The hepatitis B virus-X protein activates a phosphatidylinositol 3-kinase-dependent survival signaling cascade.
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.
Liver Cell Signal Transduction Research Unit, 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.
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D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D002352:Carrier Proteins / Q000378:metabolism; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D002461:Cell Line, Transformed; D002467:Cell Nucleus / Q000648:ultrastructure; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology*; D016895:Culture Media, Serum-Free; D005047:Etoposide / Q000494:pharmacology; D006511:Hepatitis B Antigens / Q000378:metabolism*; D006515:Hepatitis B virus / Q000502:physiology*; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000166:cytology / Q000502:physiology / Q000821:virology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D019311:Staurosporine / Q000494:pharmacology; D015534:Trans-Activators / Q000378:metabolism*; D014162:Transfection; D054334:Viral Regulatory and Accessory Proteins; D051025:bcl-Associated Death Protein
D002352:Carrier Proteins; D016895:Culture Media, Serum-Free; D005047:Etoposide; D006511:Hepatitis B Antigens; D019869:Phosphatidylinositol 3-Kinases; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D011994:Recombinant Proteins; D019311:Staurosporine; D015534:Trans-Activators; D054334:Viral Regulatory and Accessory Proteins; D051025:bcl-Associated Death Protein; C494819:BAD protein, human; C057628:hepatitis B virus X protein; C494918:AKT1 protein, human