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INTRODUCTION |
Apoptosis is an active process of self-destruction that is
important in both the development and maintenance of multicellular organisms (1). Several viral gene products affect apoptosis by
interacting directly with components of the highly conserved biochemical pathways, which regulate cell death. Most viruses have
evolved strategies to block or induce apoptosis depending upon the
cellular environment (2, 3). The human immunodeficiency virus Tat (4)
and the human T-cell leukemia virus Tax (5) induce apoptosis. On the
other hand, the IE1 and IE2 proteins of the cytomegalovirus (6) have
the ability to block apoptosis. EIA/EIB of the adenovirus (6, 7) or the
T-antigen of SV40 (8, 9) may either block or induce apoptosis.
Phosphatidylinositol 3-kinase
(PI3K)1 is recruited and
activated during the intracellular signal transduction of many growth
factor receptors and has been implicated in the signaling of survival
factors (10). Altered phosphorylation and turnover of
phosphatidylinositol (PI) has also been demonstrated in cells
transformed by DNA and RNA tumor viruses (11). PI3K phosphorylates
inositol lipids that act as second messengers for several targets,
including the serine-threonine Akt kinase (12-14). Akt was initially
described as an oncogene and is activated by serum and a variety of
growth factors sharing the ability to activate the PI3K, such as
platelet-derived growth factor, epidermal growth factor, bovine
fibroblast growth factor, insulin (12, 13, 15, 17), insulin-like
growth factor (18), and interleukin 2 (19). Activation of Akt is known
to deliver a survival signal that inhibits apoptosis induced by growth
factor withdrawal (20). Akt phosphorylates Bad, thereby blocking it
from binding and inactivating Bcl-2 and Bcl-XL, two antiapoptotic Bcl-2
family members (21), which eventually suppress apoptosis of transformed
cells. Activation of Akt ultimately leads to inhibition of the caspase
activity and protection from apoptotic cell death (21). In cardiac
muscle cells, the activation of PI3K has been identified to suppress caspase 3 activation and inhibit the occurrence of apoptosis (22). HBx
is a causative agent of hepatocellular carcinoma, and during tumorigenesis, the HBx gene product is known to play an
important role in the alteration of gene expression, sensitizing cells
to apoptotic killing and deregulating cell growth arrest (23, 24). HBx
has been shown to complex with p53, inhibiting sequence-specific DNA
binding in vitro and p53-mediated transcriptional activation in vivo (25), which eventually inhibits
p53-dependent apoptosis. HBx was also shown to block
apoptosis (26) and induce transformation in NIH3T3 cells (27). Besides
the anti-apoptotic effect, HBx has also been shown to induce apoptosis
in hepatoma cells (28). Therefore, the fate of infected cells
expressing HBx is likely to be determined by the balance between
apoptotic and anti-apoptotic signals of viral, cellular, and
environmental origin. The ability of HBx to modulate cell survival is
potentially relevant for both viral pathogenicity in acute and chronic
HBV infection as well as for the late development of hepatocellular
carcinoma. In the long term, it is the anti-apoptotic function of HBx
that is likely to be the major determinant for manifestation of the
transformed phenotype. From this perspective, it is of central
importance to define the molecular mechanisms underlying HBx-induced
dysregulation of the cell death program. In this study we show that HBx
activates PI3K and activated PI3K can lead to phosphorylation of Akt
and Bad in CHL cells in a p53-independent manner. We also demonstrate that HBx suppressed the activation of caspase 3, through PI3K signaling, and suppresses apoptosis in CHL cells. In conclusion, we
demonstrate that HBx acts as an anti-apoptosis agent through the
HBx-PI3K-Akt-Bad pathway and inactivates caspase 3 activity in a
PI3K-dependent manner that is at least partially p53 independent.
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MATERIALS AND METHODS |
Tissue Culture--
Chang liver cells (CHL) and Chang liver
cells transiently transfected with HBx (CHL-X) were cultured
in Dulbecco's modified Eagle's medium (DMEM) containing 100 units/ml
penicillin, 0.1 mg/ml streptomycin, 0.25 µg/ml amphotericin, 10 nM minimal essential medium non-essential amino acid, and
10% heat-inactivated fetal bovine serum (FBS). Cells were incubated in
a 95% air and 5% CO2 atmosphere. Chang liver cells are
undifferentiated hepatocytes of human origin.
Cell Growth Assays and Induction of Apoptosis--
Cells were
plated at a density of 5 × 105 cells per well in
6-well plates and were grown for 4 days in myo-inositol-free
DMEM supplemented with 10% fetal bovine serum, 5 µM
myo-inositol, and increasing amounts of
1-D-3-deoxy-3-fluoro-myo-inositol. Cell viability was determined by the ability of the cells to exclude trypan
blue. 1-D-3-Deoxy-3-fluoro-myo-inositol was
obtained from Moravek Biochemicals. Inc. (Brea, CA). To induce
apoptosis by serum deprivation, cells grown on 100-mm dishes (Falcon)
to 90% confluency were placed in serum free DMEM for 24 h. For
induction of apoptosis by drugs, 2 µM etoposide (Sigma
Chemical Co.) and 10 µM staurosporine (Sigma) were added
to the medium (DMEM + 10% FBS) for 24 h.
Plasmids--
All plasmids (WT (XE-1), ND1-4, CD1-4, and XE-6)
used for the expression of native and mutant X-genes in
eukaryotic cells have been described previously (29). The control
plasmid pMAMneo was purchased from CLONTECH (Palo
Alto, CA). The Akt cDNA allelic pack, which contains
four transfection grade eukaryotic expression vectors, containing
activated, dominant negative, and wild type Akt1 alleles under the
control of the cytomegalovirus promoter and the empty vector control,
for use in either transient or stable transfection of mammalian cells,
were purchased from Upstate Biotechnology Inc. (Lake Placid, NY).
Antibodies--
The antibodies used in this study include an Akt
antibody (New England BioLabs Inc., Beverly, MA), which detects total
Akt (independent of phosphorylation state) protein levels, and a
phospho-Akt antibody (Ser-473) (New England BioLabs), which detects Akt
only when phosphorylated at Ser-473. A Bad antibody was used to detect total Bad (independent of phosphorylation state) levels. The
Phospho-Bad (Ser-112) antibody which detects Bad only when
phosphorylated at Ser-112 and a Phospho-Bad (ser 136) antibody which
detects Bad only when phosphorylated at Ser-136 were used.
Apoptosis Assays--
For analysis of DNA laddering, 3-5 × 106 cells were used according to the procedure of
Hermann et al. (30). Briefly, control or treated cells were
harvested, collected by centrifugation, and washed twice in cold
phosphate-buffered saline. Pellets were then suspended in 50 µl of
lysis buffer containing 1% Nonidet P-40, 20 mM EDTA, 50 mM Tris-HCl (pH 7.5). After centrifugation for 5 min at
1600 × g, the supernatants were incubated with 5 mg/ml
RNase A for 2 h at 56 °C in the presence of 1% SDS (w/v). Then
2.5 mg/ml proteinase K were added, and the incubation continued for at
least 2 h at 37 °C. DNA fragments were precipitated with 2.5 volumes of ethanol in the presence of 0.5 volume of 10 M
ammonium acetate at
20 °C overnight. After centrifugation, samples
were washed with 70% ethanol and resuspended in loading buffer.
Electrophoresis was performed in 0.5 × TBE buffer on 1% agarose
gels containing ethidium bromide. To measure the effect of the
myo-inositol analogue on apoptosis, CHL and CHL-X were
plated on coverslips at a density of 5 × 104 cells
per well in 6-well plates. After 2 days, the medium was changed to
myo-inositol-free DMEM supplemented with 10% fetal bovine
serum, 5 µM myo-inositol, and 2 mM
analogue, and cells were grown for 2 days. To measure the effect of
serum deprivation on apoptosis, 1 × 105 to 4 × 105 cells were plated on coverslips in 8-cm2
plates and grown to 60-80% confluency in serum free DMEM for 24 h. 4',6'-Diamidino-2-phenylindole (DAPI) staining and the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay (Roche Molecular Biochemicals, Mannheim, Germany) with
attached cells were used to detect apoptotic cells. To measure apoptotic cell death by detection of bromodeoxyuracil-labeled DNA
fragments in the cytoplasm of affected cells, a photometric enzyme-linked immunosorbent assay was used (Roche Molecular
Biochemicals). Viable cells were counted after staining with trypan
blue (31).
Kinase Assays--
PI3K activity in immune complexes was assayed
as described (32). Briefly, cell lysates used for PI3K were prepared in
lysis buffer (20 mM Tris-HCl (pH 7.4), 140 mM
NaCl, 1% Nonidet P-40, 10 mM NaF, 1 mM
Na3VaO4, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, leupeptin (5 µg/ml),
and 5 mM benzamidine), and proteins were immunoprecipitated
with a phosphotyrosine antibody. Immunoprecipitates were washed twice
in lysis buffer, containing 0.5 M LiCl and 25 mM Hepes (pH 7.2) and dissolved in TNE (10 mM
Tris (pH 7.4), 100 mM NaCl, and 1 mM EDTA).
Sonicated phosphatidylinositol (at a concentration of 30 µg per
reaction) was added to the immunoprecipitates, and the kinase reaction
was started by addition to a reaction mixture of 20 mM
Hepes (pH 7.2), 5 mM MgCl2, 20 µM
ATP, and 15 µCi of [
-32P]ATP in a total volume of 50 µl. The reaction mixture was incubated for 20 min at 25 °C. The
reaction was stopped with 100 µl of 1 N HCl, and the
lipids were extracted with 200 µl of a 1:1 mixture of
MeOH:CHCl3, spotted on the oxalate-coated TLC plate, and
run in CHCl3:MeOH:4 M acetic acid (9:7:2, v/v).
PI3K activity was quantified by autoradiography. For Akt kinase assay,
immunoprecipitates were washed twice with lysis buffer, containing 25 mM Hepes (pH 7.2), 1 M NaCl, 0.1% bovine serum
albumin, 10% glycerol, and 1% Triton X-100 and twice with kinase
buffer containing 20 mM Hepes (pH 7.2), 10 mM
MgCl2, 10 mM MnCl2, 1 mM dithiothreitol, 5 µM ATP, and 0.2 mM EGTA. Reactions were done in kinase buffer supplemented with 2 µg of protein kinase A inhibitor (Upstate Biotechnology Inc.),
10 µCi of [
-32P]ATP (Amersham Pharmacia Biotech,
Buckinghamshire, England) and histone H2B (Roche Molecular
Biochemicals,) 500 ng per 40-µl assay reaction and incubated for 10 min at 30 °C in a shaking incubator. The reaction was terminated by
blotting 25 µl of the supernatant fraction on a 2- × 2-cm P81 paper
square. Squares were washed three times with 0.75% phosphoric acid and
once with acetone. The radioactivity was measured in a scintillation
counter, and the Akt activity was determined.
In Vitro Phosphorylation of Akt and Bad--
After transfection,
CHL and CHL-X were lysed in radioimmune precipitation buffer (1 × PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml aprotinin, 2 µg/ml leupeptin). Lysates were clarified by centrifugation at 10,000 × g for 10 min at 4 °C. Immunoprecipitation
was carried out by incubating the lysates with the
phosphorylation-independent Akt antibody or Bad antibody. 20 µg of
protein A/G agarose beads was added for 2 h at 4 °C.
Immunoprecipitates were washed five times with PBS containing 0.1%
Triton X-100. Immunoprecipitates were boiled with sample buffer and
electrophoresed in a 7.5% SDS-polyacrylamide gel and transferred onto
nitrocellulose paper (Amersham Pharmacia Biotech). Immunoblotting was
performed as previously described (33). Akt phosphorylation at serine
473 was detected with the phospho-specific Akt (Ser-473) antibody from
New England BioLabs, Inc. Bad phosphorylation at serine 112 or 136 was
detected with the phospho-specific Bad (Ser-112 or -136) antibody from
New England BioLabs, Inc. Anti-rabbit-IgG-horseradish peroxidase was
used as the secondary antibody. Detection of antigen-bound antibody was
carried out using enhanced chemiluminescence (ECL) (Amersham Pharmacia
Biotech) as described (34).
Detection and Quantification of Apoptotic Nuclei--
For nuclei
staining, cells were fixed with 70% ethanol for 10 min, rinsed three
times with PBS, and stained with 1 µg of Hoechst 33258 (Sigma, 1 µg/ml) in PBS for 10 min. The slides were observed with a
conventional light and fluorescence microscope. For quantification, stained nuclei in 10 randomly chosen fields were counted at 40× magnification. Only the fluorescent signals showing a typical morphology of apoptosis were selected. The apoptotic rate was expressed
as the percentage of total cells containing stained nuclei.
Caspase 3 (CPP32) Activity Assay--
Caspase 3 activity was
determined with the ApoAlet colorimetric assay kit
(CLONTECH) according to the manufacturer's
protocol. Briefly, protein extracts (150 µg) were incubated with
Asp-Glu-Val-Asp-chromophore p-nitroanilide for 1 h at 37 °C, and the chromophore p-nitroanilide was
spectrophotometrically measured at
= 405 nm. Inhibitors were
added, at the following concentration: 200 nM wortmannin (Sigma), 100 µM LY 294002 (Sigma) to the cells for
16 h.
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RESULTS |
Serum Deprivation Induces Apoptosis in CHL Cells--
We first
determined whether the number of CHL cells decreased following
withdrawal of serum from the media. 106 cells were
incubated without serum for 5 days and then counted every day after
staining with trypan blue. As shown in Fig.
1A, the percentage of CHL
surviving cells was 92 ± 2% and 61 ± 3%, respectively,
after 24- and 48-h serum deprivation compared with the 100% surviving
cells at 0-h serum. We then determined whether cell death was caused by
apoptosis following withdrawal of serum. Genomic DNA from CHL cells
were assessed for the presence of DNA fragmentation by a "ladder"
pattern on agarose gel electrophoresis, indicative of internucleosomal
cleavage, a hallmark of apoptosis. DNA fragmentation was observed in
CHL cells after 24 h of serum deprivation (Fig.
1B).

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Fig. 1.
Effect of HBx on serum deprivation-induced
and pro-apoptotic stimuli-induced apoptosis in CHL cells.
A, time course of the effect of HBx on cell survival in
serum-deprived CHL cells. 1×106 CHL and CHL(X) were
cultured in serum free DMEM for the indicated times, and viable cells
were counted by trypan blue staining. The results are expressed as
percentages of surviving cells that did not take up the trypan blue dye
in the total cell population. Each data point is the mean of triplicate
determinations. B, serum deprivation-induced apoptosis in
CHL cells. Genomic DNA was isolated from CHL cells incubated in the
absence of FBS for 0 h (lane 2) and 24 h
(lane 3) and electrophoresed on a 1.5% agarose gel as
described under "Materials and Methods" (M, molecular
weight markers: 100-bp marker (lane 1)). C,
effect of HBx on serum deprivation-induced apoptosis in CHL cells.
Genomic DNA was isolated from CHL and CHL-X cells incubated in the
absence of FBS for 0 h (lanes 2, 5), 24 h (lanes 3, 6), and 48 h (lanes
4, 7) and electrophoresed on a 1.0% agarose gel
electrophoresis. D, effect of HBx on pro-apoptotic
stimuli-induced apoptosis. CHL cells transfected with HBx or
mock-transfected were cultured for 24 h in the presence or absence
of 10 µM staurosporine or 2 µM etoposide.
Genomic DNA was isolated and fractionated on 1.0% agarose gel
electrophoresis.
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HBx Rescues Serum-deprived and Pro-apoptotic Stimuli-induced
Apoptosis--
Given that previous studies have implicated the role of
HBx as a modulator of apoptosis, we examined the effect of HBx on serum
deprivation and pro-apoptotic stimuli-induced apoptosis in CHL cells.
Transfection with HBx resulted in increased cell survival to 118 ± 3% and 112 ± 2% after 24- and 48-h serum deprivation (Fig.
1A). As shown in Fig. 1C, genomic DNA
fragmentation was not observed in CHL-X cells upon serum deprivation
for 24 and 48 h (lane 6 and 7), whereas in
CHL cells DNA fragmentation occurred (lanes 3 and
4). Treatment of CHL cells with 10 µM
staurosporine and 3 µM etoposide resulted in DNA
fragmentation (lanes 3 and 6) whereas HBx rescues
the cells from the apoptotic process elicited by the pro-apoptotic
stimuli 10 µM staurosporine or 3 µM
etoposide (Fig. 1D, lanes 4 and
7).
1-D-3-Deoxy-3-fluoro-myo-inositol Inhibits Net Growth
and Stimulates Apoptotic Death in CHL-X but Not CHL
Cells--
1-D-3-Deoxy-3-fluoro-myo-inositol,
an analogue of myo-inositol, is a potent antagonist for
cells exhibiting constitutively activated PI3K. It acts as a substrate
for mammalian phosphatidylinositol synthase and is incorporated into
phosphatidyl inositol (32, 35). The effect of the
1-D-3-deoxy-3-fluoro-myo-inositol on cell
viability is unrelated to phosphatidylinositol 4,5-biphosphate (PIP2) and phospholipase C signaling as demonstrated by the
response of intracellular calcium levels to growth factors (36, 37). To
determine the effect of
1-D-3-deoxy-3-fluoro-myo-inositol on the growth
of CHL and CHL-X cells, cells were grown with increasing amounts of
1-D-3-deoxy-3-fluoro-myo-inositol. The data in
Fig. 2A show that CHL-X cells
exhibited marked growth inhibition compared with CHL cells when grown
in the presence of 1-D-3-deoxy-3-fluoro-inositol (up to 8 mM). Because the analogue was incorporated equally well into phospholipids by both cell types, differences in viability was not
due to the differences in the cellular uptake into both cell types
(data not shown). Therefore, these results show that, although normal
and transformed cells take up the
1-D-3-deoxy-3-fluoro-myo-inositol equally, the
growth inhibitory effect was more evident in transformed cells. To
determine whether the myo-inositol analogue induced apoptosis in CHL-X cells, CHL and CHL-X cells were stained with DAPI,
and the TUNEL assay was performed after growing the cells in the
presence of 2 mM of the analogue (Fig. 2B,
I). CHL-X cells after treatment with the
myo-inositol analogue showed apoptotic phenomena with both
staining techniques (Fig. 2B, I). In the presence of
1-D-3-deoxy-3-fluoro-myo-inositol, the
percentage of apoptotic CHL-X cells rose nearly 3-fold above the
level seen in the absence of the analogue, whereas that for CHL cells
remained unchanged (Fig. 2B, II). These results show that
blocking the PI3K pathway in cells transformed by HBx drives these
cells toward apoptosis.

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Fig. 2.
Effect of
1-D-3-deoxy-3-fluoro-myo-inositol on the cell
growth and apoptosis on CHL and CHL-X cells. A, growth
of cells in the presence of
1-D-3-deoxy-3-fluoro-myo-inositol. Normal ( )
and HBx transformed Chang liver ( ) cells were
plated at subconfluency in myo-inositol-free DMEM containing
10% dialyzed calf serum, 5 µM myo-inositol
and with or without
1-D-3-deoxy-3-fluoro-myo-inositol at the
concentrations shown and grown for 4 days. Growth was measured by the
trypan blue staining method as described under "Materials and
Methods" and plotted as a percentage of the control without
1-D-3-deoxy-3-fluoro-myo-inositol. B,
effect of 1-D-3-deoxy-3-fluoro-myo-inositol on
apoptosis. I, DAPI and TUNEL staining of CHL-X cells showing
DNA condensation and fragmentation; II, percentage of
apoptotic cells in culture grown for 2 days on medium containing 5 µM myo-inositol (open bars) or 5 µM myo-inositol and 2 mM
1-D-3-deoxy-3-fluoro-myo-inositol (gray
bars) and quantitated by counting >1000 cells in duplicate
experiments.
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Wortmannin Inhibits Net Growth and Stimulates Apoptotic Cell Death
in CHL-X but Not in CHL Cells--
Further evidence that PI3K is
involved in the prevention of apoptosis in CHL-X was sought by using
wortmannin, a potent inhibitor of PI3K (38). When the CHL and CHL-X
cells were grown in the presence of increasing concentrations of
wortmannin, CHL-X cells showed marked growth inhibition compared with
CHL cells (Fig. 3A). This
inhibitory effect of wortmannin is similar to that of the
myo-inositol analogue shown in Fig. 2A. CHL and
CHL-X cells growing in 10% calf serum were treated with increasing
concentrations of wortmannin for 2 h before fixation.
TUNEL-positive CHL-X cells were observed with >10 nM
wortmannin, whereas CHL cells failed to undergo apoptosis after
treatment with wortmannin at concentrations up to 1000 nM
(Fig. 3B, I). Measurement of apoptotic cell death by detection of bromodeoxyuracil-labeled DNA fragments in the cytoplasm
of 100 nM wortmannin-treated cells using a photometric enzyme-linked immunosorbent assay also showed the same results (Fig.
3B, II). The -fold induction of apoptosis was
increased as the wortmannin treatment time increased in CHL-X cells
whereas no increase was observed in non-transformed CHL cells. These
results support the involvement of PI3K in preventing apoptosis in
cells transformed by HBx but not in non-transformed CHL
cells.

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Fig. 3.
Effect of wortmannin on the growth and
apoptosis on CHL and CHL-X cells. A, normal CHL ( )
and CHL-X ( ) cells were plated at subconfluency in DMEM containing
10% dialyzed calf serum and wortmannin at the concentrations shown and
grown for 4 days. Growth was measured by the trypan blue assay as
described under "Materials and Methods" and plotted as a percentage
of that of the control without wortmannin. B, effect of
wortmannin on apoptosis of CHL and CHL-X cells. I, the
effect of increasing amounts of wortmannin on apoptosis of CHL and
CHL-X cells. Cells grown in DMEM containing 10% calf serum were
treated with wortmannin for 2 h before fixation for DAPI staining
and the TUNEL assay. The percentage of apoptotic cells was quantitated
by counting the cells in duplicate experiments. II, effect
of incubation time with wortmannin on apoptosis of CHL and CHL-X
cells.
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Activation of PI3K and Akt in HBx-transformed CHL Cells--
To
test whether PI3K is associated with HBx-activated CHL cell survival,
we obtained phospho-tyrosine antibody immunoprecipitates of CHL cells
for determination of the PI3K activity, as described under "Materials
and Methods" (Fig. 4A).
Sonicated phosphatidyl inositol was used as substrate, and the
production of phosphoinositol phosphate was measured.
Immunoprecipitates from HBx-transfected cultures contained
three times as much PI3K activity as those from non-transfected cells
(Fig. 4A, I, lanes 1 and
2). The inhibition of PI3K by wortmannin is
dose-dependent (Fig. 4A, I,
lanes 3 and 4). Wortmannin at a concentration of
100 nM almost completely inhibited the formation of
phosphoinositol phosphate in HBx-transfected CHL cells. Fig.
4A (II) shows the quantification of the results of Fig. 4A (I), which was obtained by scanning
the autoradiographic film using a phosphorimaging analyzer.
PIP2, a product of PI3K, directly regulates Akt (39), the
serine/threonine kinase also designated protein kinase B or Rac (40).
Akt has been implicated in inhibition of apoptosis by withdrawal of
serum and of certain growth factors (13) in cell cultures. To determine
whether transformation by HBx leads to activation of Akt,
in vitro kinase assays of Akt immunoprecipitates were
performed with extracts from serum-starved cells as described under
"Materials and Methods." Akt activity, shown in Fig. 4B,
was roughly 2-fold higher in cells transformed by wild-type
transcriptional HBx and HBx-ND-1 (N-terminal
10-amino acid deletion mutant, which showed wild-type transcriptional
transactivation activity) than in non-transformed or cells transformed
by mutant HBx-ND-5 (N-terminal 70-amino acid deletion
mutant, which showed no transcriptional transactivation activity) or
HBx-CD-2 (C-terminal 17-amino acid deletion mutant, which
showed no transcriptional transactivation activity). These results
clearly showed that HBx regulates the serine/threonine kinase Akt
activity. The Akt activity was found to correlate with the PI3K lipid
product PIP2 in vivo and in vitro
(41). Binding of PIP2 occurred within the Akt Pleckstrin homology (PH) domain and facilitated dimerization of Akt (42).

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Fig. 4.
In vitro activation of PI3K and
Akt by the HBx protein. A, in vitro
activation of PI3K by the HBx protein. I. CHL and CHL-X cells were
treated with or without wortmannin in serum free media for 24 h.
PI3K was immunoprecipitated from cell lysates using tyrosine kinase
antibodies. Sonicated phosphatidyl inositol was used as substrate. The
PI3K activity was determined by measuring the product phosphoinositol
phosphate (PIP) that was visualized by autoradiography
following ascending chromatography on silica gel plates. II,
the quantification presented in the histograms was obtained by scanning
the autoradiographic film using the BAS 1500 phosphorimaging analysis
program (Fuji Co.). B, in vitro activation
of Akt by the HBx protein. Akt was immunoprecipitated from lysates
prepared from cells (CHL cells and CHL cells transfected with
HBx, ND-1, ND-5, and CD-2) that had been deprived of serum
for 24 h. Determination of Akt activity was carried out by the
method of Dudek et al. (40). Activity was quantitated by the
scintillation counting method provided by Upstate Biotechnology
Inc.
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Akt mutated in the PH domain was not activated by PI3K in
vivo or by PIP2 in vitro, and was impaired
in its binding to PIP2. Thus HBx activated PI3K production
of phosphatidylinositol 3,4-disphosphate, which eventually
activates Akt by binding to the Akt PH domain.
Effect of HBx on Akt and Bad Phosphorylations--
After
transfection with HBx or a dominant negative Akt
expression vector or treated with the PI3K inhibitor wortmannin, cell lysates were immunoblotted with Akt antibody for the total content of
Akt or Akt-P-473 the latter using an antibody to the phosphorylated form of Akt. CHL cells maintained a basal level of phosphorylations of
Akt at Ser-473 (Fig. 5A,
lane 1). However, phosphorylation of Akt by HBx was
dose-dependent (Fig. 5A, lanes 2-4)
and was inhibited by the dominant negative Akt expression
vector (Fig. 5A, lane 5). Cells treated with 20, 30, and 40 nM wortmannin showed decreased levels of
phosphorylated Akt (Fig. 5A, lanes 6,
7, and 8). Importantly, transfection of CHL cells
with either HBx alone or HBx together with
dnAkt or wortmannin produced no change in the total content
of Akt. The effect of HBx on Bad phosphorylation was also investigated
(Fig. 5B). CHL cells maintained a basal level of
phosphorylation of Bad at Ser-136 (Fig. 5B, lane
1).

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Fig. 5.
Phosphorylation of Akt and Bad by HBx and its
prevention by a PI3K inhibitor. A, induction by HBx of
the phosphorylation of Akt on Ser-473 and prevention of phosphorylation
by the PI3K inhibitor wortmannin. CHL cells were either transfected
with or without HBx and treated with 20, 30, and 40 nM wortmannin for 30 min. Cell lysates were prepared
(1.0 × 105 cells in 24-well plates), and
phosphorylated Akt (Akt-P-437) and total Akt were detected
by Western blotting as described under "Materials and Methods."
B, induction by HBx of the phosphorylation of Bad on serine
136 and its prevention by the PI3K inhibitor wortmannin. CHL cells were
either transfected with or without HBx and treated with 20, 30, or 40 nM wortmannin for 30 min. Cell lysates were
prepared and Bad-P-136 and Bad were detected by Western blotting as
described under "Materials and Methods."
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Phosphorylation of Bad by HBx was dose-dependent
(lanes 2 and 3) and was inhibited by a dominant
negative Bad expression vector (Fig. 5B,
lane 4). Cells treated with 20, 30, and 40 nM
wortmannin showed decreased levels of phosphorylated Bad (lanes
5, 6, and 7). Importantly, transfection of
CHL cells with either HBx alone or HBx together
with wortmannin produced no change in the total content of Bad (Fig.
5B, bottom panel). In addition, no staining was
detected when an antibody was used against the Ser-112 phosphorylated form of Bad, either in control, HBx, or HBx and wortmannin-treated cells (data not shown). These results clearly showed that Akt and Bad
phosphorylation by HBx was dependent on PI3K. Transient treatment with
a PI3K inhibitor or a dominant negative mutant of the serine-threonine
kinase Akt, the downstream target of PI3K, induces a blockade in cell
survival, i.e. apoptosis. The phosphorylation of Bad by Akt
kinase induced by HBx also induces cell survival, anti-apoptosis.
The HBx Protein Inhibits Caspase 3 Activity and PI3K Inhibitors
Block HBx Down-regulation of the Caspase 3 Activity--
We tested
whether HBx exerts an anti-caspase activity and how this correlates
with the anti-apoptotic function of HBx. Protein extracts were prepared
from CHL and CHL-X cells, and the caspase 3 (CPP32) activity was
analyzed. Serum deprivation caused a strong increase in the caspase
activity in the extracts from CHL cells (Fig.
6A) whereas the caspase
activity in the extracts from CHL-X cells remained at the same level as
in non-induced cells or in cells incubated with the caspase inhibitors
DEVD-fmk (1 µM) (Fig. 6A). Caspase 3 activity
was restored in CHL-X cells by adding the PI3K inhibitors, wortmannin,
or LY 294002 (Fig. 6B) whereas no effect was observed in
control cells. Taken together our results clearly demonstrate that HBx
inhibits CPP32 activity through a PI3K pathway, because PI3K inhibitors
block HBx down-regulation of caspase 3 activity.

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Fig. 6.
CPP32 activity assay. A, the
HBx protein inhibits caspase 3 activity in serum-starved cells. The
CPP32 activity was measured in cell extracts obtained from CHL and
CHL-X cells cultured in DMEM with (10%) or without (0%) FBS. Cell
extracts were preincubated with the substrate inhibitor DEVD-fmk for 30 min at 37 °C, and the CPP32 activity was measured as indicated under
"Materials and Methods." B, CPP32 reactivation by PI3K
inhibitors. CHL and CHL-X cells were incubated with either
wortmannin or LY294002 in DMEM medium containing 10% FBS for the CPP32
assay.
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HBx Blocks Apoptosis Induced by Serum Withdrawal in CHL Cells by a
p53-independent Process--
CHL cells are dependent upon serum for
survival and undergo apoptosis when treated with wortmannin. To
reconfirm the role for PI3K in the prevention of apoptosis by HBx, CHL
cells were transformed with Mam-HBx and selected with G418.
To determine whether the apoptotic response of these cells is
p53-dependent, stable expressing HBx clones were also
transfected with a temperature-sensitive p53 gene,
p53val135 (41), and selected for puromycin resistance.
p53val135 behaves like a dominant inhibitory mutant at the restrictive
temperature (38.5 °C) and exhibits wild-type function at 32 °C.
At the restrictive temperature, p53val135 is impaired in transcription
activation (42), repression (43), and nuclear translocation (44). To
determine the effect of p53 on apoptosis, cells were grown in the
presence of 10% FBS at 38.5 °C and either fixed for the assay of
apoptosis at 0 h or grown for 2 more days either at 38.5 °C in
serum-free medium or at 32 °C in medium with or without FBS and then
fixed (Fig. 7). Upon shifting to 32 °C
in the presence of serum, there was only a slight increase in the
percentage of apoptotic cells in all clones. However, when the serum
was removed from the CHL cells, there was roughly a 10-fold increase in
the percentage of apoptotic cells after 2 days at 32 °C and
38.5 °C. Cells expressing HBx showed roughly a 3-fold increase in
apoptotic cells under the same conditions, indicating the effect of
HBx-activated PI3K in partially blocking the apoptotic response brought
on by serum withdrawal. Protection against apoptotic death upon serum
withdrawal in CHL-X cells was not significantly temperature-sensitive.
The protection afforded by HBx-PI3K interaction thus appeared to be
p53-independent, at least to a larger extent.

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Fig. 7.
Serum deprivation-induced apoptosis in
Chang liver cells expressing p53val135 along with the
neomycin-resistant vector alone (MAMneo) or the
HBx-expressing vector
(MAMneo-X). Four plates of each cell type were
obtained, and cells were grown to 70% confluence in DMEM containing
10% FBS at 38.5 °C. Two plates were maintained in 10% serum, and
two plates were changed to serum-free medium. Cells in 10% serum were
either fixed at 0 h (vertically) or shifted to 32 °C
(hatched bar) and fixed at 48 h. Cells in 0% serum
either remained at 38.5 °C (stippled bar) or were shifted
to 32 °C (open bar), and then both were fixed at 48 h. Data are averages from two separate experiments.
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Identification of the Domain of the HBx Protein Involved in PI3K
Activation--
To identify the regions of HBx required for PI3K
activation, a series of HBx mutants were constructed (Fig.
8). Four N-terminal deletions, four
C-terminal deletions, and two point mutations in the HBx protein
were made (45). The PI3K activity was determined from
immunoprecipitates of mutant-X-transfected cell lysates
using tyrosine kinase antibodies. Phosphoinositol phosphate from
phosphatidylinositol was visualized by autoradiography on silica gel
plates. PI3K activities were diminished in successive deletions of the
N- and C-terminal regions of the HBx protein (Fig. 8, A and
B). In the N-terminal deletion mutant, removal of nine amino
acids did not affect the ability to activate PI3K. Rather, the
activation activity of the ND-1 mutant HBx protein was increased
compared with the wild-type HBx protein, in agreement with a recent
report that the first 20 N-terminal amino acid in the HBx sequence
might contribute to a repressor effect on X transactivation (33, 46).
Larger, internal deletions extending from amino acids 2 to 27 (ND-2), amino acids 2 to 40 (ND-3), and amino acids 2 to 52 (ND-3) resulted in
a 77-81% decrease in activity compared with the wild-type. Analysis
of C-terminal deletion mutants showed that amino acids 136~154 could
be deleted from HBx without affecting its ability to activate PI3K
(Fig. 8, A and B). However, more than a 90% loss of HBx-activating effect was obtained for CD-3 and CD-4. These results
appear to be due to the removal of an additional five amino acids
(VFVLG) from CD-2 as in the case of HBx transactivation of pSV2-CAT or
pMAMneo-CAT (33). Two point mutation constructs, XE-6 (amino acids 134)
and XE-7 (amino acids 55, 56) were used for HBx mutant activation of
the PI3K activity. Amino acid 134 is a leucine residue in the leucine
zipper-like region of amino acids 105-143 of the HBx protein whereas
amino acids 55 and 56 are leucine and arginine residues, which are well
conserved in 11 different subtypes of HBx sequences (45). Both mutants
showed a decrease of about 80% in PI3K activity compared with the
wild-type HBx (Fig. 8A). These results coincide with the
transcriptional transactivation results of HBx and mutant type HBx when
we used a pSV2-CAT vector as a reporter (Fig. 8, A and
B). Based on these experimental results, the regions
required for PI3K activation on the HBx protein overlap with the known
transcriptional domain of the HBx protein.

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Fig. 8.
Mapping of the PI3K activity activated by HBx
and HBx-mutants. A, schematic presentation of HBx
(amino acids 1-154) and the HBx deletion and insertion mutants that
were analyzed. For activation of PI3K, cells were cultured in DMEM with
10% serum and transfected with wild-type HBx or mutant HBx. For
transactivation, CHL cells were cotransfected with 10 µg of the
pSV-CAT reporter construct together with 20 µg of wild-type and
mutant type HBx expression vectors, and transient transactivation
assays were performed. B, the quantification of the PI3K
activity represented by the histograms was obtained by scanning the
autoradiographic film using the AS 1500 phosphorimaging analysis
program (Fuji Co.).
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DISCUSSION |
Phosphatidylinositol 3-kinase has been shown to mediate signaling
induced by numerous growth factors and tumor antigens (47, 48). PI3K is
a heterodimeric complex consisting of an 85-kDa regulatory subunit,
p85, and a 110-kDa catalytic subunit, p110 (48, 49). The p85 subunit
contains two Src homology 2 (SH2) domains, which bind to
tyrosine-phosphorylated receptors after stimulation of cells with
growth factors and, in this manner, recruit the p85·p110
complex to the cell membrane. PI3K appears to induce a variety
of cellular responses. These responses include the regulation of gene
expression (50, 51) and the activation of signaling kinases, which
function in different pathways (52, 53) as well as in membrane ruffling
(54), endocytosis (55), glucose transport, and DNA synthesis (56, 57).
Recently, it was demonstrated that PI3K regulates cell survival in
response to various apoptotic stimuli (58). Much evidence suggests that activation of PI3K may lead to suppress caspase 3 activity and DNA
fragmentation in a variety of cells. Most of the evidence derived from
studies using pharmacological inhibitors of PI3K (LY294002 and
wortmannin) shows that inhibition of PI3K with these compounds leads to
cell apoptosis or loss of the anti-apoptotic effect of growth factors
(59-61). Several other studies using dominant negative constructs of
PI3K to inhibit PI3K signaling confirmed that PI3K is essential in
maintaining cell survival (61, 62). Recently, Wu et al. (22)
demonstrated that activation of PI3K alone is sufficient to protect
cells from apoptotic induction and inhibition of caspase activation in
cardiac muscle cells. By using either in vivo or cell free
systems, it was shown that the phospholipids produced by PI3K mediate
PI3K signaling (53, 63). One of the generated products
phosphatidylinositol 3,4-diphosphate (53, 64), was able to
induce the kinase activity of the PI3K effector Akt (protein kinase B)
in vitro ~10-fold (39, 58). Signaling intermediates, which
bind phospholipids via domains homologous to pleckstrin such
as G-protein exchange factors and GTPase-activating proteins, are also
candidates for regulation by the products of PI3K (66, 67). Among
these, the downstream target of PI3K that mediates the anti-apoptotic
action of PI3K is Akt. The Akt pathway modulates sequential steps of
apoptotic signaling. Activation of Akt may lead to phosphorylation of
Bad (68), induction of Bcl-2 family of proteins (69), inhibition of
cytochrome c release from mitochondria (70) and
phosphorylation and inactivation of caspase 9 (71). However, some of
these observations are dependent on the experimental system used. It
appears that survival signaling may employ different antagonistic
mechanisms in different cells. In cardiomyocytes the survival signals
induced by insulin-like growth factor-I did not induce Bad
phosphorylation, although they stimulated PI3K and Akt. In this case
insulin-like growth factor-I induces anti-apoptosis in cardiomyocytes
through PI3K/Akt signaling, which is independent of Bad (22). In this study 1-D-3-deoxy-3-fluoro-myo-inositol, a
potent antagonist of cells exhibiting constitutively activated PI3K
(72), and the PI3K inhibitor wortmannin was used. Apoptosis in CHL
cells transformed by HBx is shown to be enhanced when PI3K
activity is inhibited by either growth of cells with
1-D-3-deoxy-3-fluoro-myo-inositol or after
treatment with wortmannin, indicating involvement of PI3K in blocking
apoptosis. In contrast, normal CHL cells are much less dependent on
PI3K for survival, as indicated by their resistance to death induced by
the inositol analogue and by wortmannin. These results suggest that
signal transduction via HBx through PI3K is important for
survival and protection from apoptosis. HBx also activates and
phosphorylates Akt and Bad through PI3K, because PI3K inhibitors block
HBx activation and phosphorylation of Akt and Bad. These results
suggest that HBx utilizes the PI3K/Akt/Bad survival pathway in the CHL
cell line. Caspase 3 plays a pivotal role in execution of apoptosis; ES
cells deficient in caspase 3 were resistant to apoptotic induction
(73). In human cardiomyopathy, apoptosis of cardiac muscle cells is
associated with release of cytochrome c and activation of
caspase 3 (74). Using inhibitors of caspases, Bialik et al.
(75) showed that cardiomyocyte apoptosis induced by serum and glucose
withdrawal can be attenuated. Our data show that HBx blocks caspase 3 activation during serum deprivation and caspase 3 activity is activated
by the PI3K inhibitors, wortmannin, and LY294002 as shown in Fig. 6.
These results imply that activation of caspase 3 during serum-deprived
apoptosis is attenuated by HBx through the PI3K signaling pathway. The
ability of a virus to delay host cell death is essential for viral
growth (76). Some DNA viruses, such as the adenovirus and SV40, inhibit
programmed cell death by inactivating p53. On the other hand, other DNA
tumor viruses like the polyoma virus that has no known direct
interaction with p53 (16) acts through PI3K and Akt and blocks
apoptosis through the middle T-antigen (16). Interestingly, viruses
that handle p53 directly by inactivation or degradation
(i.e. adenovirus, SV40, and papilloma virus) lack direct
mechanisms for activating PI3K. It has been proposed that a
pro-apoptotic effect facilitates viral spread and allows for evasion of
host-cell-mediated immunity (65). On the contrary, the inhibition of
apoptosis would have the effect of allowing for the accumulation of
potentially transforming mutations (65). The apparent discrepancy
between the effects of HBx on apoptosis could reflect opposing
concentration-dependent effects at different stages of
natural HBV infection. A possibility is that HBx inhibits apoptosis
during early hepatocyte infection and later on activates apoptosis to
facilitate HBV replication and spread. Either stimulation or inhibition
of apoptosis could lead to malignant transformation of hepatocytes. In
HBV, the interactions between the HBx protein and p53 have been
examined extensively (26), although the activation of PI3K by HBx
protein has not been reported. In this study, we identified that the
activation of PI3K and subsequently of Akt and Bad by the HBx protein
eventually causes anti-apoptosis in CHL cells. The PI3K activation
domains in the HBx protein are similar to the promoter transactivation domains as showed in our previous study (45). CHL cells showed marked
serum dependence for survival. In CHL cells expressing HBx proteins,
significant protection against apoptosis upon serum withdrawal was
identified. After transfection with p53val135, this
protection was temperature-independent, implying that HBx-induced anti-apoptosis is p53-independent. Consistent with our findings, p53
independence of anti-apoptosis induced by serum withdrawal has also
been observed in other cell types (47). Although some experimental
evidence showed HBx inactivation of p53 in hepatocytes, the p53
independence of anti-apoptosis by the HBx protein through the
PI3K-Akt-Bad pathway and inactivation of caspase 3 implies that HBx
functions through a p53-independent pathway in inducing an
anti-apoptosis phenomena in CHL cells.