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INTRODUCTION |
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
, 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-
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.
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EXPERIMENTAL PROCEDURES |
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 pJ418E6
F constructs were kindly provided by Dr. Greg
Matlashewski (McGill University) and consisted of the HPV-18
E6 gene and the HPV-18 E6
F mutated gene under
control of the CMV promoter, respectively. Both genes were cloned into plasmid pJ4
. The chloramphenicol reporter constructs 5'tB containing NF-
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
-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
-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
pJ6
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
pJ418E6
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
-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
-actin primer,
40 pmol of reverse primer, 5 µCi of 35S-
-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
-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
-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
-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
-actin primers, 5 µCi of
[
-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
-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
-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.
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
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 |
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).

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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.
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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).

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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 -actin mRNA were reverse transcribed followed
by PCR amplification of both cDNAs. -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.
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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
pJ418E6
F (Fig. 3B). The pJ418E6
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).

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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, pJ418E6 F, that has a
mutated E6 gene. The ratio of chloramphenicol acetylation was expressed
as % of CAT activity in pJ418E6-transfected cells compared with
pJ418E6 F-transfected cells. Each bar corresponds to the
average (±S.E.) of four independent experiments.
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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-
B responsive elements (Fig.
4).

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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.
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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-
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).

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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.
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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 -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).
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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.).
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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.

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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.
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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).

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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.
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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.
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DISCUSSION |
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-
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.