| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
J Biol Chem, Vol. 273, Issue 50, 33327-33332, December 11, 1998
,From the Department of Pathology, University Medical School, Teviot Place, Edinburgh EH8 9AG, Scotland, and the § Department of Medicine and Genetics, Stanford University School of Medicine, Stanford, California 94305-5115
 |
ABSTRACT |
|---|
Top
Abstract
Introduction
Procedures
Results
Discussion
References
|
|---|
The mechanisms by which the hepatitis B x protein
(HBx) contributes to hepatocarcinogenesis remain unclear. However,
interaction with the tumor suppressor gene p53 and
inhibition of p53-dependent cellular functions, including
nucleotide excision repair, could be central to this process. We
studied the levels of global repair (removal of cyclobutane pyrimidine
dimers (CPDs) and 6-4 photoproducts) and transcription-coupled repair
(removal of CPDs in both strands of the dihydrofolate reductase gene)
in primary wild-type and p53-null mouse hepatocytes. We
show that global repair of CPDs appears to be more efficient in mouse
hepatocytes than in other commonly studied rodent cells and approaches
the levels of human cells and that p53 is required for global genomic
DNA repair of CPDs but not for transcription-coupled repair. We then
investigated the effect of HBx expression on hepatocyte nucleotide
excision repair. We demonstrate that HBx expression affects DNA repair in a p53-dependent manner. Transient HBx expression reduces
global DNA repair in wild-type cells to the level of
p53-null hepatocytes and has no effect on the repair of a
transfected damaged plasmid. Therefore, in viral hepatitis, the
hepatitis B virus could inhibit the
p53-dependent component of global repair
leading, over time, to accumulation of genetic defects and fostering carcinogenesis.
There is compelling epidemiological evidence that chronic
hepatitis associated with hepatitis B virus
(HBV)1 infection is a major
risk factor for the development of hepatocellular carcinoma (HCC).
Among the four viral proteins produced, the small protein hepatitis B x
(HBx) is thought to be important in causing HCC, although the mechanism
of its involvement has not been defined clearly. Some strains of mice
carrying HBx as a transgene show a direct correlation between the level
of HBx expression and the likelihood to develop HCC (1, 2). In others,
HBx expression increases susceptibility to the development of HCC after
exposure to the liver carcinogen diethylnitrosamine (3, 4). A number of
mechanisms have been proposed to explain the role of HBx in hepatocarcinogenesis. HBx stimulates the transcription of many genes
(for review, see Ref. 5), including transcription factors and
oncogenes, and interacts with many cellular proteins (for review, see
Ref. 6). One important target of HBx believed to be related to
promotion of carcinogenesis is the p53 tumor suppressor protein.
Several lines of evidence suggest that functional inactivation of p53
without mutation can occur in preneoplastic liver and that, in these
circumstances, p53 mutation and allele loss are later events
(7-11), occurring after carcinogenesis and mediating tumor progression. Indeed, accumulation of wild-type p53 has been observed in
hepatocytes of patients with cirrhosis and liver cell dysplasia, both
associated with high risk for HCC development, particularly in patients
infected with HBV (10, 12-14). Observations both in vitro
and in vivo (15, 16) and in HBx-transgenic mice (17) have
suggested that HBx interacts and inactivates p53, possibly by
cytoplasmic sequestration (10, 18). In transgenic mice (17), this
functional inactivation of p53 is associated topographically with
subsequent development of HCC. Moreover, HBx and p53 can be
coprecipitated from liver samples of patients with HCC (19), supporting
this idea.
It is important to identify the functions of p53 which HBx could target
and so promote carcinogenesis. p53 is critical for the maintenance of
genomic fidelity. Current models hold that cells respond to DNA damage
by stabilization of p53 leading either to checkpoint arrest, allowing
repair, or to p53-dependent apoptosis, eliminating a
potentially mutated cell from the population. Work from our laboratory
has established the existence of p53-dependent cell cycle
arrest in hepatocytes after DNA damage but also indicates that
hepatocytes do not undergo apoptosis readily in response to DNA damage
(20). Therefore, effective DNA repair, also shown to be partially
p53-dependent in these cells (21), must be critical to
prevent development of mutations because the hepatocytes do not
self-delete in the presence of a low, but significant, level of DNA damage.
Therefore, a functional blockage of p53 by HBx could be a critical
event in cells sustaining DNA damage, leading to inhibition of
p53-dependent cell cycle arrest (20) but also alteration of
p53-dependent DNA repair (21). Interestingly, HBx binding to p53 was reported recently to inhibit the interaction between p53 and
the two DNA repair proteins XPB and XPD, part of the TFIIH complex
(22). In addition, a direct effect upon DNA repair by HBx is suggested
by the ability of HBx to bind damaged DNA in vitro (23) and
to interact directly with at least three DNA repair proteins:
XAP-1/UV-DDB (24), XPB, and XPD (25). Therefore, alteration of DNA
repair, either directly or indirectly through inhibition of p53, could
be a major contribution of HBx in carcinogenesis.
In the present study, we have used wild-type and p53-null
primary hepatocytes to investigate the effect of HBx expression on
p53-dependent and -independent DNA repair in the liver.
Cells--
Primary hepatocytes were isolated from adult
wild-type or p53-null male mice (26) 6-10 weeks of age by a
two-step EDTA/collagenase retrograde perfusion protocol and cultured as
described previously (20).
Transfection and Plasmids--
All transfections were performed
using Tfx-50 (Promega Biotech). Briefly, plasmid DNA (3 µg) and
Tfx-50 (15.5 µg) in 300 µl of warm culture medium were incubated
for 15 min at room temperature before laying the complex over the cells
in culture in 12-well plates for 24 h. After 3 h of
incubation at 37 °C, the DNA-containing medium was replaced by fresh
medium, and experiments were performed 24-72 h later. Using this
technique about 80% of hepatocytes were transfected.
The p53 reporter plasmid pRGC
The plasmids used for the host cell reactivation assay are the
chloramphenicol acetyltransferase (CAT) reporter plasmid pOP13 CAT
(Stratagene) and the LacZ-expressing plasmid p p53 Immunocytochemistry--
Primary hepatocyte monolayers on
chamber slides (LabTek) were transfected with pCMV-x1 or a control
plasmid, cultured for a further 30-36 h, then UV-C irradiated (10 J/m2) and fixed at the indicated times in acetone:methanol
(1:1 v/v). Immunocytochemistry for p53 protein was performed by a
standard avidin-biotin peroxidase technique using a primary monoclonal anti-p53 antibody pAb 421 (Oncogene Science; 1/1,000 dilution) as
described previously (28).
p53 Reporter Plasmid--
Primary hepatocytes cultured for
24 h in fibronectin-coated 12-well plates (0.3 × 105 cells/cm2) were transiently cotransfected
with a p53 reporter plasmid, pRGC Unscheduled DNA Synthesis--
Primary hepatocytes transfected
with pCMV-x1 or a control plasmid were cultured for 48 h,
irradiated with the indicated doses of UV-C, and incubated for a
further 3 h in medium supplemented with 10 µCi/ml tritiated
thymidine. The radioactive medium was removed, and cells were incubated
in fresh medium containing 1 mM nonradioactive thymidine
for an additional hour. The cultures were then fixed in Boum's
fixative, counterstained with Feulgen, then dipped in LM-1
autoradiographic emulsion (Amersham Pharmacia Biotech). Processing and
grain counting were performed as described previously (21). For each
culture, 10 high power (× 400) fields were assessed, each field
containing 20-40 nuclei. The grain density within nuclei was corrected
for background (non-nuclear) labeling by subtraction and expressed per
surface area of nuclei (= nuclear grain index). The results shown are
histograms of the nuclear grain index for one typical experiment. For
the calculation of the mean nuclear grain index, only values smaller
than 40 were considered in order to exclude cells in DNA synthesis. The
experiment was performed twice (two wild-type and two
p53-null mice) with similar results.
Reactivation of a UV-C-irradiated Reporter Plasmid--
Primary
hepatocytes were transfected with either pCMV-x1 or the control plasmid
as described above. 36 h later, a second transfection was
performed using the pOP13 CAT reporter plasmid treated with UV-C at
various doses and undamaged p
The CAT activity in transfected cells was measured by liquid
scintillation counting assay as described previously (21). The results
were corrected for the transfection efficiency, given by the
Global Genomic Nucleotide Excision Repair Immunoassay--
The
relative number of cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts in total genomic DNA was determined using an immunoblot
assay as described previously (29). Briefly, hepatocytes in culture for
8 h were UV irradiated (10 J/m2) and collected 2, 6, 12, or 24 h later and lysed, and the DNA was extracted by
phenol/chloroform and ethanol precipitation. During this period of
time, freshly isolated primary hepatocytes show no replicative DNA
synthesis (30, 31), and so it was not necessary to eliminate newly
replicated DNA.
DNA was then denatured by boiling for 5 min and placing on ice, and
equal amounts of genomic DNA from each sample were fixed to a Hybond N+
nylon membrane (Amersham Corp.) in triplicate using a slot-blot
apparatus (0.1 and 1 µg of DNA/slot for detection of CPDs and 6-4 photoproducts, respectively).
Genomic DNA from unirradiated cells was used as a control for
nonspecific DNA binding of the monoclonal antibodies. The membrane was
incubated for 1 h with mouse monoclonal antibodies specific for
either CPDs (TDM-2) or 6-4 photoproducts (64M-2), at a dilution of
1/2,000 in phosphate-buffered saline. Detection of primary antibody was
performed using horseradish peroxidase-conjugated secondary antibody
and enhanced chemoluminescence (Amersham Corp.). After antibody
detection, equal DNA loading to each slot of the membrane was confirmed
by scintillation spectrophotometry. Two separate slot blots were
performed, in triplicate, using DNA from three wild-type, one
heterozygous, and two p53-null mice.
Analysis of Strand-specific DNA Repair--
Repair of CPDs
within the transcribed and nontranscribed strand of the dihydrofolate
reductase (DHFR) gene was performed as described previously (30, 31)
using a Southern-based method. Primary hepatocytes were lysed at
various times after UV irradiation (10 J/m2), and DNA was
extracted. After digestion with BamHI, DNA samples were
treated with T4 endonuclease V or not, electrophoresed, and transferred
onto a membrane. After hybridization with a 32P-labeled
strand-specific probe to the murine DHFR gene (plasmid supplied by
Isabel Mellon) and autoradiography, the ratio of full-length restriction fragments in the T4 endonuclease V-treated and untreated samples was determined by PhosphorImager analysis (Bio-Rad model GS-363) and was used to calculate the average number of CPDs/fragment using the Poisson expression. DNA samples from each mouse (two wild-type, one heterozygous, and one p53-null) were used for
two independent Southern blots, each probed for both the transcribed and the nontranscribed strand of the DHFR gene.
Nucleotide Excision Repair in Primary Hepatocytes--
The role of
p53 in the repair of CPDs and 6-4 photoproducts in overall genomic DNA
and in the repair of CPDs in individual strands of the DHFR gene was
examined using primary hepatocytes isolated from wild-type,
p53 heterozygous, and p53-null mice.
First, the global repair of UV-induced DNA damage was determined using
monoclonal antibodies against CPDs and 6-4 photoproducts. Both
wild-type and p53 heterozygous primary hepatocytes exhibit efficient global repair of both lesions (Fig.
1). By contrast, p53-null
cells show a strong reduction in the repair of CPDs, whereas removal of
6-4 photoproducts remains normal.
Second, the repair of CPDs was studied in both the transcribed and the
nontranscribed strand of the DHFR gene. The repair of the transcribed
strand of DHFR was proficient and similar in both cell types, whereas
the repair of the nontranscribed strand, although still measurable, was
found to be slower in p53
By contrast to other mouse cell types, which inefficiently repair CPDs
in bulk DNA (33, 34), we found the global repair of CPDs in primary
hepatocytes to be very efficient and to approach the levels observed in
human cells. This finding suggests that nuclear excision repair is a
critical process in the liver.
Effect of HBx Protein on Primary Hepatocyte Responses to DNA
Damage--
Independently of UV treatment, transient expression of
pCMV-x1 had no effect on the level of cell death in either wild-type or
p53
In the absence of DNA-damaging treatment, the prevalence of p53
immunopositivity in HBx-transfected cells was similar to that of
control cells (5-10% of cells) (Fig.
3A). After UV irradiation, the
proportion of p53-immunopositive cells increased in both normal and
transfected hepatocytes. Interestingly, the proportion of hepatocytes
showing nuclear immunopositivity was significantly smaller in
HBx-transfected hepatocytes compared with controls (Fig. 3,
A and B). This difference between HBx-transfected
cells and controls was transient, lasting no longer than 24 h
after irradiation (Fig. 3, A and B). Consistent
with this result, we also found that HBx expression reduces
significantly the rise in p53 transactivation activity which normally
follows UV irradiation, especially during the first 12 h after it
(Fig. 3C). The absence of a difference between transfected
and control cells at later time points after irradiation reflects the
kinetics of expression of the transiently transfected plasmid;
expression decreases rapidly after about 60 h (28).
We next examined the effect of HBx expression on DNA repair. First, we
tested the ability of wild-type and p53-null hepatocytes transiently transfected with the HBx expression vector to repair a
reporter plasmid damaged by various doses of UV-C. There was a
dose-dependent impairment of the recovery of the reporter
expression, and this was not affected by the p53 genotype
(Fig. 4), as noted previously (21). No
differences were observed between cells transfected with pCMV-x1 or
with the control plasmid, suggesting that HBx does not affect this type
of repair (Fig. 4).
Second, we quantified the unscheduled DNA synthesis in these cells,
transiently transfected or not with the HBx expression vector. This was
measured by image analysis of grain density after autoradiography of
cultures treated with different doses of UV and subsequently allowed to
incorporate tritiated thymidine. In the absence of UV irradiation the
majority of the cells presented a low nuclear grain index (between 0 and 5), and a second group of cells exhibited a nuclear grain index
>40, representing cells undergoing DNA synthesis. After UV
irradiation, the grain index was increased in hepatocyte nuclei
regardless of genotype, showing that the cells were initiating
unscheduled DNA synthesis (Fig. 5).
However, the grain index of irradiated p53 Interspecies differences in the level of DNA repair are well
described; mouse fibroblasts typically show a lower efficiency of
global repair than human fibroblasts (33, 34). The present observation
that DNA repair efficiency is relatively high in mouse hepatocytes,
indeed approaching the level of human fibroblasts, suggests that
nuclear excision repair is particularly important in this cell type.
This observation, in association with our previous demonstration that
hepatocytes do not readily undergo apoptosis in response to DNA damage
(20), suggests an emphasis on repair mechanisms rather than
self-deletion after DNA damage. This is consistent with known
detoxifying roles of hepatocytes during which genotoxic metabolites are
likely to be generated. Indeed, mice deficient in the nucleotide repair
protein XPA (35) develop spontaneous HCC, and ERCC1 (for
excision repair
cross-complementing 1) deficiency
causes mice to die soon after birth with an unusual liver pathology
associated with nuclear p53 accumulation (36). The p53 protein is
important in regulating various responses to DNA damage, including cell
cycle arrest, apoptosis, and DNA repair, and these responses differ
among cell types. We have reported previously that in DNA-damaged
hepatocytes, p53 regulates cell cycle arrest but not apoptosis (20). We
show here, in agreement with reports in human fibroblasts (29, 31, 32),
that p53 is required for efficient global repair, especially the repair of the CPDs, but not for transcription-coupled repair. Taken together, these data strongly suggest that DNA repair is a critical process in
hepatocytes and, at least partially, depends on p53.
Interestingly, although the precise mechanisms by which the
carcinogenic viral protein HBx promotes the development of HCC remain
unclear, there is good evidence that interaction with and functional
disruption of p53 are important. The present data show that HBx
expression reduces p53 transactivation of a reporter plasmid after
irradiation, and this is direct evidence that HBx alters endogenous p53
responses to DNA damage in liver cells. Moreover, we showed that the
p53-dependent component of hepatocyte DNA repair is also
inhibited by HBx. Indeed, HBx expression in wild-type cells reduces
global DNA repair to the level of untransfected p53-null
hepatocytes. By contrast, HBx expression did not further affect global
repair in p53-null cells or the repair of a damaged plasmid
(host cell reactivation assays) which we previously demonstrated to be
p53-independent in hepatocytes (21). The precise mechanism by which HBx
interferes with p53-dependent regulation of repair was not
evaluated here but may involve direct binding of HBx to p53 (17, 18,
37). This interaction was identified in vivo by
coprecipitation of HBx and p53 from liver tumors (19) and could produce
cytoplasmic sequestration of p53 (17, 18, 37) or interference with p53
transcriptional regulation of other downstream proteins involved in
nuclear excision repair (25 and references therein). The interaction of
HBx with p53 could also inhibit p53 binding to the repair proteins XPB
and XPD as was seen previously using cell extracts (25). It is
therefore likely that direct interaction of HBx with p53 is central to
the p53-dependent effect of HBx on DNA repair observed
here. In a recent report, Becker et al. (38) used several
HBx deletion mutants to identify regions of HBx required for impairment
of DNA repair. They found a minimal region that correlates with the
region interacting with the repair protein XAP-1/UV-DDB. However, this
part of HBx protein is also required for p53 binding (39). It is
therefore difficult to rule out the possibility that the inhibition of
DNA repair observed in their study was dependent on HBx interaction
with p53.
Clearly, if HBx alters p53-dependent DNA repair through
binding to p53, it could affect other p53-regulated functions such as
p53-dependent apoptosis. Indeed, HBx expression inhibited
p53-dependent apoptosis in hepatocytes when apoptosis was
produced by microinjection of a p53-overexpressing vector (37, 40).
Under the present culture conditions where apoptosis is not
p53-dependent (20), transient expression of HBx has no
effect on hepatocyte apoptosis, as might be expected. Direct binding of
HBx to p53 could also explain the inhibition of p53 transactivation
function, either by sequestering p53 protein in the cytoplasm (37) or
by altering p53 binding to DNA (15, 16, 39) or to proteins of the
transcription machinery (25, 41). Inhibition of p53
transactivation function by HBx has been reported in cells coinjected
or cotransfected with p53 and HBx expression vectors (15, 16). The
present report extends these studies to show interference by HBx with the transactivation responses to a physiological level of endogenous p53.
Implication for Human Liver Carcinogenesis--
Numerous
biological properties for HBx have been described which could account
for its role in hepatocarcinogenesis. The present work supports the
idea that dysfunction of the p53 protein through interaction with HBx
is of biological and pathological significance for early events leading
to carcinogenesis. In our experiments, HBx expression mimics a state of
p53 deficiency or of inactivating p53 mutation. Hence
p53 mutation may be unnecessary for carcinogenesis in
HBV-infected hepatocytes that express HBx. Indeed, clinical studies
have shown an association between HBx expression and the absence of
mutation in p53 (8, 42), and by contrast, HCC carrying
p53 mutations does not express HBV products (8, 42). Moreover, there is an abnormal accumulation of p53 protein in the
hepatocytes of patients with viral-induced cirrhosis (8, 10, 19), and
HBV-associated HCC has a low frequency of p53 mutation in
non-AFB1-exposed populations (8, 10, 19, 43-45). These observations,
together with others suggesting that the p53 mutation and
loss of allele are late events in this type of cancer (7-11), strongly
support the in vitro data suggesting that HBx inactivation
of p53 functions is an authentic alternative mechanism to
p53 mutation in early carcinogenesis.
Chronic viral hepatitis is characterized by a regenerative
necroinflammatory process, typically sustained for several years. Hepatitis-related cycles of cell death and regeneration in an inflammatory environment, conditions that favor the generation of
reactive oxygen species, could be critical for a progressive accumulation of genetic defects. If, as suggested here, HBx impairs p53-dependent DNA repair, then two predictions can be made.
First, mutation incidence should be increased by HBx expression.
Second, because p53 affects global repair but not transcription-coupled repair, an increased incidence of mutation in the nontranscribed strand
would be expected in patients with HBV infection. Interestingly, HBx
infection further increases the mutation rate in patients from areas
with high dietary exposure to aflatoxin (46), and furthermore,
p53 mutations in hepatocellular carcinomas of patients from
regions where HBV is endemic show a strong bias toward the nontranscribed strand (47).
Taken together with the present demonstration that DNA repair is a very
efficient process in normal hepatocytes, these data indicate that
alteration by HBx of p53-dependent DNA repair could promote
hepatocarcinogenesis. This has implications for our understanding of
the ways in which HCC arises in chronic inflammatory liver disease and
possibly for the investigation of new treatments. Indeed, there are
additional properties of both HBx and p53 which will be interesting to
evaluate in hepatocytes: we have shown that p53 maintains hepatocyte
sensitivity to cytokines that promote growth and survival (20). There
are reports that HBx expression alters hepatocyte responses to
cytokines (48-53), and it is possible that these observations also
reflect an effect through p53. This possibility together with our
finding that deficiency in IRF-1 (21), a regulator of cellular
responses to cytokines, leads to a reduction in DNA repair, could have
implications for how antiviral treatments with cytokines might alter
hepatocyte repair efficiency.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
FosLacZ contains two copies of the RGC
p53-specific binding site placed upstream of a nonfunctional fos promoter and a lacZ gene (27). The HBx
expression vector (15) pCMV-x1 encoded for the entire HBx open reading
frame under the control of the cytomegalovirus (CMV). Efficient
expression from the HBx expression vector was monitored by reverse
transcriptase-polymerase chain reaction using two different sets of primers.
CMV
(CLONTECH).
FosLacZ, and either the HBx
expression vector (pCMV-x1) or a control plasmid (pBR322) (ratio of
10:3). 36 h after transfection cells were UV-C irradiated (10 J/m2) or not; cultured for a further 4, 8, 14, or 24 h; and then lysed in 200 µl of reporter lysis buffer (Promega
Biotech). -Galactosidase activity was determined using an
o-nitrophenyl--D-galactopyranoside substrate
assay (Promega Biotech) and is expressed relative to the amount of
protein (Bio-Rad protein assay) recovered from each well, as described
previously (28).
CMV in a 1:1 ratio. 24 h after
transfection the cells were lysed in reporter lysis buffer.
-galactosidase activity resulting from the undamaged pCMV expression/µg of protein.
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
View larger version (22K):
[in a new window]
Fig. 1.
Global genomic nucleotide excision repair in
wild-type and p53 
/ mouse primary hepatocytes.
Removal of CPDs and 6-4 photoproducts was performed on genomic DNA
extracted from wild-type, p53 /+, and p53 /
hepatocytes 2, 6, 12, or 24 h after UV treatment (10 J/m2). Two separate slot blots were performed, in
triplicate, using DNA from three wild-type, one heterozygous, and two
p53-null mice.
/ cells compared with wild-type
cells (Fig. 2). Taken together with our previous results in mouse primary hepatocytes (21) and in human fibroblasts (29, 31, 32), this suggests that p53 is required for
efficient global nucleotide excision repair but not for
transcription-coupled repair.
View larger version (25K):
[in a new window]
Fig. 2.
Time course of strand-specific removal of
CPDs from the DHFR gene in wild-type and p53 / mouse
primary hepatocytes. Quantitative Southern hybridization with
strand-specific RNA probes to the DHFR gene was performed on
BamHI-restricted DNA isolated from wild-type (
, 
),
p53/+ (
, 
), and p53/ (
, 
)
primary hepatocytes 6, 12, and 24 h after UV irradiation (10 J/m2). DNA samples from each mouse (two wild-type, one
heterozygous, and one p53-null) were used for two
independent Southern blots, each probed for both the transcribed (,
, ) and the nontranscribed strand (, , ) of the DHFR
gene.
/ cells. The rate of apoptotic cell death was
1-5%.
View larger version (22K):
[in a new window]
Fig. 3.
Effect of HBx expression on p53 stabilization
(panels A and B) and transactivation function
(panel C). Panel A, primary
wild-type hepatocytes were transfected as described under
"Experimental Procedures." 36 h after transfection, cells were
treated with UV (10 J/m2) or not, and p53
immunocytochemistry was performed. Results give the percentage of
nuclear immunopositivity in wild-type culture transfected with pCMV-x1
or a control plasmid at various times after UV treatment of one typical
experiment. The experiment was performed three times with similar
results. Panel B, percentage of cells that are p53-positive
among primary hepatocytes transfected with pCMV-x1 relative to
hepatocytes transfected with a control plasmid, averaged from three
independent experiments ± S.E. Panel C, hepatocytes in
culture for 24 h were cotransfected with the p53 reporter plasmid
(RGC FosLacZ) and either the HBx expression vector pCMV-x1 or a
control plasmid as described under "Experimental Procedures."
26 h later, cells were UV irradiated (10 J/m2), and at
various times after treatment, p53 reporter expression was measured.
Results show the average -galactosidase activity ± S.E. for
three independent transfections at various times after UV treatment.
The experiment was performed twice with similar results.
View larger version (20K):
[in a new window]
Fig. 4.
Host reactivation of a damaged plasmid.
Hepatocytes previously transfected with the HBx expression vector
(pCMV-x1) or with a control plasmid were cotransfected with an
undamaged pCMV plasmid and a CAT reporter plasmid damaged with the
indicated doses of UV-C (0, 500, 1,000 J/m2) as described
under "Experimental Procedures." The CAT activities 24 h after
transfection, corrected according to the transfection efficiency, are
given relative to the activity of an untreated plasmid transfected
under the same conditions. The figure shows the average CAT activity
from three independent transfections. The experiment was performed
twice with similar results.
/ cells was
significantly less than wild-type cells (mean ± S.E. are
23.5 ± 1.08 and 10.6 ± 0.47 for wild-type and
p53-null hepatocytes, respectively). Transfection of the
HBx-expressing plasmid in p53-null cells had no significant
effect (mean ± S.E. are 10.6 ± 0.47 for untransfected versus 11.6 ± 0.50 for pCMV-x1-transfected
hepatocytes), whereas in wild-type cells, expression of HBx resulted in
a decreased grain index (10.9 ± 0.57 and 23.5 ± 1.08 for
transfected and nontransfected, respectively) to a level similar to
that found in p53-null cells. Hence, HBx expression reduces
unscheduled DNA synthesis of wild-type cells to the level of
p53-null cells but does not further alter the unscheduled
DNA synthesis activity of p53-null cells. Taken together,
these results indicate that HBx affects p53-dependent repair, i.e. global repair, especially the repair of the
CPDs.
View larger version (28K):
[in a new window]
Fig. 5.
Unscheduled DNA synthesis. Unscheduled
DNA synthesis was quantified as described under "Experimental
Procedures" in hepatocytes treated with 0 or 20 J/m2. The
figure represents the proportion of cells of different grain index for
primary hepatocytes transfected with a control plasmid (black
columns) and with the HBx expression vector (dotted
columns). Cells with a grain index higher than 40 are undergoing
replicative DNA synthesis.
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
| |
ACKNOWLEDGEMENTS |
|---|
Many thanks to Prof. S. Friend who gave us
the p53 reporter plasmid pRGCFosLacZ, Prof. Curtis C. Harris and Dr.
X.W. Wang for the HBx expression vector pCMV-x1, and Dr. T. Mori for
the TDM-2 and 64M-2 antibodies.
| |
FOOTNOTES |
|---|
* This work was supported by the Scottish Hospital Endowments Research Trust, the Melville Trust (Edinburgh), and the NCI, National Institutes of Health.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.
To whom correspondence should be addressed: Dept. of Pathology,
University Medical School, Teviot Place, Edinburgh, EH8 9AG, Scotland.
Tel.: 44-131-650-2917; Fax: 44-131-650-6528; E-mail: s.prost{at}ed.ac.uk.
The abbreviations used are: HBV, hepatitis B virus; HCC, hepatocellular carcinoma; HBx, hepatitis B x protein; CMV, cytomegalovirus; CAT, chloramphenicol acetyltransferase; DHFR, dihydrofolate reductase.
| |
REFERENCES |
|---|
|
Top
Abstract
Introduction
Procedures
Results
Discussion
References
|
|---|
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  J. K. Jung, H. J. Kwun, J.-O. Lee, P. Arora, and K. L. Jang Hepatitis B virus X protein differentially affects the ubiquitin-mediated proteasomal degradation of beta-catenin depending on the status of cellular p53 J. Gen. Virol., August 1, 2007; 88(8): 2144 - 2154. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. T. C. Lee, J. Ren, E.-T. Wong, K. H. K. Ban, L. A. Lee, and C. G. L. Lee The Hepatitis B Virus X Protein Sensitizes HepG2 Cells to UV Light-induced DNA Damage J. Biol. Chem., September 30, 2005; 280(39): 33525 - 33535. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. J. Kwun and K. L. Jang Natural variants of hepatitis B virus X protein have differential effects on the expression of cyclin-dependent kinase inhibitor p21 gene Nucleic Acids Res., April 23, 2004; 32(7): 2202 - 2213. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Y. Ahn, E. Y. Jung, H. J. Kwun, C.-W. Lee, Y.-C. Sung, and K. L. Jang Dual effects of hepatitis B virus X protein on the regulation of cell-cycle control depending on the status of cellular p53 J. Gen. Virol., November 1, 2002; 83(11): 2765 - 2772. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. R. Madden, M. J. Finegold, and B. L. Slagle Altered DNA Mutation Spectrum in Aflatoxin B1-Treated Transgenic Mice That Express the Hepatitis B Virus X Protein J. Virol., October 11, 2002; 76(22): 11770 - 11774. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Tan and G. Chu p53 Binds and Activates the Xeroderma Pigmentosum DDB2 Gene in Humans but Not Mice Mol. Cell. Biol., May 15, 2002; 22(10): 3247 - 3254. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. R. Madden, M. J. Finegold, and B. L. Slagle Hepatitis B Virus X Protein Acts as a Tumor Promoter in Development of Diethylnitrosamine-Induced Preneoplastic Lesions J. Virol., April 15, 2001; 75(8): 3851 - 3858. [Abstract] [Full Text]
|
|
|
|
|
|
C. R. Madden, M. J. Finegold, and B. L. Slagle Expression of Hepatitis B Virus X Protein Does Not Alter the Accumulation of Spontaneous Mutations in Transgenic Mice J. Virol., June 1, 2000; 74(11): 5266 - 5272. [Abstract] [Full Text]
|
|
|
|
|
|
M. L. Smith, J. M. Ford, M. C. Hollander, R. A. Bortnick, S. A. Amundson, Y. R. Seo, C.-X. Deng, P. C. Hanawalt, and A. J. Fornace Jr. p53-Mediated DNA Repair Responses to UV Radiation: Studies of Mouse Cells Lacking p53, p21, and/or gadd45 Genes Mol. Cell. Biol., May 15, 2000; 20(10): 3705 - 3714. [Abstract] [Full Text]
|
|
|
|
|
|
Q. Zhu, M. A. Wani, M. El-mahdy, and A. A. Wani Decreased DNA Repair Efficiency by Loss or Disruption of p53 Function Preferentially Affects Removal of Cyclobutane Pyrimidine Dimers from Non-transcribed Strand and Slow Repair Sites in Transcribed Strand J. Biol. Chem., April 6, 2000; 275(15): 11492 - 11497. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Otsuka, N. Kato, K.-H. Lan, H. Yoshida, J. Kato, T. Goto, Y. Shiratori, and M. Omata Hepatitis C Virus Core Protein Enhances p53 Function through Augmentation of DNA Binding Affinity and Transcriptional Ability J. Biol. Chem., October 27, 2000; 275(44): 34122 - 34130. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Jaitovich-Groisman, N. Benlimame, B. L. Slagle, M. H. Perez, L. Alpert, D. J. Song, N. Fotouhi-Ardakani, J. Galipeau, and M. A. Alaoui-Jamali 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 J. Biol. Chem., April 20, 2001; 276(17): 14124 - 14132. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||
