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• W2058264480 abstract "Various reports have implicated the virally encoded HBx protein as a cofactor in hepatocarcinogenesis. However, direct evidence of the role of HBx as a promoter of oncogenesis in response to an initiating factor such as DNA damage remains inadequate. Here, we report the effects of HBx in HepG2 cells exposed to UV light-induced DNA damage. HBx expression was found not to affect the morphology, viability, and cell cycle/apoptotic profiles or DNA repair machinery of untreated cells. Nonetheless, upon UV treatment, HBx protein levels increased concomitantly with p53 levels. Both HBx and p53 proteins were found to interact and colocalize primarily in the nucleus. The binding of HBx to p53 modulated (but did not inhibit) the transcriptional activation function of p53. Notably, HBx-expressing cells exhibited increased sensitivity to UV damage, resulting in greater G2/M arrest and apoptosis of these cells. Additionally, these cells displayed a reduced DNA repair capacity in response to UV damage. In conclusion, this work suggests that DNA damage may be an initiating factor in hepatocarcinogenesis and that HBx may act as the promoting factor by inhibiting DNA repair. In hepatitis B virus-infected hepatocytes, a chronic infection may present the opportunity for such a DNA-damaging event to occur, and accumulated errors caused by the inhibition of DNA repair by HBx may result in oncogenesis. Various reports have implicated the virally encoded HBx protein as a cofactor in hepatocarcinogenesis. However, direct evidence of the role of HBx as a promoter of oncogenesis in response to an initiating factor such as DNA damage remains inadequate. Here, we report the effects of HBx in HepG2 cells exposed to UV light-induced DNA damage. HBx expression was found not to affect the morphology, viability, and cell cycle/apoptotic profiles or DNA repair machinery of untreated cells. Nonetheless, upon UV treatment, HBx protein levels increased concomitantly with p53 levels. Both HBx and p53 proteins were found to interact and colocalize primarily in the nucleus. The binding of HBx to p53 modulated (but did not inhibit) the transcriptional activation function of p53. Notably, HBx-expressing cells exhibited increased sensitivity to UV damage, resulting in greater G2/M arrest and apoptosis of these cells. Additionally, these cells displayed a reduced DNA repair capacity in response to UV damage. In conclusion, this work suggests that DNA damage may be an initiating factor in hepatocarcinogenesis and that HBx may act as the promoting factor by inhibiting DNA repair. In hepatitis B virus-infected hepatocytes, a chronic infection may present the opportunity for such a DNA-damaging event to occur, and accumulated errors caused by the inhibition of DNA repair by HBx may result in oncogenesis. There is compelling evidence showing that the hepatitis B virus (HBV) 2The abbreviations used are: HBV, hepatitis B virus; HCC, hepatocellular carcinoma; EGFP, enhanced green fluorescence protein; CAT, chloramphenicol acetyltransferase; CMV, cytomegalovirus; NER, nucleotide excision repair; DDB, damaged DNA-binding protein; UV-DDB, UV-damaged DNA-binding protein. is a major etiologic factor in hepatocellular carcinoma (HCC) (1Beasley R.P. Cancer. 1988; 61: 1942-1956Crossref PubMed Scopus (1171) Google Scholar). However, the association of chronic HBV infection with HCC is poorly understood. Among the four proteins translated from the HBV genome, the X gene product termed HBx has been implicated in the process of hepatocarcinogenesis. Mice carrying HBx as a transgene show a direct correlation between the level of HBx expression and the likelihood to develop HCC (2Kim C.M. Koike K. Saito I. Miyamura T. Jay G. Nature. 1991; 351: 317-320Crossref PubMed Scopus (1053) Google Scholar, 3Koike K. Moriya K. Yotsuyanagi H. Iino S. Kurokawa K. J. Clin. Investig. 1994; 94: 44-49Crossref PubMed Scopus (113) Google Scholar). However, certain lineages of HBx transgenic mice do not exhibit tumor development unless coupled with other factors such as exposure to the hepatocarcinogen diethylnitrosamine (4Slagle B.L. Lee T.H. Medina D. Finegold M.J. Butel J.S. Mol. Carcinog. 1996; 15: 261-269Crossref PubMed Scopus (163) Google Scholar) or when combined with c-myc induction (5Terradillos O. Billet O. Renard C.A. Levy R. Molina T. Briand P. Buendia M.A. Oncogene. 1997; 14: 395-404Crossref PubMed Scopus (258) Google Scholar). Furthermore, the development of hepatic neoplasia requires the expression of HBx to be above a certain threshold level (3Koike K. Moriya K. Yotsuyanagi H. Iino S. Kurokawa K. J. Clin. Investig. 1994; 94: 44-49Crossref PubMed Scopus (113) Google Scholar). In addition to the long latent periods usually observed between the time of initial HBV infection and tumor appearance, these observations collectively suggest that HBx does not directly cause cancer but plays a role in liver oncogenesis as a cofactor or tumor promoter. Chronic HBV infection may present a long-term opportunity for an initiating event to occur, and HBx may act by modifying cellular regulatory/control mechanisms facilitating the culmination of the transformation process in the cell. In this regard, a highly probable tumor-initiating event is DNA damage. Indeed, the effects of HBx on DNA damage repair mechanisms have been proposed as major oncogenic factors. By host cell reactivation assays and unscheduled DNA synthesis studies, HBx has been reported to compromise host cell DNA repair (6Becker S.A. Lee T.H. Butel J.S. Slagle B.L. J. Virol. 1998; 72: 266-272Crossref PubMed Google Scholar, 7Groisman I.J. Koshy R. Henkler F. Groopman J.D. Alaoui-Jamali M.A. Carcinogenesis. 1999; 20: 479-483Crossref PubMed Scopus (71) Google Scholar, 8Madden C.R. Finegold M.J. Slagle B.L. J. Virol. 2000; 74: 5266-5272Crossref PubMed Scopus (45) Google Scholar). The effect of HBx on host cell DNA repair is likely to be due to the interaction between HBx and proteins of the DNA repair complex or p53 (9Feitelson M.A. Zhu M. Duan L.X. London W.T. Oncogene. 1993; 8: 1109-1117PubMed Google Scholar, 10Wang X.W. Forrester K. Yeh H. Feitelson M.A. Gu J.R. Harris C.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2230-2234Crossref PubMed Scopus (635) Google Scholar, 11Takada S. Tsuchida N. Kobayashi M. Koike K. J. Cancer Res. Clin. Oncol. 1995; 121: 593-601Crossref PubMed Scopus (34) Google Scholar). Normal cells respond to cellular stress events such as DNA damage by increasing intracellular p53 concentrations to induce cell cycle arrest for the repair of damaged DNA. Cells with irreparable damage are usually eliminated by p53-dependent apoptosis (12Schwartz D. Rotter V. Semin. Cancer Biol. 1998; 8: 325-336Crossref PubMed Scopus (179) Google Scholar). However, the role of HBx in regulating apoptosis or its mechanism of regulation remains unclear. HBx has been reported to inhibit (13Su F. Schneider R.J. J. Virol. 1996; 70: 4558-4566Crossref PubMed Google Scholar, 14Gottlob K. Fulco M. Levrero M. Graessmann A. J. Biol. Chem. 1998; 273: 33347-33353Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 15Shih W.L. Kuo M.L. Chuang S.E. Cheng A.L. Doong S.L. J. Biol. Chem. 2000; 275: 25858-25864Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar) as well as induce (16Koike K. Moriya K. Yotsuyanagi H. Shintani Y. Fujie H. Tsutsumi T. Kimura S. Cancer Lett. 1998; 134: 181-186Crossref PubMed Scopus (48) Google Scholar, 17Bergametti F. Prigent S. Luber B. Benoit A. Tiollais P. Sarasin A. Transy C. Oncogene. 1999; 18: 2860-2871Crossref PubMed Scopus (73) Google Scholar, 18Sirma H. Giannini C. Poussin K. Paterlini P. Kremsdorf D. Brechot C. Oncogene. 1999; 18: 4848-4859Crossref PubMed Scopus (195) Google Scholar) apoptosis. The mechanism of regulation of apoptosis by HBx was reported to be via both p53-dependent (19Wang X.W. Gibson M.K. Vermeulen W. Yeh H. Forrester K. Sturzbecher H.W. Hoeijmakers J.H. Harris C.C. Cancer Res. 1995; 55: 6012-6016PubMed Google Scholar, 20Chirillo P. Pagano S. Natoli G. Puri P.L. Burgio V.L. Balsano C. Levrero M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8162-8167Crossref PubMed Scopus (193) Google Scholar) and p53-independent (21Terradillos O. Pollicino T. Lecoeur H. Tripodi M. Gougeon M.L. Tiollais P. Buendia M.A. Oncogene. 1998; 17: 2115-2123Crossref PubMed Scopus (155) Google Scholar) pathways. p53 was reported to bind to HBx (22Ueda H. Ullrich S.J. Gangemi J.D. Kappel C.A. Ngo L. Feitelson M.A. Jay G. Nat. Genet. 1995; 9: 41-47Crossref PubMed Scopus (328) Google Scholar) and to localize primarily in the cytoplasm (22Ueda H. Ullrich S.J. Gangemi J.D. Kappel C.A. Ngo L. Feitelson M.A. Jay G. Nat. Genet. 1995; 9: 41-47Crossref PubMed Scopus (328) Google Scholar, 23Elmore L.W. Hancock A.R. Chang S.F. Wang X.W. Chang S. Callahan C.P. Geller D.A. Will H. Harris C.C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14707-14712Crossref PubMed Scopus (307) Google Scholar, 24Takada S. Kaneniwa N. Tsuchida N. Koike K. Oncogene. 1997; 15: 1895-1901Crossref PubMed Scopus (83) Google Scholar) in HBx-expressing cells to modulate apoptosis (23Elmore L.W. Hancock A.R. Chang S.F. Wang X.W. Chang S. Callahan C.P. Geller D.A. Will H. Harris C.C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14707-14712Crossref PubMed Scopus (307) Google Scholar). However, it has also been reported that HBx does not colocalize or co-immunoprecipitate with p53 in HBV-infected human liver cells (25Su Q. Schroder C.H. Otto G. Bannasch P. Mutat. Res. 2000; 462: 365-380Crossref PubMed Scopus (33) Google Scholar). In cancers (such as HCC) that have a viral etiology, the viruses rarely act as complete oncogenic agents but instead contribute to the development of a transformed cell either as an initiator or a promoter (26Butel J.S. Carcinogenesis. 2000; 21: 405-426Crossref PubMed Scopus (277) Google Scholar). In this study, we directly demonstrate the effect of HBx as an oncogenic promoter in response to UV light-induced DNA damage. Upon UV irradiation, cells expressing HBx had decreased DNA repair capability and nuclear accumulation of functional p53. Cells expressing HBx also showed increased sensitivity to UV damage, exhibiting elevated levels of G2/M arrest and apoptosis. The ability of HBx to alter the capability of the cell to repair damaged DNA may predispose an individual with chronic HBV infection to cancer, and the long delay in onset of HCC in HBV-infected individuals may be the result of accumulated genetic lesions caused by the inhibition of DNA repair by HBx. Subsequently, a pro-apoptotic effect may exert selective pressure for apoptosis-resistant preneoplastic cells. These observations purport the view that supplementary changes (such as DNA damage) must occur to complement the pleiotropic functions of HBx to disrupt the multiple checkpoints and regulatory mechanisms of a normal cell, thus culminating in malignancy. Recombinant HBx-expressing Adenovirus Preparation—The HBx gene was amplified from pEco63. HBx-expressing (AdHBx) and control (AdControl) recombinant adenoviruses were generated as described previously (27He T.C. Zhou S. da Costa L.T. Yu J. Kinzler K.W. Vogelstein B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2509-2514Crossref PubMed Scopus (3252) Google Scholar). Briefly, the HBx gene was initially subcloned into the shuttle vector pAdTrack-CMV, and the integrity of the HBx gene in this vector was sequence-verified. Another plasmid containing the adenoviral arms (pAdEasy-1) was cotransformed with either the PmeI-linearized shuttle vector (pAdTract-CMV) or the HBx-containing shuttle vector (pAdTract-CMV-HBx) into Escherichia coli BJ5183 cells. Control (pAdControl) and HBx-expressing (pAdHBx) recombinant adenoviral vectors were then generated by homologous recombination of pAdEasy-1 and pAdTract-CMV or pAdTract-CMV-HBx in E. coli BJ5183 cells. The colonies obtained were screened for appropriate recombination events by EcoRV and PmeI restriction endonuclease analyses. The pAdControl and pAdHBx vectors (see Fig. 1A) were then digested with PacI and transfected into the 293 packaging cell line (which constitutively expresses the E1 gene product) to produce control (AdControl) and HBx-expressing (AdHBx) recombinant adenoviruses. The titer of the virus (expression-forming units/ml) was evaluated by counting the number of fluorescent cells after infection with serially diluted viruses. Recombinant HBx-expressing Adenovirus Infection and UV Treatment—HepG2 cells were obtained from American Type Culture Collection and maintained at 37 °C in a humidified atmosphere of 5% CO2 in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. These cells were seeded onto 6-well plates and grown to 40-50% confluency. Recombinant HBx-expressing or vector control adenoviruses were then added at a multiplicity of infection of 5. Twenty-four hours after adenovirus infection, the medium was replaced with fresh Dulbecco's modified Eagle's medium without virus. Cells were observed under a fluorescence microscope to detect enhanced green fluorescence protein (EGFP) fluorescence to assess infection efficiency. Efficiency was noted to be >90% and did not deviate by >5% between experiments. UVC (254 nm) irradiation of infected cells was then performed with a germicidal lamp calibrated to deliver 8 or 16 J/m2. Cell Viability—The number of viable uninfected and AdControl- and AdHBx-infected cells was determined by the Cell Titer 96 Aqueous One cell proliferation assay (Promega Corp.) according to the manufacturer's instructions. Cell growth and viability were also monitored at various time points with an Olympus Research inverted microscope (IX51), and images were captured with a QImaging Retiga 1300R digital imager. Cell Cycle and Apoptosis—Cells were harvested at various time points, washed with phosphate-buffered saline, and fixed in 1% paraformaldehyde or 70% ethanol. These fixed cells were then stained with propidium iodide (Sigma) and analyzed with a FACSCalibur flow cytometer (BD Biosciences) to determine cell cycle profiles. Apoptotic cell death was also assessed by flow cytometry after staining the cells with phycoerythrin-conjugated annexin V and 7-aminoactinomycin D using the Annexin V:PE Apoptosis Detection Kit I (BD Biosciences) or propidium iodide when the DNA content and cell granularity were assessed. Generation of Anti-HBx Antibody—The HBx open reading frame was cloned into pET16b (Invitrogen). The HBx protein was then overexpressed in bacteria and purified using nickel-nitrilotriacetic acid beads (Qiagen Inc., Hilden, Germany) according to the manufacturer's instructions. Polyclonal antibody against the recombinant HBx protein was then generated in rabbits (Zymed Laboratories Inc.) and purified using an HBx protein column (BioGenes GmbH, Berlin, Germany). Immunofluorescence Staining—Localization of p53 and HBx proteins was determined by immunofluorescence staining as described previously (28Lee C.G. Ren J. Cheong I.S. Ban K.H. Ooi L.L. Yong Tan S. Kan A. Nuchprayoon I. Jin R. Lee K.H. Choti M. Lee L.A. Oncogene. 2003; 22: 2592-2603Crossref PubMed Scopus (146) Google Scholar). Briefly, cells grown on glass coverslips were fixed in 2% paraformaldehyde solution and then permeabilized with 0.2% Triton X-100. The cells were co-stained using 1 μg/μl anti-HBx antibody and 1 μg/μl anti-p53 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for 1 h at 37°C. After three washes with phosphate-buffered saline, the coverslips were incubated with Alexa Fluor 647-labeled chicken anti-mouse and Alexa Fluor 488-labeled anti-rabbit IgG secondary antibodies (1:300 dilution; Molecular Probes, Inc., Eugene, OR) for 1 h at room temperature. Cells were also incubated with 4′,6-diamidino-2-phenylindole to distinguish the nucleus. The coverslips were then washed three times with phosphate-buffered saline and mounted on glass slides. The mounted slides were observed with a Zeiss LSM 510 laser scanning confocal microscope. Immunoprecipitation—Immunoprecipitation was performed using a protein G immunoprecipitation kit (Roche Applied Science) according to the manufacturer's instructions. Briefly, 400 μg (200 μl) of protein from cell lysates was incubated with 2 μg of anti-HBx or anti-p53 antibody at 4 °C for 6 h. A 10-μl bed volume of protein G (provided with the kit) was used per sample. Western blot analysis was performed as described below. Western Blot Analysis—Equal protein concentrations were electrophoresed on a 12% SDS-polyacrylamide gel and transferred to a polyvinylidene difluoride membrane (Bio-Rad). The blots were then probed with horseradish peroxidase-conjugated anti-p53 antibody (1:10,000 dilution; Santa Cruz Biotechnology, Inc.), goat anti-actin polyclonal antibody (1:30,000 dilution; Santa Cruz Biotechnology, Inc.), or goat anti-HBx polyclonal antibody (1:5000 dilution; generated in this laboratory) for 1 h at room temperature. Horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:10,000 dilution; Pierce) was then added to the blots probed with anti-actin or anti-HBx antibody for an additional 1 h at room temperature. Signals on the immunoblots were detected using an enhanced chemiluminescence reagent kit (ECL, Amersham Biosciences). Real-time Reverse Transcription-PCR—RNA was isolated as described previously (28Lee C.G. Ren J. Cheong I.S. Ban K.H. Ooi L.L. Yong Tan S. Kan A. Nuchprayoon I. Jin R. Lee K.H. Choti M. Lee L.A. Oncogene. 2003; 22: 2592-2603Crossref PubMed Scopus (146) Google Scholar). cDNA was synthesized from total RNA using SuperScript II reverse transcriptase (Invitrogen) according to the manufacturer's instructions. Real-time PCR was performed with a Rotor-Gene 2000 real-time thermal cycler (Corbett Research, Sydney, Australia) using the QuantiTect™ SYBR Green PCR kit (Qiagen Inc.). Amplification reactions included cDNA template (25 ng), each of the forward and reverse primers for the various genes (0.25 pmol/μl), and 2× PCR Master Mix (5 μl; Qiagen Inc.) in a total volume of 10 μl. The primers for the various genes are as follows: MDM2,5′-TGTAAGTGAACATTCAGGTG-3′ (forward) and 5′-TTCCAATAGTCAGCTAAGGA-3′ (reverse); p21, 5′-CCTCAAATCGTCCAGCGACCTT-3′ (forward) and 5′-CATTGTGGGAGGAGCTGTGAAA-3′ (reverse); bcl-2, 5′-TTGGCCCCCGTTGCTT-3′ (forward) and 5′-CGGTTATCGTACCCCGTTCTC-3′ (reverse); bax, 5′-TCCCCCCGAGAGGTCTTTT-3′ (forward) and 5′-CGGCCCCAGTTGAAGTTG-3′ (re-verse); and β-actin, 5′-ATGTTTGAGACCTTCACACC-3′ (forward) and 5′-AGGTAGTCAGTCAGGTCCCGGCC-3′ (reverse). Amplification of the transcripts involved an initial denaturation at 95 °C for 15 min, followed by 40 cycles at 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s. SYBR Green fluorescence was measured after each extension step. Standard curves were generated using serially diluted plasmids in which the respective cDNAs were cloned. The linear ranges for the expression of all respective genes were determined to be between 103 and 108 copies (r2 = 0.9997 for p21, 0.9994 for MDM2, 0.9958 for bcl-2, 0.9986 for bax, and 0.992 for β-actin). The expression levels of these various genes were then normalized against the expression levels of the β-actin housekeeping gene. All reverse transcription-PCRs were performed in triplicate. Host Cell Reactivation Assay—The β-galactosidase reporter plasmid was damaged with 800 J/m2 UVC light using a UV cross-linker (UVP, Inc., Upland, CA). Control undamaged β-galactosidase plasmids did not receive UV treatment. Cells were then transfected with the chloramphenicol acetyltransferase (CAT) plasmid and either the damaged or undamaged β-galactosidase reporter plasmid using the Superfect transfection reagent (Qiagen Inc.) according to the manufacturer's protocol. Cell extracts were harvested 48 h after transfection, and assays to detect for β-galactosidase activity and the CAT protein were performed. β-Galactosidase activity was evaluated using chlorophenol red-β-d-galactopyranoside (Roche Applied Science) as substrate to detect β-galactosidase activity in a kinetic assay at 1-min intervals at 570 nm with a SpectraMax Plus384 microplate reader (Molecular Devices Corp., Sunnyvale, CA). This β-galactosidase activity was then normalized against CAT protein expression to correct for differences in transfection efficiency. CAT expression was quantitatively measured using a CAT enzyme-linked immunosorbent assay kit (Roche Applied Science). Transfection of the plasmids and the subsequent assays for β-galactosidase activity and CAT expression were performed in control (HepG2-Control) and HBx-infected (HepG2-HBx) HepG2 cells that were either untreated or UV light-irradiated at 8 J/m2. HBx Expression Does Not Affect Cell Morphology and Viability, Apoptotic, and Cell Cycle Profiles—A major obstacle in HBx research is the difficulty encountered in obtaining high affinity antibodies to immuno-detect the HBx protein (20Chirillo P. Pagano S. Natoli G. Puri P.L. Burgio V.L. Balsano C. Levrero M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8162-8167Crossref PubMed Scopus (193) Google Scholar, 29Su Q. Schroder C.H. Hofmann W.J. Otto G. Pichlmayr R. Bannasch P. Hepatology. 1998; 27: 1109-1120Crossref PubMed Scopus (192) Google Scholar). Various groups have circumvented this by utilizing either a EGFP-HBx fusion protein (30Shirakata Y. Koike K. J. Biol. Chem. 2003; 278: 22071-22078Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar) or HBx tagged with hemagglutinin at the C terminus (31Yun C. Lee J.H. Park H. Jin Y.M. Park S. Park K. Cho H. Oncogene. 2000; 19: 5163-5172Crossref PubMed Scopus (40) Google Scholar) for immunoprecipitation or sub-cellular localization studies. A major disadvantage of these strategies is that, being a small molecule, tagging of HBx with either EGFP or hemagglutinin may alter its solubility or disrupt its normal physiological functions. Here, we report the generation of a specific anti-HBx antibody that is useful for the detection of the HBx protein in Western blots (Fig. 1, lower panels), immunoprecipitation (see Fig. 3D), and immunocytochemistry (see Fig. 3, A and B). We also developed a recombinant AdHBx adenoviral system that has an infection efficiency of >90% as assessed by EGFP fluorescence (Fig. 1A, middle panels) and that expresses the HBx protein. As shown in Fig. 1A (bottom panels), only protein extracts from HepG2 cells infected with AdHBx (HepG2-HBx), but not protein extracts from cells infected with AdControl (HepG2-Control), showed a single distinct band at ∼17 kDa, the size of HBx, upon Western blotting. Previous studies have reported that expression of HBx induces spontaneous apoptotic cell death when expressed in mouse fibro-blasts (20Chirillo P. Pagano S. Natoli G. Puri P.L. Burgio V.L. Balsano C. Levrero M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8162-8167Crossref PubMed Scopus (193) Google Scholar), Chang liver cells (18Sirma H. Giannini C. Poussin K. Paterlini P. Kremsdorf D. Brechot C. Oncogene. 1999; 18: 4848-4859Crossref PubMed Scopus (195) Google Scholar), HepG2 cells (32Pollicino T. Terradillos O. Lecoeur H. Gougeon M.L. Buendia M.A. Biomed. Pharmacother. 1998; 52: 363-368Crossref PubMed Scopus (47) Google Scholar), and HBx transgenic mouse liver (16Koike K. Moriya K. Yotsuyanagi H. Shintani Y. Fujie H. Tsutsumi T. Kimura S. Cancer Lett. 1998; 134: 181-186Crossref PubMed Scopus (48) Google Scholar). However, we found that expression of HBx was not deleterious to HepG2 cells. Under a phase-contrast microscope, HepG2-HBx cells had a similar morphology and number of doubling mitotic or rounded phase-bright apoptotic cells as the HepG2-Control cells (Fig. 1C, upper right panel). When cell viability was assessed, there was no statistical difference between the uninfected HepG2 cells and HepG2-Control or HepG2-HBx cells (Fig. 1B). Additionally, the apoptotic and cell cycle profiles of HepG2-Control and HepG2-HBx cells were similar (Fig. 1C). p53 and HBx Accumulate in Response to UV Damage—Because our data show that HBx expression did not affect proliferation or induce spontaneous cell death, we hypothesized that the role of HBx as a promoter would be more evident following an initiating event such as DNA damage. DNA damage may precede cellular transformation if DNA repair mechanisms are disrupted (9Feitelson M.A. Zhu M. Duan L.X. London W.T. Oncogene. 1993; 8: 1109-1117PubMed Google Scholar, 10Wang X.W. Forrester K. Yeh H. Feitelson M.A. Gu J.R. Harris C.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2230-2234Crossref PubMed Scopus (635) Google Scholar, 11Takada S. Tsuchida N. Kobayashi M. Koike K. J. Cancer Res. Clin. Oncol. 1995; 121: 593-601Crossref PubMed Scopus (34) Google Scholar, 33de Vries A. van Oostrom C.T. Hofhuis F.M. Dortant P.M. Berg R.J. de Gruijl F.R. Wester P.W. van Kreijl C.F. Capel P.J. van Steeg H. Verbeek S.J. Nature. 1995; 377: 169-173Crossref PubMed Scopus (361) Google Scholar, 34Sands A.T. Abuin A. Sanchez A. Conti C.J. Bradley A. Nature. 1995; 377: 162-165Crossref PubMed Scopus (218) Google Scholar). To evaluate this, we examined the effect of HBx in HepG2 cells exposed to UVC irradiation. Twenty-four hours after infection with AdControl or AdHBx, HepG2-Control or HepG2-HBx cells were exposed to UV damage at a dose of 8 J/m2. The cells were then harvested at various time points after UV irradiation, and the HBx protein levels in these cells were determined by Western blot analyses. As shown in Fig. 2A, left panels, in the absence of UV irradiation, only low levels of HBx proteins were observed at the various time points. However, when the cells were UV light-irradiated, the HBx protein was found to accumulate in a time-dependent manner (Fig. 2A, middle panels). The HBx protein level was also found to increase when the UV dose was increased to 16 J/m2 (Fig. 2B). These results suggest that UV irradiation affects HBx protein levels in a time-as well as dose-dependent manner. Genotoxic stress (including DNA damage) induces post-transcriptional modification and stabilization of p53, resulting in accumulation of p53 (35Chresta C.M. Arriola E.L. Hickman J.A. Behring. Inst. Mitt. 1996; 97: 232-240PubMed Google Scholar). Because p53 is a well known target of HBx (9Feitelson M.A. Zhu M. Duan L.X. London W.T. Oncogene. 1993; 8: 1109-1117PubMed Google Scholar, 10Wang X.W. Forrester K. Yeh H. Feitelson M.A. Gu J.R. Harris C.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2230-2234Crossref PubMed Scopus (635) Google Scholar, 11Takada S. Tsuchida N. Kobayashi M. Koike K. J. Cancer Res. Clin. Oncol. 1995; 121: 593-601Crossref PubMed Scopus (34) Google Scholar), we evaluated whether the accumulation of p53 in UV light-damaged cells is altered by HBx. As shown in Fig. 2A, upon UV irradiation, p53 accumulated in both HepG2-Control and HepG2-HBx cells. However, although p53 protein levels in HepG2-Control cells gradually decreased after an initial peak at 24 h, p53 protein levels continued to increase throughout the experiment in HepG2-HBx cells. Additionally, p53 increased concomitantly with HBx in a dose-dependent manner. Hence, UV irradiation causes an accumulation of both p53 and HBx proteins in HBx-expressing HepG2 cells. HBx Colocalizes and Interacts with p53 in the Nucleus upon UV Treatment—The subcellular localization of HBx and p53 in HBx-expressing cells and their biological significance remain unclear. HBx seems to be a nucleocytoplasmic protein (36Nomura T. Lin Y. Dorjsuren D. Ohno S. Yamashita T. Murakami S. Biochim. Biophys. Acta. 1999; 1453: 330-340Crossref PubMed Scopus (24) Google Scholar, 37Hoare J. Henkler F. Dowling J.J. Errington W. Goldin R.D. Fish D. Mc-Garvey M.J. J. Med. Virol. 2001; 64: 419-426Crossref PubMed Scopus (51) Google Scholar, 38Doria M. Klein N. Lucito R. Schneider R.J. EMBO J. 1995; 14: 4747-4757Crossref PubMed Scopus (275) Google Scholar), although it was reported to localize primarily to the cytoplasmic/perinuclear compartment of the cells (24Takada S. Kaneniwa N. Tsuchida N. Koike K. Oncogene. 1997; 15: 1895-1901Crossref PubMed Scopus (83) Google Scholar, 25Su Q. Schroder C.H. Otto G. Bannasch P. Mutat. Res. 2000; 462: 365-380Crossref PubMed Scopus (33) Google Scholar, 31Yun C. Lee J.H. Park H. Jin Y.M. Park S. Park K. Cho H. Oncogene. 2000; 19: 5163-5172Crossref PubMed Scopus (40) Google Scholar, 39Shintani Y. Yotsuyanagi H. Moriya K. Fujie H. Tsutsumi T. Kanegae Y. Kimura S. Saito I. Koike K. J. Gen. Virol. 1999; 80: 3257-3265Crossref PubMed Scopus (69) Google Scholar). p53 was reported to bind to HBx (22Ueda H. Ullrich S.J. Gangemi J.D. Kappel C.A. Ngo L. Feitelson M.A. Jay G. Nat. Genet. 1995; 9: 41-47Crossref PubMed Scopus (328) Google Scholar) and to localize primarily in the cytoplasm (22Ueda H. Ullrich S.J. Gangemi J.D. Kappel C.A. Ngo L. Feitelson M.A. Jay G. Nat. Genet. 1995; 9: 41-47Crossref PubMed Scopus (328) Google Scholar, 23Elmore L.W. Hancock A.R. Chang S.F. Wang X.W. Chang S. Callahan C.P. Geller D.A. Will H. Harris C.C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14707-14712Crossref PubMed Scopus (307) Google Scholar, 24Takada S. Kaneniwa N. Tsuchida N. Koike K. Oncogene. 1997; 15: 1895-1901Crossref PubMed Scopus (83) Google Scholar) in HBx-expressing cells. However, HBx was also reported not to colocalize or co-immunoprecipitate with p53 in HBV-infected human liver cells (25Su Q. Schroder C.H. Otto G. Bannasch P. Mutat. Res. 2000; 462: 365-380Crossref PubMed Scopus (33) Google Scholar). Because HBx and p53 protein levels were found to increase concomitantly in UV light-treated HepG2-HBx cells (Fig. 2A), we assessed whether these two proteins occur in the same subcellular compartment and interact. The immunofluorescence images in Fig. 3A (upper two rows) show weak HBx staining in both the nuclear and cytoplasmic compartments of untreated HepG2-HBx cells. Upon UV exposure, there was an increase in HBx staining primarily in the nucleus (Fig. 3A, lower four rows), suggesting nuclear accumulation of HBx in t" @default.
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• W2058264480 title "The Hepatitis B Virus X Protein Sensitizes HepG2 Cells to UV Light-induced DNA Damage" @default.
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