Transcriptional Regulation of the TFIIH Transcription Repair Components XPB and XPD by the Hepatitis B Virus x Protein in Liver Cells and Transgenic Liver Tissue*

Iris Jaitovich-GroismanDagger , Naciba BenlimameDagger , Betty L. Slagle§, Maite Hernandez PerezDagger , Lesley AlpertDagger , Daniel J. SongDagger , Nasser Fotouhi-ArdakaniDagger , Jacques GalipeauDagger , and Moulay A. Alaoui-JamaliDagger

From the Dagger  Lady Davis Institute of the Sir Mortimer B. Davis Jewish General Hospital, Departments of Medicine, Pharmacology and Therapeutics, Pathology, and Oncology, Faculty of Medicine, McGill University, Montreal H3T 1E2, Canada, and the § Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas 77030

Received for publication, December 1, 2000, and in revised form, January 23, 2001




    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Human hepatitis B virus is a risk factor for the development of hepatocellular carcinoma. The hepatitis B virus x protein (HBx) has been shown to inactivate the p53 tumor suppressor protein and impair DNA repair, cell cycle, and apoptosis mechanisms. Herein we report that HBx represses two components of the transcription-repair factor TFIIH, XPB (p89), and XPD (p80), both in p53-proficient and p53-deficient liver cells. This inhibition is observed while HBx maintains its transactivation function. Expression of HBx in liver cells results in down-regulation of endogenous XPB and XPD mRNAs and proteins; this inhibition is not observed with other TFIIH subunits, XPA or PCNA. In liver tissue from HBx transgenics, XPB and XPD proteins are down-regulated in comparison to matched normal liver tissue. HBx has been shown to interact with Sp1 transcription factor and affects its DNA binding activity. Sp1 is essential for the basal promoter activity of XPB in liver cells and Drosophila SL2 cells. In the Sp1-deficient SL2 cells, HBx-induced XPB and XPD inhibition is Sp1-dependent. In summary, our results provide evidence that HBx represses the expression of key TFIIH proteins at least in part through Sp1 elements; this repression may impair TFIIH function in DNA repair mechanisms.




    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Chronic infection with human hepatitis B virus (HBV)1 is a risk factor for the development of hepatocellular carcinoma (reviewed in Ref. 1). However, there is substantial evidence that infection with HBV is not always sufficient for cell transformation to occur and that there are other cofactors that allow for the progression of the HBV-infected cells to hepatocellular carcinoma (HCC). This progression evolves through a multistep and multifactorial process, demonstrated in several hepatocarcinogenesis animal models (2-4). This is consistent with two main features of human HCC: its long latency and its large regional variation. More important is the evidence of synergistic interaction between HBV and exposure to environmental factors in the high incidence of HCC in humans and transgenic mice (3-8). Therefore, understanding molecular mechanisms by which HBV affects cellular susceptibility to hepatocarcinogenesis is essential to identify preventive strategies for liver cancer.

HBV is a double-stranded DNA virus that contains 4 open reading frames, three of which code for surface and core proteins and a DNA polymerase. The fourth open reading frame, which is highly conserved among all mammal-specific members of the hepadnaviridae family, encodes a small 16.5-kDa polypeptide of 154 amino acids referred to as X protein (HBx). It is localized in the cytoplasm and nucleus, and can therefore interact with both cell signal transduction and transcription machineries. Extensive studies have demonstrated that HBx acts as a promiscuous transactivator of cellular and viral promoters of several genes including human interferon beta , c-Myc, IGFII, simian virus 40, and the Rous sarcoma virus LTR (9-13). The cellular function of HBx has been attributed to its ability to transactivate transcription factors such as NF-kappa B, AP1, AP2, ATF2/cAMP-response element-binding protein, basal transcription factor TATA box-binding, RNA polymerase subunit RPB5, components of TFIIB and TFIIH transcription complexes, as well as a physical association with p53 (reviewed in Ref. 1). Although the biological significance of these interactions is still not fully established, the interaction of HBx with p53, which results in inactivation of certain p53 function, is reported to be a major player for HBV-induced liver carcinogenesis (14-17).

HBx has been found to enhance cell susceptibility to carcinogen-induced DNA damage both in cell lines and transgenic mice (3, 4, 18-21). Several mechanisms have been proposed to account for this synergistic interaction including HBx interaction with a DNA-damage recognition protein (18), binding to damaged DNA (22, 23), and inhibition of the G1-S checkpoint (24). HBx has been shown to inhibit nucleotide excision repair (NER), a major mechanism by which cells repair DNA bulky adducts induced by carcinogens such as aflatoxins and UV radiation (25-27). Interestingly, altered NER activity has been observed in liver cells from HBx-transgenic mice (8). The observation that apoptotic signals are impaired in the presence of HBx (14, 28, 29) also suggests that HBx may alter the balance between DNA repair and apoptosis, two important cellular defense mechanisms.

NER is a multistep process during which damaged bases are recognized and removed in the form of an oligomer. It requires several proteins that act in a sequential manner (reviewed in Ref. 30). The recognition step involves XPC-hHR23B, TFIIH, XPA, RPA, and ERCC1·XPF complex. The incision/excision steps involve XPG, ERCC1/XPF, and possibly XPC. The final step involves synthesis of a new strand by several replication proteins including PCNA, replication factor-C, polymerases, and a DNA ligase. NER can perform repair in both nontranscribed genes (global NER) as well as actively transcribed genes (transcription-coupled NER). TFIIH is a multisubunit protein complex essential for both global NER and transcription-coupled NER, as well as the initiation of RNA polymerase II transcription, cell cycle regulation, and possibly apoptosis (reviewed in Refs. 30 and 31). During NER, TFIIH is required for melting the DNA double helix as well as DNA damage recognition and recruitment of other DNA repair proteins. Nine TFIIH subunits have been characterized: p34, p44, p52, p62, and XPD (p80, also referred to as ERCC2), XPB (p89, also referred to as ERCC3), cyclin-dependent kinase 7 (cdk7), cyclin H, and MAT1. This multiprotein complex possesses several enzymatic activities. Cdk7, cyclin H, and MAT1 form a ternary complex with a Cdk-activating kinase activity (reviewed in Refs. 32 and 33). The p34 and p44 subunits contain putative DNA-binding sites and have been implicated in DNA repair mechanisms while the biological function of p52 and p62 proteins in DNA repair is not fully established (reviewed in Ref. 30). During NER, XPB and XPD act as 3'-5' and 5'-3' ATP-dependent helicases, respectively. This bidirectional helicase activity is believed to unwind the DNA double helix around the damage before incision takes place during the process of NER (34). An intriguing finding is the physical and functional interaction of p53 with both XPB and XPD (35). The association of HBx with p53 has been shown to alter p53 interaction with XPB (17). We report herein transcriptional repression of XPB and XPD genes by HBx in vitro and in HBx-transgenic liver tissue. This transcriptional repression by HBx occurs in both p53-proficient and p53-deficient cells, and is distinct from HBx transactivation function. We also provide data indicating that HBx regulation of XPB and XPD is Sp1-dependent.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Plasmid Constructs-- pXPB-CAT and pXPD-CAT vectors were constructed by ligating XPB or XPD promoters between the XbaI and the HindIII site of the pCAT basic vector (Promega, Madison, WI). XPB and XPD promoters were amplified by PCR using the following primers: XPB forward primer: 5'-ACACACATTTATTGCCCACATGG-3'; XPB reverse primer: 5'-TTTTGCCCATGGCAGCTACAGCAGC-3'; XPD forward primer: 5'-GCTATCTTGCTCAAGCTGATCT-3'; XPD reverse primer: 5'-GTGACATCCTCGAGGGCTCGC-3'. The amplifications of XPD and XPB promoters were carried out as follows: 33 pmol of both forward and reverse primers were mixed with 1 × Taq DNA polymerase buffer (Pharmacia Biotech, Quebec), 200 mM dNTP, 10 µg of genomic DNA from HeLa cells, 2.5 units/ml of Taq DNA polymerase (Amersham Pharmacia Biotech) in a final volume of 100 µl. The PCR reaction was set up for 2 min at 94 °C, followed by 35 cycles of 30 s at 94 °C, 1 min at 58 °C, 45 s at 72 °C, and a final cycle of 7 min at 72 °C. The constructs were confirmed by DNA sequence analysis using Sequenase version 2.0 (U. S. Biochemical Corp., Cleveland, OH). The analyzed sequences corresponded to a fragment of 346 base pairs (positions -346 to +1) for XPB (based on sequence GeneBankTM M31899) and a fragment of 309 base pairs (positions -300 to +9) for XPD (based on sequence GeneBankTM X52470).

The HBx expression vector, pCMV-HBx, consisted of the HBx gene from the HBV ayw subtype (nucleotides 1241 to 1991) ligated into the HindIII site of the pRc-CMV vector (Invitrogen, La Jolla, CA) (16). The pRc-CMV plasmid was used as a negative control and referred to as pCMV. pAP2 is a bicistronic, nonsplicing retroviral expression vector which incorporates a multiple cloning site allowing insertion of sequences linked by an internal ribosomal entry site to the enhanced green fluorescent protein reporter (36). The HBx gene from the HBV subtype ayw (nucleotides 1241 to 1991) (16) was subcloned into the XhoI-BamHI sites of the pAP2 vector and referred to as pAP2-HBx. The pAP2 vector was used as a negative control. The pJ418E6 and the pJ418E6Delta F constructs were kindly provided by Dr. Greg Matlashewski (McGill University) and consisted of the HPV-18 E6 gene and the HPV-18 E6Delta F mutated gene under control of the CMV promoter, respectively. Both genes were cloned into plasmid pJ4Omega . The chloramphenicol reporter constructs 5'tB containing NF-kappa B responsive elements, and pERE3CAT containing the AP1 responsive elements were kindly provided by Dr. John Hiscott from McGill University and Dr. Sylvie Mader from Université de Montreal, respectively. The Drosophila Sp1 expression vector pPac-Sp1 was kindly provided by Dr. Robert Tjian (University of California, Berkeley, CA).

Cell Lines-- HepG2 is a hepatoblastoma cell line expressing wild type p53. HepG2 cells were grown in alpha -MEM supplemented with 10% fetal bovine serum (FBS) and gentamicin (50 µg/ml). CCL13 cells are human liver epithelial cells and contain wild type p53. These cells were maintained in DMEM, 10% FBS, and 50 µg/ml gentamicin. Saos-2 is a human osteosarcoma cell line which lacks endogenous p53. These cells were grown in McCoy's 5A medium supplemented with 15% FBS and penicillin-streptomycin (50 units/ml and 5 mg/ml, respectively). All cells were maintained at 37 °C in an atmosphere of 5% CO2. The p53-deficient hepatoma cell line, Hep3B, was grown in alpha -MEM supplemented with 10% FBS and gentamicin (50 µg/ml). The 293GPG packaging cell line used to produce HBx was kept in DMEM medium containing 10% FBS, 1.0 µg/ml tetracycline, 2.0 µg/ml puromycine, and 0.3 mg/ml geneticin. Drosophila SL2 cells were cultured in Grace's insect cell culture medium (Life Technologies) at 25 °C.

Retroviral Production of HBx-- The 293GPG cells were plated at a concentration of 4 × 106 cells per 60-mm dish the night before transfection in the appropriate medium. Vesicular stomatitus virus-G pseudotyped retroparticles AP2 and AP2-HBx were collected as described (20) and utilized to transduce target cells. Five µg of linear DNA containing either pAP2 or pAP2-HBx were co-transfected in a ratio 50:1 with the Zeocin-resistant plasmid pJ6Omega 2bleo (36) using LipofectAMINE according to the recommendations of the manufacturer. Selection for cells stably transfected with pAP2 or pAP2-X was performed for 2-3 weeks in 293 cell culture medium containing 100 µg/ml Zeocin (Invitrogen). Fluorescein-activated cell sorter analysis was performed to determine the percentage of producer cells that expressed the GFP reporter protein. Cells expressing GFP were selected using a fluorescence-activated cell sorter, STAR PLUS TURBO (530-30; FL-1), and immediately frozen or kept in culture. When cells reached 60% confluence, the selection medium was removed and replaced with DMEM, 10% FBS, penicillin-streptomycin (50 units/ml and 50 µg/ml, respectively). Three days after tetracycline withdrawal, the supernatant was collected, filtered, and frozen daily for 6 to 7 days. Supernatants were thawed, pooled, and retroviral particles AP2 and AP2-HBx were concentrated 20 to 30 times (v/v) by ultracentrifugation as described (37).

Transfection and Chloramphenicol Acetyltransferase (CAT) Assay-- Cells were seeded at 2.7 × 105 cells per 35-mm diameter dish using 6-well plates and grown overnight in the appropriate media. The following day, the cells were transiently transfected, using LipofectAMINE (Life Technologies, Inc., Ontario), with pXPB-CAT or pXPD-CAT together with pCMV-HBx or pCMV, pJ418E6 or pJ418E6Delta F. As controls 5'tB or pERE3CAT were co-transfected with pCMV-HBx or pCMV. LipofectAMINE was used at a concentration of 3 µg/1 µg of DNA. pCMV was added to equalize the amount of DNA transfected in each well whenever necessary. Cells were incubated with DNA-LipofectAMINE complexes for 6 h, after which cells were washed gently and cultured in fresh serum-supplemented medium. Cells were harvested 48 h after transfection and protein extracts were used to determine CAT activity as described (38). For retroviral infection, 8 × 105 cells/35-mm diameter dish were plated and grown overnight in appropriate medium. The following day, cells were transiently transfected with pXPB-CAT, pXPD-CAT, 5'kB, or pERE3CAT reporter plasmids as described above. After 6 h incubation with LipofectAMINE-DNA complexes, the medium was removed and cells were transduced with ×20 concentrated retroviral supernatant obtained from AP2 or AP2-HBx 293GPG producer cells and 6 µg/ml Polybrene (Sigma). Samples were collected 24 h later and CAT assays were performed as described above. For Drosophila SL2 cells, transfection was carried out by calcium phosphate precipitation (Promega). Briefly, 1 × 106 cells were plated 24 h prior transfection and complexes were incubated 4 to 6 h with the cells. The pPac plasmid was used as a negative control for Sp1 and pPac or pCMV were added to equalize the amount of DNA transfected in each well whenever necessary. Cell extracts were harvested 48 h after transfection. The quantification of the reaction products in the CAT assay was performed using the Bio-Rad Gelscan PhosphorImager and the Molecular Analyst (Bio-Rad) software program. The percentage of chloramphenicol conversion to its acetylated metabolites was determined for each sample in at least three independent experiments in duplicate. Statistical comparison was performed according to Student's test.

Semi-quantitation of CAT mRNA by Polymerase Chain Reaction-- CAT mRNA expression was quantitated by method of PCR based on a previously described protocol (39). Total RNA was isolated 48 h post-transfection using the RNeasy Total RNA Kit (Qiagen Inc., Chastworth, CA). The RNA was treated with RNase-free DNase I and further cleaned (RNeasy Total RNA Kit). Each RNA sample (1 µg) was mixed with 1× reverse transcription buffer (Life Technologies, Inc.) and 50 pmol of both 3'-CAT primer and beta -actin primer. A solution containing 0.025 mM dNTP, 0.5 mM dithiothreitol, 1 × reverse transcriptase buffer, and 10 units/ml Moloney murine leukemia virus reverse transcriptase (Life Technologies, Inc.) was added and the reaction was allowed to proceed for 1 h at 37 °C. Each sample was mixed together with 5 mM dCTP, dGTP, and dTTP and 0.5 mM dATP, 1 × Taq polymerase buffer, 50 pmol of forward CAT or beta -actin primer, 40 pmol of reverse primer, 5 µCi of 35S-alpha -dATP (Amersham Pharmacia Biotech) and 40 units/ml Taq polymerase in a final volume of 50 µl. PCR amplification of CAT cDNA was performed for 30 cycles as described (39). Amplification of beta -actin was performed as follows: one cycle at 94 °C for 3 min, 30 cycles at 94 °C for 1 min, 60 °C for 1 min, 72 °C for 1 min with a final extension at 72 °C for 7 min. The appropriate cDNA volumes were 0.16 µl and 5 × 10-4 µl, for CAT and beta -actin, respectively. These dilutions were determined to be in the linear range of each standard curve. Samples were run on a 10% acrylamide gel, dried, and exposed to x-ray film. The quantification of the bands was performed using the Bio-Rad Gelscan PhosphorImager and the Molecular Analyst (Bio-Rad) software program.

RT-PCR for Endogenous Gene Expression-- Total RNA was isolated using the RNeasy Total RNA Kit (Qiagen Inc., Chastworth, CA). The RNA was treated with RNase-free DNase I and further cleaned (RNeasy Total RNA Kit). Each RNA sample (5 µg) was mixed with 100 pmol of random hexamer primer. A mixture containing 1 mM dNTP, 20 µl of ribonuclease inhibitor, 1 × reverse transcriptase buffer, and 40 µl of Moloney murine leukemia virus reverse transcriptase (MBI Fermentas) were added and the reaction was allowed to proceed for 1 h at 37 °C. The cDNA mixtures were amplified by PCR. The appropriate cDNA volumes used were 0.1 and 1 × 10-3 µl for each NER gene analyzed and beta -actin, respectively. These dilutions were determined to be in the linear range of each standard curve. Each sample was mixed with 0.25 mM dATP, dGTP, dTTP and 0.025 mM dCTP, 1 × Taq polymerase buffer, 50 pmol of both forward and reverse NER gene specific primers or beta -actin primers, 5 µCi of [alpha -32P]dCTP (Amersham Pharmacia Biotech), and 2.5 units of Taq polymerase in a final volume of 50 µl. PCR amplification of NER genes was performed for 30 cycles as follows: 94 °C for 1 min, 60 °C for 30 s, and 72 °C for 1 min with a final extension at 72 °C for 7 min. Amplification of beta -actin was performed for 25 cycles. Samples were run on a 5% acrylamide gel and exposed to x-ray film. The quantification of the bands was performed using the Bio-Rad Gelscan PhosphorImager and the Molecular Analyst (Bio-Rad) software program. The primer sequences of NER genes analyzed are indicated in Table I. The cDNA of human beta -actin was amplified as a control gene using the specific primers 5'- TCCTGTGGCATCCACGAAACT-3' and 5'-GAAGCATTTGCGGTGGACGAT-3'. To confirm for the presence of HBx, a sense (5'-GGCTGCTAGGCTGTGCTGCC-3') and antisense (5'-GTTCCGGTGGGCGTTCACGG-3') oligonucleotide primers were used.


                              
View this table:
[in this window]
[in a new window]
 
Table I
Primer sequences used for NER genes

Western Blot Analysis-- Total cell extracts from cells transduced with AP2 and AP2-HBx retroviral particles were used to examine the expression of the endogenous TFIIH proteins by Western blot analysis. For Sp1 expression in Drosophila SL2 cells, cells were transfected with pPac (negative control) or pPac-Sp1 using calcium phosphate complexes. Cell extracts were obtained following resuspension in lysis buffer (10 mM Tris-HCl, pH 8.0, 60 mM KCL, 1 mM EDTA, 1 mM dithiothreitol, 0.5% Nonidet P-40, 0.5 mg/ml leupeptin, 0.5 mg/ml pepsin, 0.5 mg/ml aprotinin per 4 × 107 cells). Polyacrylamide gel electrophoresis was performed using a 4% polyacrylamide stacking gel layered over a 10% resolving gel for XPB and XPD. 30 µg of protein extract were run at 50 V for 16 h and transferred onto nitrocellulose membrane (Costar, Cambridge, MA). The membranes were blocked with 10% low fat milk in PBS and incubated overnight with the corresponding antibody, mouse monoclonal anti-Sp1 (clone 1C6 from Santa Cruz Biotechnology, Inc.), mouse monoclonal anti-XPB, or mouse monoclonal anti-XPD (kindly provided by Dr. J. M. Egly, Université Louis Pasteur, Strasbourg, France). Enhanced chemiluminescence (ECL) detection was performed using the corresponding detection reagents (Amersham Pharmacia Biotech). Blots were subsequently stripped in 100 mM 2-mercaptoethanol, 2% SDS, 62.5 mM Tris-HCl, pH 6.7, 50 °C for 30 min, then immunoblotted with a monoclonal anti-GAPDH antibody (clone 6C5, Cedarlane Laboratories, Ontario, Canada).

Transgenic Mice and Immunohistochemistry-- Transgenic mice harboring the X gene (nucleotides 1376-1840) under the control of the human alpha 1-antitrypsin regulatory gene (ATX mice) were developed as described earlier (40). Male ATX mice (ICRxB6C3) were bred with nontransgenic females (purchased from Jackson Laboratory). Both transgenic and nontransgenic littermates, males and females from the F1 generation, were sacrificed at 3-7 weeks of age and their livers were removed and numbered blindly. A portion of liver tissues from founder animals and progeny was fixed in 10% buffered formalin and embedded in paraffin wax for immunohistochemical study. Immunoperoxidase staining for XPB, XPD, and XPA was performed by the avidin-biotin complex method (Vector Laboratories, Burlingame, CA). Six-µm sections of liver were deparafinized in toluene and rehydrated through graded alcohols to water. Endogenous peroxidase activity was quenched by incubation in 1.5% hydrogen peroxide for 30 min at room temperature. Sections were immersed in 10 mM sodium citrate buffer, pH 6.0, and subjected to heat-induced antigen retrieval. Endogenous biotin was blocked by incubation for 10 min with the Avidin/Biotin blocking kit (Zymed Laboratories Inc., San Francisco, CA). To block nonspecific protein binding, sections were then treated with 5% normal goat serum or normal horse serum (Vector Laboratories) in PBS, pH 7.4, for 60 min at room temperature. Sections were incubated overnight at 4 °C with primary antibodies at appropriate dilutions (ranging from ×100 to 250). The antibodies that were used consisted of rabbit polyclonal antibody against XPB or XPA, and goat polyclonal antibody against XPD (Santa-Cruz Biotechnology, Inc.). All dilutions were made in PBS with 5% normal serum. After rinsing with PBS, sections were incubated with biotinylated antibodies (goat anti-rabbit or horse anti-goat) (Vector laboratories) containing 2% normal serum for 30 min at room temperature. The sections were then incubated for 30 min with avidin-biotin-horseradish peroxidase complex (Vector Laboratories) in PBS, pH 7.4, followed by final color development with hydrogen peroxide-activated 0.1% diaminobenzidine tetrahydrochloride in 0.1 M Tris buffer, pH 7.2, for 2-5 min. A negative control was performed by the omission of the primary antibody. Sections were then lightly counterstained with Harris' hematoxylin, dehydrated in graded alcohols, cleared in toluene, and coverslipped. Sections were analyzed by conventional light microscopy and photographed using Kodak color slides.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Effect of HBx on XPB and XPD Promoters-- To examine the effect of HBx on the transcriptional regulation of XPB and XPD, we constructed plasmids in which the expression of the CAT coding sequence was driven by either the XPB (pXPB-CAT) or XPD (pXPD-CAT) promoter. HepG2 cells were co-transfected with either pXPB-CAT or pXPD-CAT and the HBx expression plasmid, pCMV-HBx also referred to as HBx. The empty expression vector pCMV was used as a negative control. Expression of HBx resulted in ~65 and 37% reduction in pXPB-CAT or pXPD-CAT activity, respectively, as compared with control cells transfected with pCMV (Fig. 1, A and B). Under similar conditions, expression of HBx in CCL13 liver cells induced ~50% inhibition of pXPB-CAT or pXPD-CAT (Fig. 1, A and B).



View larger version (29K):
[in this window]
[in a new window]
 
Fig. 1.   Effect of HBx on XPB and XPD promoter activities in p53-proficient cells. Subconfluent HepG2 and CCL13 cells were transfected with pCMV or pCMV-HBx and pXPB-CAT (A) or pXPD-CAT (B). Cell extracts were used to determine CAT activity as described under "Experimental Procedures." The ratio of chloramphenicol acetylation was expressed as % of CAT activity in HBx-transfected compared with pCMV-transfected cells. Each bar corresponds to the average (±S.E.) of at least five independent experiments each in duplicate. Inserted are representative autoradiograms of duplicates for each condition.

We further investigated whether decreased CAT activity occurs at the transcriptional level. Cells were harvested 48 h after transfection following similar conditions used to determine CAT activity in co-transfection experiments. Total RNA was isolated and used to examine CAT mRNA expression by semi-quantitative PCR. The concentrations of CAT and actin cDNA used were determined to be in the linear range based on standard curves (data not shown). As compared with cells transfected with the control pCMV plasmid, expression of pCMV-HBx resulted in inhibition of CAT mRNA expression in both HepG2 and CCL13 cell lines co-transfected with either pXPD-CAT or pXPB-CAT plasmid (Fig. 2, A and B). In the presence of HBx, ~70 and 85% reduction of CAT mRNA was observed from pXPB-CAT in HepG2 and CCL13 cells, respectively. CAT mRNA from pXPD-CAT was reduced by ~80 and 40% in HepG2 and CCL13 cells, respectively (Fig. 2, A and B).



View larger version (21K):
[in this window]
[in a new window]
 
Fig. 2.   Effect of HBx on CAT mRNA expression of pXPB-CAT and pXPD-CAT in p53 proficient cells. Subconfluent HepG2 and CCL13 cells were co-transfected with pCMV or pCMV-HBx and XPB-CAT or XPD-CAT. Total RNA was isolated 48 h post-transfection. CAT mRNA and the beta -actin mRNA were reverse transcribed followed by PCR amplification of both cDNAs. beta -Actin cDNA was used as an internal control. After overnight exposure, densitometric analysis was performed using a LKB Ultrascan Laser Densitometer. A shows a representative RT-PCR experiment. B is a summary of mRNA CAT/actin (mean ± S.E.), from two independent experiments, expressed as % of corresponding control cells transfected with pCVM plasmid.

To determine whether XPB and XPD promoter inhibition by HBx is p53-dependent, we examined CAT activity in the p53-deficient osteosarcoma cell line Saos-2 and hepatoblastoma cell line Hep3B. Compared with control cells transfected with pCMV, expression of HBx in Saos-2 cells resulted in ~65 and 45% inhibition for pXPB-CAT or pXPD-CAT activity, respectively, while expression of HBx in Hep3B cells resulted in ~50 and 35% inhibition with XPB-CAT and XPD-CAT, respectively (Fig. 3A). Co-transfection of pXPB-CAT or pXPD-CAT with the CMV driven expression vector for the E6 protein of the human papilloma virus, pJ418E6, in HepG2 cells did not impair CAT activity when compared with cells transfected with pJ418E6Delta F (Fig. 3B). The pJ418E6Delta F expression vector, used as a negative control, expresses a mutated form of HPV-18 E6 protein which does not promote p53 degradation due to a deletion in amino acids 113 through 117 (41).



View larger version (23K):
[in this window]
[in a new window]
 
Fig. 3.   p53-independent regulation of XPB and XPD promoter activities by HBx. A, the p53-deficient cell lines Saos-2 and Hep3B cells were co-transfected with the expression vectors pCMV or pCMV-HBx and XPB-CAT or XPD-CAT. Cell extracts were used to determine CAT activity as described under "Experimental Procedures." The ratio of chloramphenicol acetylation was expressed as % of CAT activity in HBx-transfected compared with pCMV-transfected cells. B, the p53-proficient cell line HepG2 was co-transfected with the HPV-18 E6 expression vector pJ418E6 or a control vector, pJ418E6Delta F, that has a mutated E6 gene. The ratio of chloramphenicol acetylation was expressed as % of CAT activity in pJ418E6-transfected cells compared with pJ418E6Delta F-transfected cells. Each bar corresponds to the average (±S.E.) of four independent experiments.

HBx is a promiscuous transactivator affecting a large number of cellular and viral genes. We examined whether HBx-induced transactivation is maintained while XPB/XPD transcription is repressed by HBx. Under our experimental conditions, HBx-induced inhibition of XPB and XPD promoters was observed under conditions that allow HBx transactivation function on Ap1 and NF-kappa B responsive elements (Fig. 4).



View larger version (18K):
[in this window]
[in a new window]
 
Fig. 4.   Effect of HBx on transactivation of AP1 and Nf-kB responsive elements. HepG2 cells were co-transfected with pCMV-HBx or pCMV and reporter vectors containing AP1 or Nf-kB responsive elements. CAT activity was determined as described under "Experimental Procedures" and expressed as % of control cells transfected with pCMV. Each bar corresponds to the average (±S.E.) of four independent experiments.

Effect of HBx on Endogenous mRNA and Protein Expression Levels of TFIIH Factors-- To examine the effect of HBx on the expression of endogenous XPB and XPD mRNAs and proteins, we used a retroviral system to express HBx in a polyclonal population. Such an experiment was not possible using the LipofectAMINE transfection assay because of the very low transfection efficiency in liver cells (<5-8%). We reported previously that the HBx retroviral vector used in this study is efficient in expressing HBx (19, 20). We confirmed the presence of functional HBx by its ability to transactivate AP1 and NF-kappa B elements. Although our control retroviral vectors infected equivalent numbers of cells (Fig. 5A), the transactivation effect was not observed in cells transduced with control viral particles (empty AP2) (Fig. 5B). As indicated in Fig. 6, expression of HBx induced a significant decrease in the endogenous level of XPB, and to a lesser extent XPD mRNA expression. No significant effect of HBx was observed on p34, p44, p52, p62, XPA, and PCNA. Inhibition of XPB and XPD mRNA by HBx correlated with a decrease in the level of endogenous proteins as revealed by Western blot analysis (Fig. 7).



View larger version (25K):
[in this window]
[in a new window]
 
Fig. 5.   Expression of HBx in polyclonal cell population using a retroviral expression system. A is a representative flow cytometry analysis of HepG2 cells transduced with bicistronic retroviral particles expressing GFP alone (AP2) or GFP and the HBx coding sequence (AP2-HBx). Nontransduced cells were used as a control. B shows the transactivation function of HBx expressed by the retroviral bicistronic system. CAT assays were performed on HepG2 cells transduced with AP2 or AP2-HBx retroviral particles and transfected with the reporter plasmids containing AP1 and Nf-kB responsive elements driving CAT expression. CAT activity was determined as described under "Experimental Procedures." Bars correspond to the average (±S.E.) of two independent experiments.



View larger version (28K):
[in this window]
[in a new window]
 
Fig. 6.   Effect of HBx on the expression of endogenous mRNA of TFIIH subunits, XPA, and PCNA. HepG2 cells were transduced with AP2 or AP2-HBx viral particles as described under "Experimental Procedures." Total RNA was isolated and XPB, XPD, p34, p44, p52, p62, XPA, PCNA, and beta -actin mRNA were reverse transcribed followed by PCR amplification of the cDNAs. After overnight exposure, densitometric analysis was performed using a LKB Ultrascan Laser Densitometer. The figure shows the average ratio (±S.E.) of the expression of each tested mRNA to actin from three independent experiments. *, statistically significant from AP2 control cells using Student's test (p < 0.001).



View larger version (37K):
[in this window]
[in a new window]
 
Fig. 7.   Effect of HBx on endogenous XPB and XPD protein expression. The expression of XPB and XPD proteins in HepG2 cells transduced with retroviral particles producing AP2 (control) or AP2-HBx was determined by Western blot analysis using XPB and XPD monoclonal antibodies. The membranes were stripped and probed with GAPDH monoclonal antibody. A representative autoradiogram is shown on the top of the figure. The bands were scanned (LKB Ultrascan Laser Densitometer) and the ratio of the density XPB/GAPDH and XPD/GAPDH for each lane was determined. The results correspond to 3 independent experiments (±S.E.).

Expression of TFIIH in HBx Transgenic Mice-- To investigate the in vivo relevance of XPB and XPD down-regulation by HBx, we examined the expression of these proteins in HBx transgenic liver tissue using the ATX-transgenic mice generated by Lee et al. (40). The expression of HBx in the livers of these ATX mice has been reproducibly confirmed at all ages (6, 8). The transgenic mice used in our study were of the F1 generation and were sacrificed at 3-7 weeks. The presence of HBx mRNA was confirmed by RT-PCR analysis (data not shown). All the mice appeared normal at the time of sacrifice and we did not notice any gross abnormalities in their livers by histopathology. Paraffin sections from liver tissues from HBx transgenics and their matched controls (5 mice each) were used to examine the expression of XPB and XPD proteins by histochemical analysis. XPA was included as an additional control. All the proteins tested were found to be homogenously distributed throughout liver tissue and were specifically localized to the nucleus of hepatocytes; this is in agreement with the nuclear localization of these DNA repair proteins (Fig. 8, E-G). As shown in Fig. 8, a clear inhibition was observed in the expression of XPB and XPD between control (left) and HBx transgenic (right) liver tissues. Unlike XPB and XPD, the expression of XPA (Fig. 8) was not affected in HBx-transgenic mice.



View larger version (62K):
[in this window]
[in a new window]
 
Fig. 8.   Expression of XPB and XPD in control and HBx-transgenic mice liver. Liver tissue from control transgenic and HBx-transgenic mice were fixed, embedded in paraffin, and stained with hematoxylin and eosin (H and E) or immunolabeled for XPB and XPD. The nuclei were lightly counterstained with Harris' hematoxylin. As it is shown for the control transgenic mouse, the immunoreactivity for XPB and XPD is very strong and is mainly localized in the nucleus of the hepatocytes, whereas very low immunoreactivity for XPB and XPD was detected in HBx-transgenic mouse liver. Insets represent high magnification. In contrast, the expression of XPA was not affected in HBx-transgenic mouse liver. A negative control (-) was made by omission of the primary antibody. Scale bar, 100 µm.

Involvement of Sp1 Transcription Factor on the Regulation of XPB and XPD by HBx-- To understand potential mechanisms by which both XPB and XPD are affected by HBx, we examined common features of regulation in the XPB and XPD promoters. A common consensus Sp1 regulatory sequence is present both in XPB and XPD promoters (Fig. 9A). HBx has been shown to interact with Sp1 and to affect its DNA binding activity (9). Studies with Sp1 mutated promoter sequences in human liver cells were not possible as we observed that these sites were indispensable for XPB basal expression; this is in agreement with an earlier study (42). To examine a possible role of Sp1 in HBx-mediated transcriptional repression of XPB and XPD, we used the Drosophila cell line SL2 which has an Sp1-deficient background and thus provides a valuable model. We confirmed that the Sp1 expression vector used was able to produce Sp1 protein that is detectable by Western blot analysis (Fig. 9B), and that expression of HBx was able to transactivate Ap1 elements (Fig. 9C). Basal XPB promoter activity in SL2 cells was similar to the background expression of the control vector, and coexpression of HBx did not modify the basal activity of the XPB promoter (Fig. 10A). These results support our observation in human liver cells on the essential role of Sp1 for the basal activity of XPB. Expression of Sp1 significantly increased XPB promoter activity, while coexpression of HBx decreased pXPB-CAT activity by ~80% (Fig. 10). In the absence of Sp1, XPD basal expression was high in SL2 cells, and was inhibited by HBx. However, coexpression of Sp1 significantly decreased XPD basal activity, and enhanced HBx-induced XPD transcriptional inhibition (Fig. 10).



View larger version (13K):
[in this window]
[in a new window]
 
Fig. 9.   Sp1 and HBx expression in Drosophila SL2 cells. A indicates the localization of Sp1 elements in the XPB and XPD promoters. In B and C, SL2 cells were co-transfected with pPac-Sp1, pAP1-CAT, and pCMV, or pCMV-HBx using LipofectAMINE. CAT activity was determined as described under "Experimental Procedures." B indicates the capacity of pPac-Sp1 vector to express Sp1, which is detectable by Western blot analysis. C indicates the capacity of HBx expressed in SL2 to transactivate a reporter vector containing an AP1 element.



View larger version (20K):
[in this window]
[in a new window]
 
Fig. 10.   Effect of Sp1 on HBx-induced transcriptional repression of XPB and XPD in Drosophila SL2 cells. SL2 cells were co-transfected with pCMV, pCMV-HBx, and XPB-CAT or XPD CAT vectors in the absence and presence of pPac-Sp1, as indicated in the figures. Cell extracts were used to determine CAT activity as described under "Experimental Procedures." Results are represented as % CAT activity in HBx-transfected compared with pCMV-transfected cells. Each bar corresponds to the average (±S.E.) of three independent experiments.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Molecular mechanisms by which HBV contributes to the development of hepatocellular carcinoma have been the subject of extensive investigations. The HBx protein, encoded by the HBV genome, has been proposed to play a role in HCC development. HBx sequence is conserved within integrated HBV DNA in HCC and cirrhotic livers infected with HBV. In addition, the presence of HBx mRNA in liver tissues from HBV-infected patients, the presence of circulating antibodies, and the frequent cellular immune response specific to HBx are consistent with a possible implication of this protein to liver carcinogenesis (reviewed in Refs. 1 and 3). HBx has also been shown to possess pleiotropic functions including impairment of cell cycle progression (24), interaction with transcription machinery (9-13), and cell signal transduction and apoptosis mechanisms (14, 43-45). Furthermore, HBx associates physically with p53 via its C terminus resulting in the sequestration of p53 in the cytoplasm (28), inhibition of p53 function including its DNA binding and transactivation activities (1, 17), as well as p53 interaction with XPB protein (17). The biological relevance of these interconnected mechanisms is not fully established.

Our study provides evidence that expression of HBx can inhibit the endogenous expression of XPB and XPD. This inhibition correlates with repression of transcriptional activity in the transient system as revealed by inhibition of XPB or XPD promoter activities by HBx. HBx has been shown to enhance cell susceptibility to the cytotoxic effect of genotoxic agents, e.g. UVC and aflatoxins, that induce bulky adducts. This effect has been linked to impaired regulation of DNA repair and the associated cell cycle checkpoint mechanisms (24-27), and/or the proapoptotic effect of HBx (45). DNA damage induced by bulky adducts are preferred substrates for the NER mechanism, where the TFIIH repair complex plays an essential role (30).

We demonstrate that expression of HBx inhibits the endogenous expression of XPB and XPD. In a previous study (19), we have shown that HBx expressed by the mean of our retroviral bicistronic system can induce apoptosis in a small subset of cells, giving rise to the possibility that down-regulation of XPB and XPD may be a consequence of the toxic effect of HBx. However, this is unlikely under our experimental conditions since (i) HBx has no inhibitory effect on other promoters, such as ERCC1 and XPA (not shown), and (ii) HBx-induced XPB and XPD mRNA inhibition was not observed on the other TFIIH subunits p34, p44, p52, and p62. It is important to note that TFIIH itself is suggested to regulate apoptosis (17, 33, 46). In addition, inhibition of TFIIH activity by HBx may inhibit DNA repair and hence promote cells to undergo apoptosis. While several studies have focused on the transactivation capacity of the HBx protein in carcinogenesis, our data indicates that HBx is capable of transcriptional repression while maintaining its transactivation functions on NF-kappa B and AP1 responsive elements. The biological relevance of transactivation versus repression properties of HBx is not fully understood. The implication of transactivation in carcinogenesis is demonstrated primarily in transient systems and there is evidence that HBx-induced transactivation is not sufficient for cell transformation (47).

The observation that HBx suppresses XPB and XPD in liver tissue from HBx-transgenic mice supports the biological relevance of our findings. XPB and XPD helicase and ATPase activities, but not the TFIIH kinase, are required for NER function (30, 33). During the transcription-initiation process, where the TFIIH complex plays a key role, the XPB helicase is required to open the promoter around the start site, while XPD, which is dispensable, stimulates transcription and allows the cdk-activating kinase complex to be anchored to TFIIH (48). Mutations in the XPD protein have been shown to produce weaker interaction between XPD and the p44 component of the TFIIH complex, leading to reduced XPD helicase activity (49). Changes in the stoichiometry, expression, or composition of the complex may affect the functions of TFIIH (50, 51). Therefore, HBx mediated reduction of XPB and XPD mRNA and protein expression may result in the impairment of TFIIH functions including NER.

Among the various subunits of TFIIH, XPB and XPD protein are the most affected. Both XPB and XPD proteins interact with p53 (35), suggesting that HBx/p53 interaction accounts for HBx-induced XPB and XPD transcriptional repression. However, HBx-induced transcriptional repression was observed in both p53-proficient and p53-deficient cell lines. We also observed that expression of the HPV E6 protein, which promotes degradation of wild type p53 (52), did not result in inhibition of XPB or XPD promoter activity. Our observations suggest a p53-independent mechanism for HBx-induced repression of XPB and XPD, and support earlier studies showing that (i) HBV replication does not interfere with known cellular functions of p53 (53), (ii) the transactivation function of HBx take place in p53-negative cells (54), and (iii) interaction of HBx with the apoptotic pathway occurs in both p53-dependent (29, 43, 44) and p53-independent pathways (14, 19, 45). However, we cannot rule out a contribution of p53 inactivation by HBx on the regulation of TFIIH function. The TFIIH helicases, XPB and XPD, were shown to be members of the p53-dependent apoptotic pathway (55-57), while involvement in both p53-independent (25, 26) and p53-dependent mechanisms of DNA repair are postulated (26, 27). In addition, wild-type p53 negatively regulates a variety of genes that lack p53 consensus binding sites (58, 59) as is the case with the XPB and XPD promoters. Wild type p53 has been reported to be required for repair in both nontranscribed (33, 61) and actively transcribed genes (62), as well as in cell cycle checkpoints and apoptosis (63, 64). In addition to XPB and XPD, p53 protein also associates directly or indirectly with several proteins involved in cellular response to DNA damage and DNA repair including p62, RPA, BRCA1, and human Rad 51 (reviewed in Refs. 64 and 65). Additional functional studies are necessary to understand the contribution of HBx-p53 interaction through various p53-dependent and p53-independent regulatory mechanisms.

Mechanisms by which HBx affects both XPB and XPD promoters are still unclear. Our study implicates a common promoter regulatory element in HBx-mediated XPB and XPD inhibition. Several transcription factor responsive elements are present in both XPB and XPD promoters including Sp1, Oct, TATA box-binding protein, and ETS-1. The Sp1 transcription factor has been shown to be a specific target for HBx resulting in impairment of its DNA binding properties (9). To address a possible role for Sp1, we used the Drosophila cell line SL2 which has an Sp1-deficient background. Our results clearly show that HBx-induced XPB inhibition is Sp1 dependent. Also, the presence of Sp1 potentiates HBx-induced XPD transcriptional inhibition. This observation sheds new insight into the molecular mechanism by which HBx may affect TFIIH function. The Sp1 transcription factor is capable of both stimulating and down-regulating gene transcription (66-68). Also, an important feature of the transcriptional regulation by the Sp1 protein is its requirement for cofactors including Ets, p53, and HDACs (60, 69), and their synergistic interaction with other members of the Sp family. Ongoing studies will determine how HBx/Sp1 interacts in the regulation of TFIIH.


    ACKNOWLEDGEMENTS

We are grateful to Dr. Jean-Marc Egly (Université Louis Pasteur, Strasbourg, France) for kindly providing XPB and XPD antibodies, Dr. James Cromlish (IRCM, Montreal) for providing HBx constructs, Dr. Robert Tjian for providing the Drosophila Sp1 expression vector pPac-Sp1, and John Cho for technical help.


    FOOTNOTES

* This work was supported by the Cancer Research Society of Canada (to M. A. A.-J.) and National Institutes of Health Grant 54SS7 (to B. L. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Senior Scientist of the "Fond de Recherches en Santé du Québec" (FRSQ). To whom correspondence should be addressed: Lady Davis Institute for Medical Research, Rm. 523, 3755 Chemin Cote-Ste-catherine, Montreal (Quebec) H3T 1E2, Canada. Tel.: 514-340-8260 (ext. 3438); Fax: 514-340-7576; E-mail: malaou@po-box.mcgill.ca.

Published, JBC Papers in Press, January 25, 2001, DOI 10.1074/jbc.M010852200


    ABBREVIATIONS

The abbreviations used are: HBV, hepatitis B virus; HCC, hepatocellular carcinoma; PCNA, proliferating cell nuclear antigen; RT-PCR, reverse transcriptase-polymerase chain reaction; CMV, cytomegalovirus; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; GFP, green fluorescent protein; CAT, chloramphenicol acetyltransferase; PBS, phosphate-buffered saline; TFIIH, transcription factor II H; HBx, hepatitis B virus × protein; XPB/D, xeroderma pigmentosum complementation group B/D; NER, nucleotide excision repair; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


1. Feitelson, M. A. (1999) J. Cell. Physiol. 181, 188-202[CrossRef][Medline] [Order article via Infotrieve]
2. Farber, E., and Sarma, D. S. R. (1987) in Concepts and Theories in Carcinogenesis (Maskens, A. P. , Ebbesen, P. , and Burny, A., eds) , pp. 185-220, Elsevier Press Inc., Amsterdam
3. Chisari, F. V., Klopchik, K., Moriuama, T., Pasquinelli, C. C., Dunsdorf, H. A., Sell, S., Pinkert, C. A., Brinster, R. L., and Palmiter, R. D. (1989) Cell 59, 1145-1156[Medline] [Order article via Infotrieve]
4. Chisari, F. V., Pinkert, C. A., Milich, D. R., Filippi, P., Mclachlan, A., Palmiter, R. D., and Brinster, R. L. (1985) Science 230, 1157-1160[Medline] [Order article via Infotrieve]
5. Unsal, H., Jakicier, C., Markais, C., Kew, M., Volkmann, M., Zentgraf, H., Isselbacker, K. J., and Ozturk, M. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 822-826[Abstract]
6. Slagle, B. L., Lee, T.-H., Medina, D., Finegold, M. J., and Butel, J. S. (1996) Mol. Carcinogen. 15, 261-269[CrossRef][Medline] [Order article via Infotrieve]
7. Dragani, T. A., Manenti, G., Farza, H., Della Porta, G., Tiollais, P., and Pourcel, C. (1990) Carcinogenesis 11, 953-956[Abstract]
8. Madden, C. R., Finegold, M. J., and Slagle, B. L. (2000) J. Virol. 74, 5266-5272[Abstract/Free Full Text]
9. Lee, Y. I., Lee, S., Lee, Y., Bong, Y. S., Hyun, S. W., Yoo, Y. D., Kim, S. J., Kim, Y. W., and Poo, H. R. (1998) Oncogene 16, 2367-2380[CrossRef][Medline] [Order article via Infotrieve]
10. Twu, J. S., and Shloemer, R. H. (1987) J. Virol. 61, 3448-3453[Medline] [Order article via Infotrieve]
11. Koike, K., Shirakata, Y., Yaginuma, K., Arii, M., Takada, S., Nakamura, I., Hayashi, Y., Kawada, M., and Kobayashi, M. (1989) Mol. Biol. Med. 6, 151-160[Medline] [Order article via Infotrieve]
12. Twu, S. J., and Robinson, W. S. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 2046-2050[Abstract]
13. Zahm, P., Hofscneider, P., and Koshy, R. (1988) Oncogene 3, 169-177[Medline] [Order article via Infotrieve]
14. Shintani, Y., Yotsuyanagi, H., Moriya, K., Fujie, H., Tsutsmi, T., Kanegae, Y., Kimura, S., Saito, I., and Koike, K. (1999) J. Gen. Virol. 80, 3257-3265[Abstract/Free Full Text]
15. Feitelson, M. A., Zhu, M., Duan, L. X., and London, W. T. (1993) Oncogene 8, 1109-1117[Medline] [Order article via Infotrieve]
16. Truant, R., Antunovic, J., Greenblatt, J., Prives, C., and Cromlish, J. (1995) J. Virol. 69, 1851-1859[Abstract]
17. Wang, X. W., Forrester, K., Yeh, H., Feitelson, M. A., Gu, J. R., and Harris, C. C. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 2230-2234[Abstract]
18. Lee, T.-H., Elledge, S. J., and Butel, J. S. (1995) J. Virol. 69, 1107-1114[Abstract]
19. Sohn, S., Jaitovitch Groisman, I., Benlimame, N., Galipeau, J., Batist, G., and Alaoui-Jamali, M. A. (2000) Mutat. Res. 460, 17-28[Medline] [Order article via Infotrieve]
20. Jaitovitch-Groisman, I., Fotouhi-Ardakani, N., Schecter, R. L., Woo, A., Alaoui-Jamali, M. A., and Batist, G. (2000) J. Biol. Chem. 275, 33395-33403[Abstract/Free Full Text]
21. Sell, S., Hunt, J. M., Dunsford, H. A., and Chisari, F. V. (1991) Cancer Res. 51, 1278-1285[Abstract]
22. Capovilla, A., Carmona, S., and Arbuthnot, P. (1997) Biochem. Biophys. Res. Commun. 232, 255-260[CrossRef][Medline] [Order article via Infotrieve]
23. Quadri, I., Ferrari, M. E., and Siddiqui, A. (1996) J. Biol. Chem. 271, 15443-15450[Abstract/Free Full Text]
24. Benn, J., and Schneider, R. J. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 11215-11219[Abstract]
25. Jaitovich, G., roisman, I., Koshy, R., Henkler, F., Groopman, J. D., and Alaoui-Jamali, M. A. (1999) Carcinogenesis 20, 479-483[Abstract/Free Full Text]
26. Jia, L., Wang, X. W., and Harris, C. C. (1999) Intl. J. Cancer 80, 875-879[CrossRef][Medline] [Order article via Infotrieve]
27. Prost, S., Ford, J. M., Taylor, C., Doig, J., and Harrison, D. J. (1998) J. Biol. Chem. 273, 33327-33332[Abstract/Free Full Text]
28. 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) Proc. Natl. Acad. Sci. U. S. A. 94, 14707-14712[Abstract/Free Full Text]
29. Kim, H., Lee, H., and Yun, Y. (1998) J. Biol. Chem. 273, 381-385[Abstract/Free Full Text]
30. de Laat, W. L., Jaspers, N. G. J., and Hoeijmakers, J. H. J. (1999) Genes Dev. 13, 768-785[Free Full Text]
31. Bhatia, P., Wang, Z., and Friedberg, E. C. (1996) Curr. Opin. Genet. Dev. 6, 146-150[CrossRef][Medline] [Order article via Infotrieve]
32. Nigg, E. (1996) Curr. Opin. Cell Biol. 8, 312-317[CrossRef][Medline] [Order article via Infotrieve]
33. Hoeijmakers, J. H. J., Egly, J. M., and Vermeulen, W. (1996) Curr. Opin. Genet. Dev. 6, 26-33[Medline] [Order article via Infotrieve]
34. Marinoni, J.-C, Roy, R., Vermeulen, W., Miniou, P., Lutz, Y., Weeda, G., Seroz, T., Molina Gomez, D., Hoijmakers, J. H. J., and Egly, J. M (1997) EMBO J. 16, 1093-1102[Abstract/Free Full Text]
35. Leveillard, T., Andera, L., Bissonnette, N., Schaeffer, L., Bracco, L., Egly, J.-M., and Wasylyk, B. (1996) EMBO J. 15, 1615-1624[Abstract]
36. Galipeau, J., Li, H., Paquin, A., Sicilia, F., Karpati, G., and Nalbantoglu, J. (1999) Cancer Res. 59, 2383-2394
37. Morgenstern, J. P., and Land, H. (1990) Nucleic Acids Res. 18, 3587-3596[Abstract]
38. Yen, L., Nie, Z.-R., You, X.-L., Richard, S., Langton-Webster, B. C., and Alaoui-Jamali, M. A. (1997) Oncogene 14, 1827-1835[CrossRef][Medline] [Order article via Infotrieve]
39. Koromilas, A. E., Lazaris-Karatzas, A., and Sonenberg, N. (1992) EMBO J. 11, 4153-4198[Abstract]
40. Lee, T. H., Finegold, M. J., Shen, R. F., DeMayo, J. L., Woo, S. L., and Butel, J. S. (1990) J. Virol. 64, 5939-5947[Medline] [Order article via Infotrieve]
41. Gu, Z., Pim, D., Labrecque, S., Banks, L., and Matlashewski, G. (1994) Oncogene 9, 629-633[Medline] [Order article via Infotrieve]
42. Ma, L., Weeda, G., Jochemsen, A. G., Bootsma, D., Hoeijmakers, J. H. J., and van der Eb, A. J. (1991) Nucleic Acids Res. 20, 217-224[Abstract]
43. Chirillo, P., Pagano, S., Natoli, G., Puri, P. L., Burgio, V. L., Balsano, C., and Levrero, M. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 8162-8167[Abstract/Free Full Text]
44. Bergametti, F., Prigent, S., Luber, B., Benoit, A., Tiollais, P., Sarasin, A., and Transy, C. (1999) Oncogene 18, 2860-2871[CrossRef][Medline] [Order article via Infotrieve]
45. Terradillos, O., Pollicino, T., Lecoeur, H., Tripodi, M., Gougeon, M. L., Tiollais, P., and Buendia, M. A. (1998) Oncogene 17, 2115-2123[CrossRef][Medline] [Order article via Infotrieve]
46. Shirasaki, F., Makhluf, H. A., LeRoy, C., Watson, D. K., and Trojanowska, M. (1999) Oncogene 18, 7755-7764[CrossRef][Medline] [Order article via Infotrieve]
47. Gottlob, K., Pagano, S., Levrero, M., and Graessmann, A. (1998) Cancer Res. 58, 3566-3570[Abstract]
48. Tirode, F., Busso, D., Coin, F., and Egly, J. M. (1999) Mol. Cell. 3, 87-95[Medline] [Order article via Infotrieve]
49. Coin, F., Marinoni, J. C., Rodolfo, C., Pedrini, M. A., and Egly, J. M. (1998) Nat. Genet. 20, 184-188[CrossRef][Medline] [Order article via Infotrieve]
50. Schultz, P., Fribourg, S., Poterszman, A., Mallouh, V., Moras, D., and Egly, J. M. (2000) Cell 102, 599-607[Medline] [Order article via Infotrieve]
51. Winkler, G. S., Araujo, S. J., Fiedler, U., Vermeulen, W., Coin, F., Egly, J. M., Hoeijmakers, J. H., Wood, R. D., Timmers, H. T., and Weeda, G. (2000) J. Biol. Chem. 275, 4258-4266[Abstract/Free Full Text]
52. Aboussekhra, A., Biggerstaff, M., Shivji, K., Vilpo, J. A., Moncollin, V., Podust, V. N., Protic, M., Hubscher, U., Egly, J. M., and Wood, R. D. (1995) Cell 80, 859-868[Medline] [Order article via Infotrieve]
53. Puisieux, A., Ji, J., Guguillot, C., Legros, Y., Soussi, T., Isselbacher, K., and Ozturk, M. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 1342-1346[Abstract]
54. Lin, Y., Nomura, T., Cheong, J., Dorjsuren, D., Iida, K., and Murakami, S. (1997) J. Biol. Chem. 272, 7132-9[Abstract/Free Full Text]
55. Wang, X. W., Vermeulen, W., Coursen, J. D., Gibson, M., Lupold, S. E., Forrester, K., Xu, G., Elmore, L., Yeh, H., Hoeijmakers, J. H., and Harris, C. C. (1996) Genes Dev. 10, 1219-1232[Abstract]
56. Spillar, E. A., Robles, A. I., Wang, X. W., Shen, J. C., Yu, C. E., Schellenberg, G. D., and Harris, C. C. (1999) Genes Dev. 13, 1355-1360[Abstract/Free Full Text]
57. Robles, A. I., Wang, X. W., and Harris, C. C. (1999) Oncogene 18, 4681-4688[CrossRef][Medline] [Order article via Infotrieve]
58. Ravi, R., Mookerjee, B., Van Hensbergen, Y., Bedi, G. C., Giordano, A., El-Deiry, W. S., Fuchs, E. J., and Bedi, A. (1998) Cancer Res. 58, 4531-4536[Abstract]
59. Giebler, H. A., Lemasson, I., and Nyborg, J. K. (2000) Mol. Cell. Biol. 20, 4849-4858[Abstract/Free Full Text]
60. Zaid, A., Li, R., Luciakova, K., Barath, P., Nery, S., and Nelson, B. D. (1999) J. Bioenerg. Biomembr. 31, 129-135[CrossRef][Medline] [Order article via Infotrieve]
61. Ford, J. M., and Hanawalt, P. C. (1997) J. Biol. Chem. 272, 28073-28080[Abstract/Free Full Text]
62. Therrien, J. P., Drouin, R., Baril, C., and Drobetsky, E. A. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 15038-15043[Abstract/Free Full Text]
63. Smith, M. L., Ford, J. M., Hollander, M. C., Bortnick, R. A., Amundson, S. A., Seo, Y. R., Deng, C. X., Hanawalt, P. C., and Fornace, A. J., Jr. (2000) Mol. Cell. Biol. 10, 3705-3714[CrossRef]
64. Levine, A. J. (1997) Cell 88, 323-331[Medline] [Order article via Infotrieve]
65. Kohn, K. W. (1999) Mol. Biol. Cell 10, 2703-2734[Abstract/Free Full Text]
66. Haviv, I., Shamay, M., Doitsh, G., and Shaul, Y. (1998) Mol. Cell. Biol. 18, 1562-1569[Abstract/Free Full Text]
67. Wang, H. D., Trivedi, A., and Johnson, D. L. (1998) Mol. Cell Biol. 18, 7086-7094[Abstract/Free Full Text]
68. Noti, J. D., Reinemann, B. C., and Petrus, M. N. (1996) Mol. Cell. Biol. 16, 2940-2950[Abstract]
69. Ye, J., Xu, R. H., Taylor-Papadimitriou, J., and Pitha, P. M. (1996) Mol. Cell. Biol. 16, 6178-6189[Abstract]


Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.