Carcinogenesis

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Carcinogenesis, Vol. 20, No. 3, 479-483, March 1999
© 1999 Oxford University Press

Downregulation of DNA excision repair by the hepatitis B virus-x protein occurs in p53-proficient and p53-deficient cells

Iris Jaitovich Groisman, Rajen Koshy¹, Frank Henkler¹, John D. Groopman² and Moulay A. Alaoui-Jamali³

Lady Davis Institute of the Sir Mortimer B.Davis Jewish General Hospital, Departments of Medicine and Oncology and the McGill Centre for Translational Research in Cancer, McGill University, 3755 Chemin de la Cote-Ste-Catherine, Montreal H3T 1E2, Canada,
¹ Department of Virology, Royal Postgraduate Medical School, London W12 0NN, UK and
² School of Hygiene and Public Health, Johns Hopkins Universiy, Baltimore, MD 21205, USA

	Abstract

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Synergism between exposure to chemical carcinogens and infection with the hepatitis B virus (HBV) has been implicated in the high incidence of hepatocellular carcinoma. In this study we report that the HBV protein HBx, inhibits cellular DNA repair capacity in a p53-independent manner. Two alternative assays were used: the host cell reactivation assay, which measures the cell's capacity to repair DNA damage in a reporter plasmid, and unscheduled DNA synthesis, which measures the overall DNA repair capacity in damaged cells. Two p53-proficient cell lines, the hepatocellular carcinoma cell line HepG2 and liver epithelial cell line CCL13, were co-transfected with the pCMV–HBx reporter plasmid and the pCMV–CAT plasmid damaged with UVC radiation. Compared with cells transfected with control plasmid, the presence of HBx resulted in ~50% inhibition of the cell's capacity to reactivate CAT activity of UVC-damaged plasmid, and ~25% inhibition of unscheduled DNA synthesis in cells treated with either aflatoxin B1 epoxide or UVC radiation. Using the p53-deficient cell line Saos-2, we demonstrated that expression of HBx also resulted in diminished overall cellular DNA repair of damage induced by both aflatoxin B1 epoxide and UVC radiation, using both the host cell reactivation and unscheduled DNA synthesis assays. In summary, this study provides evidence for p53-independent regulation of DNA repair by HBx.

Abbreviations: CAT, chloramphenicol acetyl transferase; CMV, cytomegalovirus; HBV, hepatitis B virus; HBx, hepatitis B virus-x protein; HCC, hepatocellular carcinoma; HCR, host cell reactivation; NER, nucleotide excision repair; UDS, unscheduled DNA synthesis.

	Introduction

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Genes mediating DNA repair play an important role in the maintenance of gene integrity and stability in situations of genomic stress. Activities of these genes are intimately linked to cell cycle checkpoint mechanisms, which coordinate the timing of repair processes. Genotoxicity of chemical carcinogens depends on the balance between DNA damaging events and DNA repair mechanisms. Failure to repair DNA damage can enhance genotoxicity, lead to genomic instability or trigger apoptosis.

Inherited deficiencies in specific DNA repair genes are associated with various human genetic diseases, including xeroderma pigmentosum and Cockayne syndrome (1), and individual predisposition to familial non-polyposis colorectal cancer (2), ovarian (3), breast (4) and lung (5) cancers. Whereas most DNA repair studies have focused on the inherited gene defects associated with human diseases, there is mounting evidence that DNA repair can be altered by acquired factors, such as viruses and chemicals, leading to enhanced susceptibility to carcinogenesis (6). For example, the p53 tumor suppressor gene, which plays a role in several mechanisms including the regulation of cell cycle checkpoint mechanisms required for DNA repair (7), can be inactivated by many viruses as well as chemical carcinogens. The large T antigen of SV40, the E1B 55 kDa protein of adenovirus, the E6 protein of human papilloma virus (HPV) and the hepatitis B virus-x protein (HBx) (8–12) inactivate p53. The liver carcinogen aflatoxin B1 induces a GT transversion in codon 249 of the p53 gene (13), whereas benzo[a]pyrene induces transversions and substitutions in the p53 gene (14).

The most characterized model in which a synergistic association between viral infection and chemical carcinogens seems to play an important role in cancer development is hepatocellular carcinoma (HCC). HCC has a high incidence in specific geographic areas such as southern China and central Africa (15). The concomitant exposure to liver carcinogens such as aflatoxins and HBV has been associated with a high incidence of HCC in endemic regions (16), and in laboratory animal models (17,18). One of the most documented mechanisms by which HBV contributes to HCC involves the hepatitis B-x protein (HBx) (15,19). HBx has been shown to associate with the tumor suppressor gene product p53 and inhibits its function (10,12,20). Considering the evidence that p53 interacts with several nucleotide excision repair (NER) proteins, including RPA, XPB (ERCC3) and XPD (ERCC2) (7,10,11,21), and it is involved in cell cycle regulation; its inactivation by HBx may lead to altered DNA repair, cell cycle checkpoint and/or apoptotic mechanisms. HBx itself was previously described to interfere with these cellular processes (22–27).

In this study we have shown that expression of HBx, in both p53-proficient and -deficient cells, inhibits DNA repair following DNA damage induced by aflatoxin B1 epoxide or UV radiation.

	Materials and methods

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Plasmid constructs
The HBx protein expression vector pCMV–HBx consists of the HBx gene ligated into the HindIII site of the pRc–CMV vector (Invitrogen, La Jolla, CA) (28). The pRc–CMV plasmid was used as a negative control. The pCMV–CAT chloramphenicol reporter construct used for the host cell reactivation assay was obtained by subcloning the CAT gene into the HindII and EcoRI sites of the pRc–CMV plasmid. The pCMV–CAT reporter plasmid (50 µg/ml), which was irradiated with 1000 J/m² from a UVC lamp at 1 J/s/m², was used in the host cell reactivation assay.

Cell lines
HepG2 is a hepatoblastoma cell line expressing wild-type p53, as determined by DNA sequence analysis using Sequenase version 2.0 (United States Biochemical, Cleveland, OH). HepG2 cells were grown in -minimum essential medium (-MEM; Mediatech, Washington, DC) supplemented with 10% fetal bovine serum (FBS) and gentamicin (50 µg/ml; Mediatech). CCL13 (also known as Chang cells) is a human liver epithelial cell line expressing wild-type p53, as determined by immunoprecipitation coupled western blot analysis using an antibody that only recognized wild-type p53 (Ab5; Oncogene Laboratories, Cambridge, MA). These cells were maintained in D-MEM (Mediatech), 10% FBS, and 50 µg/ml of gentamicin. Saos-2 is a human osteosarcoma cell line that lacks endogenous p53. These cells were grown in McCoy's 5A medium (Mediatech) supplemented with 15% FBS and penicillin–streptomycin (Mediatech) (50 U/ml and 50 µg/ml, respectively). All cells were maintained at 37°C in an atmosphere of 5% CO₂. The three cell lines were obtained from the American Tissue Culture Collection (ATCC, Rockville, MD).

Western blot analysis
Cells were washed twice in cold phosphate-buffered saline (PBS) and then lysed directly using lysis buffer (1% Triton X-100, 10 mM Tris–HCl, pH 8.0, 60 mM KCl, 1 mM EDTA, 1 mM DTT, 0.5% NP-40, 0.5 mM phenylmethyl-sulfonylfluoride, 0.01 µg/ml leupeptin, 0.01 µg/ml pepstatin, 0.01 µg/ml aprotonin, 5 mM sodium orthovanadate and 10 mM sodium PPi). Total cell extracts from cells transfected with pCMV–HBx and pRc–CMV were used to examine the expression of HBx by western blot analysis. Polyacrylamide gel electrophoresis was performed using a 4% polyacrylamide stacking gel layered over a 12% resolving gel for HBx. Aliquots of 20 µg of protein extract were run at 50 V for 16 h and transferred onto nitrocellulose membrane (Costar, Cambridge, MA). The membranes were blocked overnight at 4°C with 10% low fat milk in PBS and incubated overnight with the corresponding antibody. HBx protein was detected using the monoclonal antibody 16F1 (28) and an enhanced-chemiluminescence (ECL) reagent kit (Amersham, Oakville, Ontario).

Host cell reactivation assay (HCR)
Cells were seeded at 2.7–3.0 x10⁵ cells per well, in six-well plates, and grown overnight in the appropriate media. The following day, the cells were transiently transfected using Lipofectamine (Gibco BRL, Burlington, Ontario) with the corresponding plasmids as described in the figure legends. Lipofectamine was used at a concentration of 3 µg/1 µg DNA. Salmon sperm DNA was added to equalize the amount of DNA transfected in each well when necessary. Cells were incubated with DNA–Lipofectamine complexes for 8 h at 37°C, 5% CO₂ and harvested 12 h after transfection. Protein extracts were used to determine chloramphenicol acetyl transferase (CAT) activity essentially as described (29). The quantification of the reaction products in the CAT assay was performed using a Bio-Rad Gelscan Phosphoimager and a Molecular Analyst (Bio-Rad, Richmond, CA) software program. The results were expressed as the percentage of chloramphenicol conversion to its acetylated metabolites.

Unscheduled DNA repair synthesis
Cells were seeded at a density of 5x10⁵/well in six-well plates and cultured in complete medium until cells reached full confluence. Cells were then transfected with 5µg pCMV–HBx or pRc–CMV DNA, using 15µg Lipofectamine. Control samples were transfected with 5 µg of salmon sperm DNA per well. After 16 h incubation at 37°C, 5% CO₂, transfection medium was removed and cells were incubated for an additional 24 h in arginine-free medium (MEM Select-Amine; Gibco BRL) containing 1% dialyzed FBS, at 37°C, 5% CO₂. Under these conditions, cell viability was >90% as determined by Trypan Blue staining. Cells were then treated with 50 J/m² UV (254 nm using a 60 Hz, 0.16 A UV lamp at a distance of 19 cm), or with 100 ng/ml of aflatoxin active metabolite, aflatoxin B1 epoxide, for 2 h. Aflatoxin B1 epoxide was synthesized as previously described (30) and its structure confirmed by HPLC and mass spectrometry analysis. After each respective treatment, cells were incubated for an additional 4 h in regular medium containing 2% serum and 10 µCi/ml [³H]methyl-thymidine ([³H]dThd, sp. act. 20 Ci/mmol; Du Pont-NEN, Missisauga, Ontario), at 37°C, 5% CO₂. Cells were then washed twice with PBS, chased with 0.5 mM cold thymidine for 30 min, and DNA was extracted using DNAzol reagent (Gibco BRL). Untreated cells transfected with either pRc–CMV or pCMV–HBx were used as a control to determine the background level of [³H]dThd incorporation. Unscheduled DNA synthesis (UDS) was estimated as (d.p.m. incorporated/µg treated DNA)/(d.p.m. incorporated/µg control DNA)x100.

	Results

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To investigate the effect of HBx on DNA repair, we first confirmed the presence of HBx in the three cell lines examined in this study, HepG2, CCL13 and Saos-2. Figure 1 shows that following transient transfection of pCMV–HBx, HBx protein was specifically identified by western blot analysis. Protein extracts of cells transfected with the negative control vector demonstrate the absence of HBx.

View larger version (33K): [in this window] [in a new window]   Fig. 1. Expression of HBx in transfected cells. Cells at 70% confluence grown in 60 mm plates were transfected with 20 µg of pCMV–HBx or pRc–CMV vectors. Cells were harvested 12 h after transfection and protein extracts were then used for western blot analysis as described in Materials and methods.

 
To determine if HBx modifies the ability of the cell to repair DNA damage, we analyzed the cellular DNA repair capacity by UDS (Figures 2 and 4), following HBx expression and treatment with the DNA-damaging agents aflatoxin B1 epoxide or UVC radiation. The assay detects [³H]dThd incorporation into DNA whereas repair takes place following DNA damage. Since the three cell lines investigated do not possess the adequate enzymatic machinery (cytochrome P450s) required to activate aflatoxin B1 (data not shown), we used the aflatoxin B1 active metabolite, aflatoxin B1 epoxide. Cell viability was examined by Trypan Blue staining, after transfection and carcinogen treatments, and was >90% in cells transfected with HBx or the negative controls (pRc–CMV or salmon sperm) following 24 h incubation of transfected cells in arginine-free medium as well as 4 h after treatment with UV radiation or aflatoxin B1 epoxide. Results of UDS show that HepG2 and CCL13 cells transfected with pCMV–HBx demonstrate 17–25% inhibition of repair of DNA damage induced by aflatoxin B1 epoxide or UVC radiation (Figure 2). The expression of pRc–CMV, which serves as a negative control for HBx, had no significant effect on DNA repair capacity.

View larger version (19K): [in this window] [in a new window]   Fig. 2. Unscheduled DNA synthesis assay. Confluent cells, in which semi-conservative DNA synthesis was completely inhibited, were transfected with pCMV–HBx or pRc–CMV. Cells were then treated with (a) 100 ng/ml aflatoxin B1 epoxide or (b) 50 J/m2 UVC, and [3H]dThd was added in medium containing 2% serum. DNA was then extracted and the incorporation of [3H]dThd was determined. UDS was determined as described in Materials and methods. Results are expressed as percentage inhibition compared with untreated cells. Each value corresponds to the mean ± SE of three independent experiments, each in triplicate. The background value (no treatment) was 87 ± 8.9 and 53 ± 4.7 of DNA for HepG2 and CCL13, respectively. *Significantly different from control pRc–CMV transfected cells at P < 0.01, using Student's t-test.

 

View larger version (20K): [in this window] [in a new window]   Fig. 4. Effect of HBx on DNA repair in p53-deficient cells. (a) Unscheduled DNA synthesis assay: confluent cells, in which semi-conservative DNA synthesis was completely inhibited, were transfected with salmon sperm DNA, pCMV–HBx or pRc–CMV. The assay was performed as explained in Materials and methods and in the legend of Figure 2. Each value corresponds to the mean ± SE of three independent experiments, and the background value (no treatment) was 77 ± 10.5 d.p.m./µg for this cell line. (b) HCR assay was performed on Saos-2 following the same conditions as for HepG2 and CCL13 cells, as explained in Figure 3 and Materials and methods. The results obtained using the UV-treated reporter plasmid are shown and expressed as percentage of acetylation, corresponds to the means of at least three independent experiments. *Significantly different from control pRc–CMV transfected cells at P < 0.01, using Student's t-test.

 
To confirm and further support the results obtained by UDS, we performed the HCR assay. This procedure measures the cell's capacity to repair a UV damaged reporter plasmid following the expression of the HBx protein. The pCMV–HBx expression or the negative control (pRc–CMV) vector was co-transfected with the pCMV–CAT reporter plasmid or the same plasmid irradiated with UVC light (1000 J/m²) into HepG2 and CCL13 cell lines. Cell extracts were harvested 12 h after transfection and CAT assays were performed (Figure 3). As compared with the non-irradiated plasmid, a decreased CAT activity was observed following transfection of the UV-treated plasmid as a consequence of DNA damage. Co-transfection of the pCMV–HBx plasmid was shown to inhibit the recovery of CAT activity from the irradiated plasmid whereas no effect of HBx was observed on the non-irradiated plasmid. Cell viability >90% was observed by Trypan Blue staining at the time of collecting the cells for CAT assay in HBx transfected cells as well in the control. HBx expression decreased cellular DNA repair capacity while promoting transactivation of Ap1 and Nf-B responsive elements (unpublished data).

View larger version (47K): [in this window] [in a new window]   Fig. 3. Host cell reactivation assay. HepG2 (a) and CCL13 (b) cell lines were transfected with pCMV–CAT or pCMV–CAT UV treated plasmids. HCR assay was carried out as described in Materials and methods. (a) CAT activity from non-irradiated and irradiated plasmid co-transfected with salmon sperm (control), the expression vector for HBx (pCMV–HBx) or its negative control. (b) CCL13 cells where only the results using the UV-treated reporter plasmid are shown. Results are expressed as percentage of acetylation, and the means of at least three independent experiments are shown. *Significantly different from control pRc–CMV transfected cells at P<0.01, using Student's t-test.

 
To determine whether DNA repair inhibition was p53-dependent, we tested UDS on the Saos-2 cell line, which lacks endogenous p53 due to gene deletion. These cells showed the same extent of DNA repair inhibition by HBx as the p53-proficient cell lines HepG2 and CCL13 (Figure 4a). We also subjected Saos-2 cells to the HCR assay following the same conditions used for HepG2 and CCL13. Figure 4b shows decreased CAT activity from pCMV–CAT that was UV-treated following HBx expression when compared with the negative control.

	Discussion

Top Abstract Introduction Materials and methods Results Discussion References

 
There is mounting evidence that individual susceptibility to carcinogenesis is affected by the interaction of several factors including genetic predisposition, exposure to genotoxic agents as well as acquired factors such as infection with viruses. Earlier epidemiological studies reported a synergistic association between exposure to environmental pollutants, such as aflatoxin B1 and chronic infection with viruses such as the hepatitis B virus (HBV), in the high incidence of hepatocellular carcinoma in endemic areas where both chronic HBV infection and exposure to aflatoxins prevail (31,32). This was also demonstrated in cell lines and transgenic mice expressing HBV products (17,18).

One of the most documented mechanisms by which HBV enhances carcinogenesis involves the HBx protein. The HBx protein is expressed in chronic hepatitis, cirrhotic liver and HCC from individuals infected with HBV (15). It is localized in both the cytoplasm and nucleus (33), and can therefore interact with cell signal transduction pathways and transcription machinery (33–35). HBx has been reported to transactivate a variety of cellular genes (36–39). Furthermore, HBx associates with the p53 tumor suppressor protein in vitro and in vivo (10,12,15), leading to p53 inhibition of its functions. Moreover, p53 inactivation by HBx has been implicated in liver carcinogenesis (12,20).

The p53 protein has been implicated in several functions including the regulation of DNA repair and the associated cell cycle checkpoint mechanisms (40). p53 associates with XPB, XPD and p62 subunits of the TFIIH complex, which is involved in both nucleotide excision repair and transcription coupled repair mechanisms (7,25). The p53 protein also interacts with RPA, human Rad51 and BRCA1, which has been implicated in DNA repair (21,41,42). A recent study demonstrates that p53 is phosphorylated in vitro by the TFIIH-associated kinase (CDK7–cyclin H–p36 trimeric complex) enhancing its ability to bind sequence-specific p53-responsive elements (43). All of these interactions support a pivotal role of p53 in many cellular functions such as DNA repair, cell cycle checkpoint controls and/or apoptosis.

DNA repair is an important mechanism by which cells cope with DNA damage. In this study we examined repair of DNA damage induced by two carcinogens that trigger the nucleotide excision repair (NER) pathway: aflatoxin B1 and UVC light. UVC radiation induces pyrimidine dimers. Aflatoxin B1 is metabolized to aflatoxin-8,9-epoxide, the ultimate genotoxic metabolite that binds to DNA, predominantly at guanine residues, to form the trans-8,9-dihydro-(N⁷-guanyl)-9-hydroxy-aflatoxin B1 adduct (30). The incidence of carcinogen-induced mutations is dependent on the balance between the level of DNA damage and DNA repair capacity. Our results indicate that HBx expression was associated with inhibition of the overall DNA repair capacity in p53-proficient cells, which is in agreement with a previous study (22). Furthermore, we have found the same extent of DNA repair inhibition in the p53-deficient cell line Saos-2, which supports the idea that HBx affects the regulation of DNA repair through a p53-independent pathway. Transactivation of multiple cellular genes, another mechanism where HBx is involved, was also reported to be independent of the p53-inhibiting functions by HBx (44). The evidence supporting a role for p53 in DNA repair include: (i) the association of p53 with several DNA repair proteins (7,21,41,42); (ii) p53 can recognize and bind to both irradiated DNA and mismatch DNA (45,46); and (iii) disruption of wild-type p53 results in selective loss of global genomic nucleotide excision repair (47). However, there are some discrepancies because it was also reported that Li-Fraumeni cells exhibit defective global DNA repair but are normal for transcription coupled repair; p53^–/– mouse fibroblasts display normal rates of repair; and p53 does not influence DNA repair capacity in vitro (48–50). Our study does not rule out a p53-dependent mechanism because the assays used estimate the overall DNA repair capacity but not other DNA repair mechanisms such as transcription-coupled repair. The XPB and XPD NER proteins of the TFIIH complex are also involved in transcription-coupled repair (51), and there is evidence that p53 is involved in the regulation of this process (40). Further studies are required to understand the biological significance of these multiple interactions in relation to DNA repair and the associated cell cycle checkpoint mechanisms.

Whereas the mechanisms by which HBx interferes with DNA repair are unknown, studies by other groups have demonstrated a direct interaction of HBx with XAP-1/UVDDB, XPB and XPD proteins as well as binding of HBx to damaged DNA (22,51–53). Although the in vivo relevance of these interactions is still not known, they may account for the impaired DNA repair activity observed in our study. In addition, HBx has been reported to inhibit cell cycle checkpoint mechanisms required for DNA repair (24). However, differences in cell cycle checkpoints cannot fully explain our results. The DNA repair inhibition observed in the UDS assay was carried out on cells arrested at G₀/G₁ and where DNA semi-conservative synthesis was negligible, suggesting a cell cycle-independent interaction of HBx with NER pathway.

In summary, we report the first evidence that HBx-induced DNA repair inhibition occurs through a p53-independent regulatory pathway and suggests that inhibition of DNA repair mechanisms by HBV products may contribute to the observed synergistic interaction between chronic infection with HBV and exposure to liver carcinogens. Further studies are required to determine the proteins involved and the in vivo implications of these findings.

	Acknowledgments

 
We are grateful to Dr Robyn Schecter (McGill University) for carefully reading this manuscript. This work was supported by the Cancer Research Society and Medical Research Council. M.A.A.-J. is a recipient of a Senior Scholarship from the Fond de Recherches en Santé du Québec, Canada.

	Notes

 
³ To whom correspondence should be addressed Email: mdaj{at}musica.mcgill.ca

	References

Top Abstract Introduction Materials and methods Results Discussion References

 

1. Hoeijmakers,J.H.J., Egly,J.-M. and Vermeulen,W. (1996) TFHIIH: a key component in multiple DNA transactions. Curr. Opin. Genet. Dev., 6, 26–33.[Web of Science][Medline]
2. Liu,B., Nicolaides,N.C., Markowitz,S. et al. (1995) Mismatch repair gene defects in sporadic colorectal cancers with microsatellite instability. Nature Genet., 9, 48–55.[Web of Science][Medline]
3. Orth,K., Hung,J., Gazdar,A., Bowcock,A., Mathis,J.M. and Sambrook,J. (1994) Genetic instability in human ovarian cancer cell lines. Proc. Natl Acad. Sci. USA, 91, 9495–9499.[Abstract/Free Full Text]
4. Zhang,H., Tombline,G. and Weber,B.L. (1998) BRCA1, BRCA2 and DNA damage response: collision or collusion? Cell, 92, 433–436.[Web of Science][Medline]
5. Fong,K.M., Zimmerman,P.V. and Smith,P.J. (1995) Microsatellite instability and other molecular abnormalities in non-small cell lung cancer. Cancer Res., 55, 28–30.[Abstract/Free Full Text]
6. Griffin,S. (1996) DNA damage, DNA repair and disease. Curr. Biol., 6, 497–499.[Web of Science][Medline]
7. Leveillard,T., Andera,L., Bissonnette,N., Schaeffer,L., Bracco,L., Egly,J.-M. and Wasylyk,B. (1996) Functional interactions between p53 and the TFIIH complex are affected by tumour-associated mutations. EMBO J., 15, 1615–1624.[Web of Science][Medline]
8. Scheffner,M., Werness,B.A., Huibregtse,J.M., Levine,A.J. and Howley,P.M. (1990) The E6 oncoprotein encoded by human papilloma virus types 16 and 18 promotes the degradation of p53. Cell, 63, 1129–1136.[Web of Science][Medline]
9. Bargonetti,J., Reynisdorf,I., Friedman,P. and Prives,C. (1992) Site-specific binding of wild-type p53 to cellular DNA is inhibited by SV40 T antigen and mutant p53. Genes Dev., 6, 1886–1898.[Abstract/Free Full Text]
10. Wang,X.W., Forrester,K., Yeh,H., Feitelson,M.A., Gu,J.R. and Harris,C.C. (1994) Hepatitis B virus-x protein inhibits p53 sequence-specific DNA binding, transcriptional activity and association with transcription factor ERCC3. Proc. Natl Acad. Sci. USA, 91, 2230–2234.[Abstract/Free Full Text]
11. Wang,X.W., Yeh,H., Schaeffer,L. et al. (1995) p53 modulation of TFIIH-associated nucleotide excision repair activity. Nature Genet., 10, 188–195.[Web of Science][Medline]
12. Truant,R., Antunovic,J., Greenblatt,J., Prives,C. and Cromlish,J. (1995) Direct interaction of the hepatitis B virus HBx protein with p53 leads to inhibition by HBx of p53 response element-directed transactivation. J. Virol., 69, 1851–1859.[Abstract]
13. Aguilar,F., Perwez,H.S. and Cerutti,P. (1993) Aflatoxin B1 induces the transversion of GT in codon 249 of the p53 tumour suppressor gene in human hepatocytes. Proc. Natl Acad. Sci. USA, 90, 8586–8590.[Abstract/Free Full Text]
14. Cherpillod,P. and Amstad,P.A. (1995) Benzo[a]pyrene-induced mutagenesis of p53 hot-spot codons 248 and 249 in human hepatocytes. Mol. Carcinogen., 13, 15–20.[Web of Science][Medline]
15. Feitelson,M.A., Zhu,M., Duan,L.-X. and London,W.T. (1993) Hepatitis B-x antigen and p53 are associated in vitro and in liver tissues from patients with primary hepatocellular carcinoma. Oncogene, 8, 1109–1117.[Web of Science][Medline]
16. Sun,C.H.-A., Farzadegan,H., You,S.-L. et al. (1996) Mutual confounding and interactive effects between hepatitis C and hepatitis B infections in hepatocellular carcinogenesis: a population-based case-control study in Taiwan. Cancer Epidemiol. Biomarkers Prev., 5, 173–178.[Abstract/Free Full Text]
17. Slagle,B.L., Lee,T.-H., Medina,D., Finegold,M.J. and Butel,J.S. (1996) Increased sensitivity to the hepatocarcinogen diethylnitrosamine in transgenic mice carrying the hepatitis B virus-x gene. Mol. Carcinogen., 15, 261–269.[Web of Science][Medline]
18. Dandri,M., Schirmacher,P. and Rogler,C.E. (1996) Woodchuck hepatitis virus-x protein is present in chronically infected woodchuck liver and woodchuck hepatocellular carcinomas which are permissive for viral replication. J. Virol., 70, 5246–5254.[Abstract/Free Full Text]
19. Zhu,M., London,W.T., Duan,L.X. and Feitelson,M.A. (1993) The value of hepatitis B-x antigen as a prognostic marker in the development of hepatocellular carcinoma. Int. J. Cancer, 55, 571–576.[Web of Science][Medline]
20. Elmore,L.W., Hancock,A.R., Chang,S.F., Wang,X.W., Chang,S., Callahan,C.P., Geller,D.A., Will,H. and Harris,C.C. (1997) Hepatitis B virus-x protein and p53 tumor suppressor interactions in the modulation of apoptosis. Proc. Natl Acad. Sci. USA, 94, 14707–14712.[Abstract/Free Full Text]
21. Abramova,N.A., Russell,J., Botchan,M. and Li,R. (1997) Interaction between replication protein A and p53 is disrupted after UV damage in a DNA repair-dependent manner. Proc. Natl Acad. Sci. USA, 94, 7186–7191.[Abstract/Free Full Text]
22. Becker,S.A., Lee,T.H., Butel,J.S. and Slagle,B.L. (1998) Hepatitis B virus-x protein interferes with cellular DNA repair. J. Virol., 72, 266–272.[Abstract/Free Full Text]
23. Wang,X.W., Gibson,M.K., Vermeulen,W., Yeh,H., Forrester,K., Sturzbecher,H.-W., Hoeijmakers,J.H.J. and Harris,C.C. (1995) Abrogation of p53 induced apoptosis by the hepatitis B virus-x gene. Cancer Res., 55, 6012–6016.[Abstract/Free Full Text]
24. Benn,J. and Schneider,R.J. (1995) Hepatitis B virus HBx protein deregulates cell cycle checkpoint controls. Proc. Natl Acad. Sci. USA, 92, 11215–11219.[Abstract/Free Full Text]
25. Wang,X.W., Vermeulen,W., Coursen,J.D. et al. (1996) The XPB and XPD DNA helicases are components of the p53-mediated apoptosis pathway. Genes Dev., 10, 1219–1232.[Abstract/Free Full Text]
26. Kim,H., Lee,H. and Yun,Y. (1998) X-gene product of hepatitis B virus induces apoptosis in liver cells. J. Biol. Chem., 273, 381–385.[Abstract/Free Full Text]
27. Su,F. and Schneider,R.J. (1997) Hepatitis B virus HBx protein sensitizes cells to apoptotic killing by tumor necrosis factor alpha. Proc. Natl Acad. Sci. USA, 94, 8744–8749.[Abstract/Free Full Text]
28. Henkler,F., Waseem,N., Golding,M.H., Alison,M.R. and Koshy,R. (1995) Mutant p53 but not hepatitis B virus-x protein is present in hepatitis B virus-related human hepatocellular carcinoma. Cancer Res., 55, 6084–6091.[Abstract/Free Full Text]
29. Yen,L., Nie,Z.-R., You,X.-L., Richard,S., Langton-Webster,B.C. and Alaoui-Jamali,M.A. (1997) Regulation of cellular response to cisplatin-induced DNA damage and DNA repair in cells overexpressing p185 (ErbB-2) is dependent on the ras signaling pathway. Oncogene, 14, 1827–1835.[Web of Science][Medline]
30. Levy,D.D., Groopman,J.D., Lim,S.E., Seidman,M.M. and Kraemer,K.H. (1992) Sequence specificity of aflatoxin B1-induced mutations in a plasmid replicated in xeroderma pigmentosum and DNA repair proficient human cells. Cancer Res., 52, 5668–5673.[Abstract/Free Full Text]
31. Bresac,B., Kew,M., Wands,J. and Ozturk,M. (1991) Selective G to T mutations of p53 gene in hepatocellular carcinoma from southern Africa. Nature, 350, 429–431.[Medline]
32. Hsu,I.C., Tokiwa,T., Bennett,W., Metcalf,R.A., Welsh,J.A., Sun,T. and Harris,C.C. (1993) p53 gene mutation and integrated hepatitis B viral DNA sequences in human liver cancer cell lines. Carcinogenesis, 14, 987–992.[Abstract/Free Full Text]
33. Doria,M., Klein,N., Lucito,R. and Schneider,R.J. (1995) The hepatitis B virus HBx protein is a dual specificity cytoplasmic activator of ras and nuclear activator of transcription factors. EMBO J., 15, 101–111.
34. Klein,N.P. and Schneider,R.J. (1997) Activation of Src family kinases by hepatitis B virus HBx protein and coupled signaling to ras. Mol. Cell. Biol., 17, 6427–6436.[Abstract]
35. Maguire,H.F., Hoeffler,J.P. and Siddiqui,A. (1991) HBVx protein alters the DNA binding specificity of CREB and ATF-2 protein–protein interactions. Science, 252, 842–844.[Abstract/Free Full Text]
36. Natoli,G., Avantaggiati,M.L., Chirillo,P., Costanzo,A., Artini,M., Balsano,C. and Levrero,M. (1994) Induction of the DNA binding activity of c-jun/c-fos heterodimers by the hepatitis B virus transactivator pX. Mol. Cell. Biol., 14, 989–998.[Abstract/Free Full Text]
37. Cheong,J.H., Yi,M.K., Lin,Y. and Murakami,S. (1995) Human RPB5, a subunit shared by eukaryotic nuclear RNA polymerases, binds human hepatitis B virus-x protein and may play a role in x transactivation. EMBO J., 14, 143–150.[Web of Science][Medline]
38. Chirillo,P., Falco,M., FriPuri,P.L., Artini,M., Balsano,C., Levrero,M. and Natoli,G. (1996) Hepatitis B virus pX activates NF-kappa B-dependent transcription through a Raf-independent pathway. J. Virol., 70, 641–646.[Abstract]
39. Lin,Y., Nomura,T., Cheong,J., Dorjsuren,D., Iida,K. and Murakami,S. (1997) Hepatitis B virus-x protein is a transcriptional modulator that communicates with transcription factor IIB and the RNA polymerase II subunit 5. J. Biol. Chem., 272, 7132–7139.[Abstract/Free Full Text]
40. Ko,L.J. and Prives,C. (1996) p53: puzzle and paradigm. Genes Develop., 10, 1054–1072.[Free Full Text]
41. Buchhop,S., Gibson,M.K., Wang,X.W., Wagner,P., Sturzbecher,H.W. and Harris,C.C. (1997) Interaction of p53 with the human Rad51 protein. Nucleic Acid Res., 25, 3868–3874.[Abstract/Free Full Text]
42. Zhang,H., Somasundaram,K., Peng,Y., Tian,H., Bi,D., Weber,B.L. and El-Deiry,W.S. (1998) BRCA1 physically associates with p53 and stimulates its transcription activity. Oncogene, 16, 1713–1721.[Web of Science][Medline]
43. Ko,L.J., Shieh,S.Y., Chen,X., Jayaraman,L., Tamai,K., Taya,Y., Prives,C. and Pan,Z.Q. (1997) p53 is phosphorylated by CDK7–cyclin H in a p36MAT1-dependent manner. Mol. Cell Biol., 17, 7220–7229.[Abstract]
44. Lin,Y., Nomura,T., Yamashita,T., Dorjsuren,D., Tang,H. and Murakami,S. (1997) The transactivation and p53-interacting functions of hepatitis B virus-x protein are mutually interfering but distinct. Cancer Res., 57, 5137–5142.[Abstract/Free Full Text]
45. Lee,S., Elenbaas,B., Levine,A. and Griffith,J. (1995) p53 and its 14 kDa C-terminal domain recognize primary DNA damage in the form of insertion/deletion mismatches. Cell, 81, 1013–1020.[Web of Science][Medline]
46. Reed,M., Woelker,B., Wang,P., Wang,Y., Anderson,M.E. and Tegtmeyer,P. (1995) The C-terminal domain of p53 recognizes DNA damage by ionizing radiation. Proc. Natl Acad. Sci. USA, 92, 9455–9459.[Abstract/Free Full Text]
47. Ford,J.M. and Hanawalt,P.C. (1997) Expression of wild-type p53 is required for efficient global genomic nucleotide excision repair in UV-irradiated human fibroblasts. J. Biol. Chem., 272, 28073–28080.[Abstract/Free Full Text]
48. Ford,J.M. and Hanawalt,P.C. (1995) Li-Fraumeni syndrome fibroblasts homozygous for p53 mutations are deficient in global DNA repair but exhibit normal transcription-coupled repair and enhanced UV resistance. Proc. Natl Acad. Sci. USA, 92, 8876–8880.[Abstract/Free Full Text]
49. Sands,A.T., Suraokar,M.B., Sanchez,A., Marth,J.E., Donehower,L.A. and Bradley,A. (1995) p53 deficiency does not affect the accumulation of point mutations in a transgene target. Proc. Natl Acad. Sci. USA, 92, 8517–8521.[Abstract/Free Full Text]
50. Sancar,A. (1995) Excision repair in mammalian cells. J. Biol. Chem., 270, 15915–15918.[Free Full Text]
51. Qadri,I., Conaway,J.W., Conaway,R.C., Schaack,J. and Siddiqui,A. (1996) Hepatitis B virus transactivator protein, HBx, associates with the components of TFIIH and stimulates the DNA helicase activity of TFIIH. Proc. Natl Acad. Sci. USA, 93, 10578–10583.[Abstract/Free Full Text]
52. Capovilla,A., Carmona,S. and Arbuthnot,P. (1997) Hepatitis B virus x-protein binds damaged DNA and sensitizes liver cells to ultraviolet irradiation. Biochem. Biophys. Res. Commun., 232, 255–260.[Web of Science][Medline]
53. Qadri,I., Ferrari,M.E. and Siddiqui,A. (1996) The hepatitis B virus transactivator protein, HBx, interacts with single-stranded DNA (ssDNA). Biochemical characterizations of the HBx–ssDNA interactions. J. Biol. Chem., 271, 15443–15450.[Abstract/Free Full Text]

Received May 29, 1998; revised October 20, 1998; accepted November 4, 1998.

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