe H. Schlegelberger B. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 1309-1314Crossref PubMed Scopus (80) Google Scholar). Herein, employing the HBV X protein (pX) as the oncogenic signal, we investigate whether pX expression induces chromosomal abnormalities, resulting in HCC pathogenesis. The link between HBV-mediated HCC and pX is derived both from clinical evidence (8Su Q. Schroder C.H. Hofmann W.J. Otto G. Pichlmayr R. Bannasch P. Hepatology. 1998; 27: 1109-1120Crossref PubMed Scopus (191) Google Scholar) as well as animal and cell culture transformation studies (9Andrisani O.M. Barnabas S. Int. J. Oncol. 1999; 15: 373-379PubMed Google Scholar). Specifically, integration of HBV DNA into the host genome occurs at early steps of clonal tumor expansion, with most tumors displaying sustained expression of pX (8Su Q. Schroder C.H. Hofmann W.J. Otto G. Pichlmayr R. Bannasch P. Hepatology. 1998; 27: 1109-1120Crossref PubMed Scopus (191) Google Scholar). Importantly, pX, which is essential for the viral life cycle (10Zoulim F. Saputelli J. Seeger C. J. Virol. 1994; 68: 2026-2030Crossref PubMed Google Scholar), is a multifunctional protein inducing activation of the cellular mitogenic ras-raf-MAPK, c-Jun NH2-terminal kinase, and p38MAPK pathways (11Bouchard M.J. Schneider R.J. J. Virol. 2004; 78: 12725-12734Crossref PubMed Scopus (402) Google Scholar) and transcription of select viral and cellular genes (9Andrisani O.M. Barnabas S. Int. J. Oncol. 1999; 15: 373-379PubMed Google Scholar). These pX activities deregulate cellular gene expression, resulting either in unscheduled cell cycle progression (12Lee S. Tarn C. Wang W.H. Chen S. Hullinger R.L. Andrisani O. J. Biol. Chem. 2002; 277: 8730-8740Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar) or apoptosis (13Wang W.H. Gregori G. Hullinger R.L. Andrisani O.M. Mol. Cell. Biol. 2004; 24: 10352-10365Crossref PubMed Scopus (81) Google Scholar), depending on the growth conditions. Specifically, pX expression sensitizes the less-differentiated 4pX-1 hepatocyte cell line (14Tarn C. Bilodeau M.L. Hullinger R.L. Andrisani O.M. J. Biol. Chem. 1999; 274: 2327-2336Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar) to p53-mediated apoptosis only when X-expressing cells are challenged with additional pro-apoptotic stimuli (13Wang W.H. Gregori G. Hullinger R.L. Andrisani O.M. Mol. Cell. Biol. 2004; 24: 10352-10365Crossref PubMed Scopus (81) Google Scholar, 15Wang W.H. Hullinger R.L. Andrisani O.M. J. Biol. Chem. 2008; 283: 25455-25467Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). By contrast, in optimal growth factor conditions, pX induces unscheduled cell cycle progression, a transient S phase pause, activation of the G2/M checkpoint, and eventual progression through the cell cycle (12Lee S. Tarn C. Wang W.H. Chen S. Hullinger R.L. Andrisani O. J. Biol. Chem. 2002; 277: 8730-8740Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Studies by others have demonstrated that overexpression of cyclin E, Cdc25A, and E2F1 leads to unscheduled entry into the S-phase and activation of the ATM/ATR kinases (16Bartkova J. Horejsi Z. Koed K. Kramer A. Tort F. Zieger K. Guldberg P. Sehested M. Nesland J.M. Lukas C. Orntoft T. Lukas J. Barted J. Nature. 2005; 434: 864-870Crossref PubMed Scopus (2197) Google Scholar). Likewise, overexpression of the cellular oncogene c-myc (17Pusapati R.V. Rounbehler R.J. Hong S. Powers J.T. Yan M. Kiguchi K. McArthur M.J. Wong P.K. Johnson D.G. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 1446-1451Crossref PubMed Scopus (121) Google Scholar) or expression of pX in apoptotic conditions activate ATM or ATR, respectively, leading to p53 activation and p53-mediated apoptosis (15Wang W.H. Hullinger R.L. Andrisani O.M. J. Biol. Chem. 2008; 283: 25455-25467Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Interestingly, pre-neoplastic human specimen exhibit markers of DNA damage and apoptosis, including phosphorylated ATM, Chk2, H2AX, and p53 (16Bartkova J. Horejsi Z. Koed K. Kramer A. Tort F. Zieger K. Guldberg P. Sehested M. Nesland J.M. Lukas C. Orntoft T. Lukas J. Barted J. Nature. 2005; 434: 864-870Crossref PubMed Scopus (2197) Google Scholar, 18Gorgoulis V.G. Vassiliou L.-V. F. Karakaidos P. Zacharatos P. Kotsinas A. Liloglou T. Venere M. Ditullio Jr., R.A. Kastrinakis N.G. Levy B. Kletsas D. Yoneta A. Herlyn M. Kittas C. Halazonetis T.D. Nature. 2005; 34: 907-913Crossref Scopus (1673) Google Scholar). These results suggest that unscheduled S-phase entry induces via ATM/ATR the activation of p53, which acts as a barrier to cancer development. In agreement with these observations, our hypothesis is that in response to pX expression, the hepatocyte undergoes unscheduled S-phase entry, leading to replication stress and DNA damage, activation of ATR, and induction of p53-mediated apoptosis, thereby constraining oncogenesis. Conversely, deregulation of the G2/DNA damage checkpoint, inhibition of DNA repair, or inactivation of p53 would rescue infected hepatocytes from X-induced apoptosis, allowing the generation of genomic aberrations and progression to malignancy. In all cell types DNA replication and segregation of the chromosomes is a highly regulated process, ensuring that both daughter cells inherit a complete and intact complement of the genome (19Blow J.J. Dutta A. Nat. Rev. Mol. Cell Biol. 2005; 6: 476-486Crossref PubMed Scopus (521) Google Scholar, 20Diffley J.F.X. Curr. Biol. 2004; 14: 778-786Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar). The process ensuring that DNA replication occurs only once per cell cycle is called replication licensing. Deregulation of replication licensing results in re-replication or partial replication of the genome, leading to chromosomal abnormalities characteristic of cancer (21Mihaylov I.S. Kondo T. Jones L. Ryzhikov S. Tanaka J. Zheng J. Higa L.A. Minamino N. Cooley L. Zhang H. Mol. Cell. Biol. 2002; 22: 1868-1880Crossref PubMed Scopus (167) Google Scholar, 22Nishitani H. Lygerou Z. Front. Biosci. 2004; 9: 2115-2132Crossref PubMed Scopus (48) Google Scholar). Replication licensing involves assembly of the pre-replicative complex (pre-RC) at the origin of replication, comprised of the origin recognition complex, Cdc6, Cdt1, and MCM2-7 proteins (19Blow J.J. Dutta A. Nat. Rev. Mol. Cell Biol. 2005; 6: 476-486Crossref PubMed Scopus (521) Google Scholar, 23Prasanth S.G. Mendez J. Prasanth K.V. Stillman B. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 2004; 359: 7-16Crossref PubMed Scopus (69) Google Scholar). The binding of MCM2-7 proteins to pre-RC completes the process of replication licensing, limiting DNA replication to once per cell cycle. The functional licensing of the origin, i.e. the initiation of replication, occurs once cyclin-dependent kinases become active at the onset of S phase (22Nishitani H. Lygerou Z. Front. Biosci. 2004; 9: 2115-2132Crossref PubMed Scopus (48) Google Scholar). In metazoans, regulation of replication licensing or pre-RC assembly is mediated by down-regulating Cdt1 activity, required for the recruitment of the MCM2-7 proteins to the replication origin. Cdt1 is expressed in early G1 and is degraded at the late G1 and early S phase (24Nishitani H. Sugimoto N. Roukos V. Nakanishi Y. Saijo M. Obuse C. Tsurimoto T. Nakayama K.I. Nakayama K. Fujita M. Lygerou Z. Nishimoto T. EMBO J. 2006; 25: 1126-1136Crossref PubMed Scopus (308) Google Scholar, 25Nishitani H. Lygerou Z. Nishimoto T. J. Biol. Chem. 2004; 279: 30807-30816Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). In addition, Cdt1 interacts directly with geminin, the main inhibitor of replication licensing in S and G2 phases (26Hodgson B. Li A. Tada S. Blow J. Curr. Biol. 2002; 12: 678-683Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 27Tada S. Li A. Maiorano D. Mechali M. Blow J.J. Nat. Cell Biol. 2001; 3: 107-113Crossref PubMed Scopus (389) Google Scholar, 28Wohlschlegel J.A. Dwyer B.T. Dhar S.K. Cvetic C. Walter J.C. Dutta A. Science. 2000; 290: 2271-2273Crossref PubMed Scopus (573) Google Scholar), resulting in dissociation of the MCM2-7 complex from chromatin (29Lee C. Hong B. Choi J.M. Kim Y. Watanabe S. Ishimi Y. Enomoto T. Tada S. Kim Y. Cho Y. Nature. 2004; 430: 913-917Crossref PubMed Scopus (117) Google Scholar). Geminin is absent in G1, accumulating during the S and G2/M phases. Geminin is degraded at the end of mitosis, consistent with being a substrate of the anaphase-promoting complex (30McGarry T.J. Kirschner M.W. Cell. 1998; 93: 1043-1053Abstract Full Text Full Text PDF PubMed Scopus (719) Google Scholar), thus permitting the onset of a new round of replication (22Nishitani H. Lygerou Z. Front. Biosci. 2004; 9: 2115-2132Crossref PubMed Scopus (48) Google Scholar). Cyclin-dependent kinases also regulate replication licensing, demonstrated by the induction of re-replication after elimination of the mitotic Cdc2 kinase (27Tada S. Li A. Maiorano D. Mechali M. Blow J.J. Nat. Cell Biol. 2001; 3: 107-113Crossref PubMed Scopus (389) Google Scholar, 31Ballabeni A. Melixetian M. Zamponi R. Masiero L. Marinoni F. Helin K. EMBO J. 2004; 23: 3122-3132Crossref PubMed Scopus (114) Google Scholar, 32Fujita M. Yamada C. Tsurumi T. Hanaoka F. Matsuzawa K. Inagaki M. J. Biol. Chem. 1998; 273: 17092-17101Google Scholar, 33Itzhaki J.E. Gilbert C.S. Porter A.C. Nat. Genet. 1997; 15: 258-265Crossref PubMed Scopus (138) Google Scholar). Cyclin-dependent kinase inactivation promotes re-accumulation of Cdt1 on chromatin (34Sugimoto N. Tatsumi Y. Tsurumi T. Matsukage A. Kiyono T. Nishitani H. Mujita M. J. Biol. Chem. 2004; 279: 19691-19697Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). Moreover, overexpression of both Cdt1 and Cdc6 in p53-negative cells induces re-replication and polyploidy (35Vaziri D. Saxena S. Geon Y. Lee C. Murata K. Machida Y. Wagle N. Hwang D.S. Dutta A. Mol. Cell. 2003; 11: 997-1008Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar, 36Melixetian M. Ballabeni A. Masiero L. Gasparini P. Zamponi R. Bartek J. Lukas J. Helin K. J. Cell Biol. 2004; 165: 473-482Crossref PubMed Scopus (213) Google Scholar). Likewise, inhibition of geminin expression induces re-replication (35Vaziri D. Saxena S. Geon Y. Lee C. Murata K. Machida Y. Wagle N. Hwang D.S. Dutta A. Mol. Cell. 2003; 11: 997-1008Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar, 36Melixetian M. Ballabeni A. Masiero L. Gasparini P. Zamponi R. Bartek J. Lukas J. Helin K. J. Cell Biol. 2004; 165: 473-482Crossref PubMed Scopus (213) Google Scholar, 37Zhu W. Chen Y. Dutta A. Mol. Cell. Biol. 2004; 24: 7140-7150Crossref PubMed Scopus (203) Google Scholar), supporting that failure to control pre-RC formation results in re-replication, leading to chromosomal abnormalities and cancer development (21Mihaylov I.S. Kondo T. Jones L. Ryzhikov S. Tanaka J. Zheng J. Higa L.A. Minamino N. Cooley L. Zhang H. Mol. Cell. Biol. 2002; 22: 1868-1880Crossref PubMed Scopus (167) Google Scholar, 22Nishitani H. Lygerou Z. Front. Biosci. 2004; 9: 2115-2132Crossref PubMed Scopus (48) Google Scholar). Herein, employing the less-differentiated 4pX-1 hepatocyte cell line, a tetracycline regulated pX-expressing cell line (14Tarn C. Bilodeau M.L. Hullinger R.L. Andrisani O.M. J. Biol. Chem. 1999; 274: 2327-2336Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar), we demonstrate that pX expression induces DNA re-replication, DNA damage, and polyploidy, identifying a likely mechanism for the genomic instability characteristic of HBV-mediated HCC (2Cougot D. Neuveut C. Buendia M.A. J Clin Virol. 2005; 34: S75-S78Crossref PubMed Scopus (160) Google Scholar, 3Marchio A. Meddeb M. Pineau P. Danglot G. Tiollais P. Bernheim A. Dejean A. Genes Chromosomes Cancer. 1997; 18: 59-65Crossref PubMed Scopus (260) Google Scholar, 4Zhang S.-H. Cong W.-M. Xian Z.H. Dong H. Wu M.-C. J. Cancer Res. Clin. Oncol. 2004; 130: 757-761Crossref PubMed Scopus (19) Google Scholar). Cell Culture—Cell lines 4pX-1 (14Tarn C. Bilodeau M.L. Hullinger R.L. Andrisani O.M. J. Biol. Chem. 1999; 274: 2327-2336Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar) and 4pX-1-p53kd (15Wang W.H. Hullinger R.L. Andrisani O.M. J. Biol. Chem. 2008; 283: 25455-25467Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar) were used. pX expression was initiated by removal of tetracycline (14Tarn C. Bilodeau M.L. Hullinger R.L. Andrisani O.M. J. Biol. Chem. 1999; 274: 2327-2336Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). The following reagents were used: nocodazole (250 ng/ml), Sigma; SB 202190 (5 μm), Calbiochem; and BrdUrd (20 mm), Invitrogen. Construction of Clonal 4pX-1-gemininkd Cell Line—4pX-1 cells were transfected with shRNAmir for geminin in retroviral vector derived from pSM2C, purchased from Open Biosystems. Clonal stable cell lines were isolated by puromycin (1.0 μg/ml) selection and screened by Western blot assays employing geminin antibody (Santa Cruz Biotechnology, Inc.). Flow Cytometry and Live Cell Sorting—4pX-1 and 4pX-1-p53kd cell lines were growth factor-deprived for 18 h as described (14Tarn C. Bilodeau M.L. Hullinger R.L. Andrisani O.M. J. Biol. Chem. 1999; 274: 2327-2336Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar) and subsequently grown for 10 h with 10% fetal calf serum followed by removal of tetracycline for an additional 10 h; nocodazole (250 ng/ml) was added for the last 6 h. Cells harvested by trypsin were washed in PBS, fixed in 70% ethanol containing 0.1% Triton X-100 for 20 min, and incubated for 2 h in Vindelov reagent containing PBS, 3.5 units/ml RNase A, 75 mg/ml propidium iodide, and 0.1% Nonidet P-40. Cells were analyzed by Cytomics FC-500 (Beckman-Coulter) at a flow rate of <1000 cells/s using WinList 5.0 software (Verity Software House). Calibration (not shown) was performed using chick erythrocytes for each assay. For live cell sorting, 4pX-1 cells were grown with and without tetracycline as described above, harvested by trypsin treatment, resuspended in Dulbecco's modified Eagle's medium/F-12 medium, and stained with the Hoechst 33422 (500 ng/ml) 1 h before cell sorting. Cells were sorted for DNA content employing the Epics Altra cell sorter (Beckman-Coulter). For dual parameter flow cytometry, 4pX-1 cells grown as described above were incubated with 20 mm BrdUrd for 30 min before fixation in 70% ethanol, Triton X-100. Cells were washed 3 times with PBS and incubated in blocking buffer containing PBS and 10% goat serum (Sigma) for 30 min followed by a 2-h incubation with BrdUrd-fluorescein isothiocyanate-conjugated antibody (1:300) and propidium iodide (75 mg/ml). Cells were analyzed by Cytomics FC-500 (Beckman-Coulter) with compensation adjustments standardized for each experiment. Double Thymidine Block—4pX-1 and 4pX-1-gemininkd cell lines at 20% confluence were grown for 19 h in growth medium (14Tarn C. Bilodeau M.L. Hullinger R.L. Andrisani O.M. J. Biol. Chem. 1999; 274: 2327-2336Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar) containing 5 μg/ml tetracycline and 2 mm thymidine (Sigma) followed by an additional incubation for 9 h in the same growth medium without thymidine. The second thymidine block was performed for an additional 16 h of incubation ± 5 μg/ml tetracycline. After release from the double thymidine block, cells were grown for 0-14 h and processed for flow cytometry, cell sorting, immunofluorescence microscopy, or whole cell extract (WCE) preparation. Immunofluorescence Microscopy—Cells were fixed in 4% paraformaldehyde (Sigma) for 20 min, washed 3 times in PBS, and incubated for 30 min in blocking buffer containing 130 mm NaCl, 7 mm Na2HPO4, 3.5 mm NaH2PO4, 7.7 mm NaN3, 0.1% bovine serum albumin, 1.0% Triton-X-100, 0.05% Tween 20, and 10% goat serum. Incubation with primary antibody was for at least 2 h at room temperature at the following dilutions: 1:1000 for α-tubulin (Sigma), 1:1000 for Cdc6 (MBL International), 1:700 for MCM5 (Abcam), 1:700 for geminin (Santa Cruz), 1:500 for phosphohistone 3 (Upstate Biotechnology), and 1:1000 for γ-H2AX (Calbiochem). The BrdUrd kit was from Roche Applied Science. Incubation with secondary antibodies AlexaFluor488 (Invitrogen) and AlexaFluor568 (Invitrogen) was for 1 h. Cells were visualized by Nikon TE300 at 60× or by Nikon E800 confocal microscope. Real-time PCR Analysis—Total RNA was isolated by the Trizol method and processed for real-time PCR quantification as described (12Lee S. Tarn C. Wang W.H. Chen S. Hullinger R.L. Andrisani O. J. Biol. Chem. 2002; 277: 8730-8740Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). PCR primers are listed: Cdc6, forward, 5′-TCCCAGACACAAGCTACC-3′, reverse, 5′-ATTTTACGTCCACACACG-3′; Cdt1, forward, 5′-CCATGTGTCGAGAAAGCTCC-3′, reverse, 5′-CAATGGTGTCCATGC TGC-3′; geminin, forward, 5′-CCATCGGAAGAGGAAGACAC-3′, reverse, 5′-AAGTGGCTGAGCACGTACA; MCM4, forward, 5′-TCAAGTCAGACCTTTTAATGCG-3′, reverse, 5′-TCTGATGACCATGCCACTG-3′; MCM5, forward, 5′-ACATGCAGCTTTATTGTGACAG-3′, reverse, 5′-TTCAAGCCAAACTTCTTGATGG-3′; MCM6, forward, 5-′ AATGATGAAGTAAAACGCGGTG-3′, reverse, 5-TGCAAACATTTATATCCCCACG-3′; proliferating cell nuclear antigen, forward, 5′-GTATTCGAAGCACCAAATCAAG-3′, reverse, 5′-AGCTGTACTCCTGTTCTGG-3′; thymidylate synthase, forward, 5′-GGCCCAGTTTATGGTTTCC-3′, reverse, 5′-GTTGTTTTGATGGTGTCAATC-3′. Comet Assay—4pX-1 cultures were trypsinized for obtaining single-cell suspension and embedded in 1% agarose on a slide covered by a coverslip and chilled at 4 °C. Slides were placed in lysis buffer (10 mm Tris-HCl, pH 10, 2.5 m NaCl, 0.1 m EDTA, 1% Triton-X) at 4 °C and electrophoresed for 40 min at 25 V. After electrophoresis, slides were incubated for 15 min in neutralization buffer (0.4 m Tris-HCl, pH 7.5), stained with SYBRGREEN (1:1000), and visualized by fluorescence microscopy. Cells were scored for DNA damage when the DNA tailing was discernable at 20×. Quantification is derived from three independent experiments. Western blot analyses employed WCE isolated from synchronized 4pX-1 cells, sorted by DNA content using the Epics Atra cell sorter (Beckman-Coulter) or in a time course after release from double thymidine block. WCEs (20 μg) were prepared in radioimmune precipitation assay buffer containing PBS, pH 7.4, 0.5% deoxycholate, 1% Nonidet P-40, 1 mm EDTA, 0.4 mm EGTA, 10% glycerol, 0.1% SDS, 1 mm phenylmethylsulfonyl fluoride, 1 μg/ml aprotinin, 1 μg/ml leupeptin, 1 μg/ml of pepstatin A, 1 mm sodium orthovanadate, and 1 mm sodium fluoride were analyzed by SDS-PAGE. Antibodies used: phospho-ATR (1:1000) and total ATR (1:1000) from Cell Signaling; p-Cdc2 (1:750) and Cdc2 (1:1000) from Santa Cruz Research Biotechnology; p53 (CM5) (1:1000) from Vector Laboratories; γ-H2AX (1:1000) and H2AX from Cell Signaling; Geminin (1:400) from Santa Cruz Research Biotechnology; phospho-Rad17 (1:700) and Rad17 (1:700) from Cell Signaling; Chk1 (1:1000) and phospho-Chk1 (1:600) from Cell signaling; Cdc6 (1:800) and Cdt1 (1:500) both from Santa Cruz Research Biotechnology. HBV pX Induces Polyploidy—Expression of pX in the immortalized mouse hepatocyte 4pX-1 cell line (14Tarn C. Bilodeau M.L. Hullinger R.L. Andrisani O.M. J. Biol. Chem. 1999; 274: 2327-2336Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar) induces accelerated cell cycle progression, unscheduled S phase entry followed by a transient S phase pause, activation of the G2/M checkpoint, and eventual progression through the cell cycle (12Lee S. Tarn C. Wang W.H. Chen S. Hullinger R.L. Andrisani O. J. Biol. Chem. 2002; 277: 8730-8740Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Similar to human T-cell lymphotrophic virus, type I Tax-expressing cells (38Jin D.Y. Spencer F. Jeang K.T. Cell. 1998; 93: 81-91Abstract Full Text Full Text PDF PubMed Scopus (459) Google Scholar), pX-expressing 4pX-1 cells become multiand micro-nucleated upon treatment for 24 h with a low concentration of nocodazole (250 ng/ml) (39Kurata S.-I. J. Biol. Chem. 2000; 275: 23413-23416Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). To understand how pX causes these chromosomal abnormalities, the tetracycline-regulated pX-expressing 4pX-1 cell line was synchronized by growth factor withdrawal as described (14Tarn C. Bilodeau M.L. Hullinger R.L. Andrisani O.M. J. Biol. Chem. 1999; 274: 2327-2336Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar); to re-enter the cell cycle, 4pX-1 cells were first treated for 10 h with 10% fetal calf serum followed by removal of tetracycline for 10 h to allow pX expression; nocodazole was added for the last 6 h of cell growth (Fig. 1A). Employing flow cytometry, we quantified that 20% of the cells were in the G2/M phase at 20 h after growth in 10% FCS in the presence of pX expression; nocodazole treatment for an additional 6 h increased the cell number in the G2/M phase to 45% (see the table in Fig. 1. Employing the growth protocol shown in the diagram of Fig. 1A, we quantified by flow cytometry the percent of cells progressing through the cell cycle (Fig. 1, table) and those displaying >4N DNA (Fig. 1A). pX expression increases by 2.5-fold the cell population containing >4N DNA, which we refer to as partially polyploid. Importantly, nocodazole treatment increased this pX-dependent polyploid cell population by 3-fold. Nocodazole acts as a mitotic stressor when used at low concentration by activating the p38MAPK pathway (39Kurata S.-I. J. Biol. Chem. 2000; 275: 23413-23416Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). Because pX also activates the p38MAPK pathway, which inhibits the Cdc25 phosphatase (40Tarn C. Zou L. Hullinger R.L. Andrisani O.M. J. Virol. 2002; 76: 9763-9772Crossref PubMed Scopus (47) Google Scholar) required for activation of the mitotic Cdc2 kinase (12Lee S. Tarn C. Wang W.H. Chen S. Hullinger R.L. Andrisani O. J. Biol. Chem. 2002; 277: 8730-8740Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar), we examined the effect of inhibition of the p38MAPK pathway on pX-induced polyploidy. Treatment of 4pX-1 cells with the p38MAPK inhibitor SB202190 (41Nemoto S. Xiang J. Huang S. Lin A. J. Biol. Chem. 1998; 273: 16415-16420Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar, 42Karahashi H. Nagata K. Ishii K. Amano F. Biochim. Biophys. Acta. 2000; 1502: 207-223Crossref PubMed Scopus (35) Google Scholar) resulted in only a 1.6-fold increase in pX-induced polyploidy in the presence of nocodazole (Fig. 1A), an increase that is statistically insignificant (p < 0.1). We interpret these results to mean that pX expression together with nocodazole treatment synergistically activate the p38MAPK pathway, enhancing the pX-induced polyploidy. In supplemental Fig. 1 we show that nocodazole (250 ng/ml) increases the inhibitory phosphorylation of Cdc2 on Tyr-15 in the presence of pX via a p38MAPK-dependent mechanism (supplemental Fig. 1a) without disrupting microtubule polymerization and mitotic spindle assembly (supplemental Fig. 1b). To further confirm the existence of the pX-induced polyploid cell population, we employed fluorescence-activated cell sorting and isolated these pX-induced polyploid cells (Fig. 1B). The increased nuclear size of the sorted cells containing >4N DNA further demonstrates that these pX-expressing cells have aberrant DNA content (Fig. 1C). The integrity of the genome is maintained by activation of p53, which induces cell cycle arrest or apoptosis (43Fei P. El-Deiry W.S. Oncogene. 2003; 2: 5774-5783Crossref Scopus (408) Google Scholar). Because our results indicated that pX induces polyploidy, we examined the level of p53 in 4pX-1 cells. A pX-dependent induction in the p53 protein level is detected in 4pX-1 cells with or without nocodazole treatment, suggesting that this p53 increase is the cellular response to pX-induced polyploidy (Fig. 1D). p53 Antagonizes pX-mediated Polyploidy—To determine whether p53 negatively regulates pX-mediated polyploidy, we employed the 4pX-1-p53kd cell line displaying more than 80% depletion of endogenous p53 (Ref. 15Wang W.H. Hullinger R.L. Andrisani O.M. J. Biol. Chem. 2008; 283: 25455-25467Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar and Fig. 2A) and quantified the polyploid cell population as a function of pX expression (Fig. 2" @default.
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• W2169182876 title "Hepatitis B Virus X Protein Increases the Cdt1-to-Geminin Ratio Inducing DNA Re-replication and Polyploidy" @default.
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