The Hepatitis B Virus-X Protein Activates a Phosphatidylinositol 3-Kinase-dependent Survival Signaling Cascade*

Yoon Ik Lee, Sukmi Kang-Park, Su-Il DoDagger , and Young Ik Lee§

From the Liver Cell Signal Transduction Research Unit, Dagger  Animal Cell and Medical Glycobiology Research Unit, Bioscience Research Division, Korea Research Institute of Bioscience and Biotechnology, P. O. Box 115, Yusong, Taejon 305-600, Korea

Received for publication, December 14, 2000, and in revised form, February 27, 2001


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The hepatitis B virus-X (HBx) protein is known as a multifunctional protein that not only coactivates transcription of viral and cellular genes but coordinates the balance between proliferation and programmed cell death, by inducing or blocking apoptosis. In this study the role of the HBx protein in activation of phosphatidylinositol 3-kinase (PI3K) was investigated as a possible cause of anti-apoptosis in liver cells. HBx relieved serum deprivation-induced and pro-apoptic stimuli-induced apoptosis in Chang liver (CHL) cells. Treatment with 1-D-3-deoxy-3-fluoro-myo-inositol, an antagonist to PI3K, which blocks the formation of 3'-phosphorylated phosphatidyl inositol in CHL cells transformed by HBx (CHL-X) but not normal Chang liver (CHL) cells, showed a marked loss of viability with evidence of apoptosis. Similarly, treatment with wortmannin, an inhibitor of PI3K, stimulated apoptosis in HBx-transformed CHL cells but not in normal cells, confirming that HBx blocks apoptosis through the PI3K pathway. The serine 47 threonine kinase, Akt, one of the downstream effectors of PI3K-dependent survival signaling was 2-fold higher in HBx-transformed CHL (CHL-X) cells than CHL cells. Phosphorylation of Akt at serine 473 and Bad at serine 136 were induced by HBx, which were specifically blocked by wortmannin and dominant negative mutants of Akt and Bad, respectively. We also demonstrated that HBx inhibits caspase 3 activity and HBx down-regulation of caspase 3 activity was blocked by the PI3K inhibitor. Regions required for PI3K phosphorylation on the HBx protein overlap with the known transactivation domains. HBx blocks apoptosis induced by serum withdrawal in CHL cells in a p53-independent manner. The results indicate that, unlike other DNA tumor viruses that block apoptosis by inactivating p53, the hepatitis B virus achieves protection from apoptotic death through a HBx-PI3K-Akt-Bad pathway and by inactivating caspase 3 activity that is at least partially p53-independent in liver cells. Moreover, these data suggest that modulation of the PI3K activity may represent a potential therapeutic strategy to counteract the occurrence of apoptosis in human hepatocellular carcinoma.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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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 [gamma -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 [gamma -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 lambda  = 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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INTRODUCTION
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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.

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 (black-square) 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.

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 (open circle ) 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.

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.

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."

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.

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.

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.).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

    FOOTNOTES

* This work was supported by grants from the Korean Science and Engineering Foundation (Science Research Center Fund) and Korea Ministry of Science and Technology (grant NBM0080031).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: Tel.: 82-42-860-4150; Fax: 82-42-860-4597; E-mail: yilee@mail.kribb.re.kr.

Published, JBC Papers in Press, March 1, 2001, DOI 10.1074/jbc.M011263200

    ABBREVIATIONS

The abbreviations used are: PI3K, phosphatidylinositol 3-kinase; HBV-X, hepatitis B virus-X; HCC, hepatocellular carcinoma; CAT, chloramphenicol acetyl transferase; CHL, Chang liver cell; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP end labeling; CHL-X, Chang liver cell-X; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; DAPI, 4',6'-diamidino-2-phenylindole; PBS, phosphate-buffered saline; PIP2, phosphatidylinositol 4,5-biphosphate; PH, pleckstrin homology; SH2, Src homology 2 domain; CPP32, caspase 3; bp, base pair(s).

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