title: Overexpression of Bcl-2 protects human hepatoma cells from Fas-antibody-mediated apoptosis.
abstract: OBJECTIVE
  Fas is a cell surface antigen, that triggers apoptosis upon specific ligand or antibody binding. The proto-oncogene bcl-2 prevents apoptosis induced by various treatments. The aim of our study was to evaluate whether Bcl-2 protects hepatoma cells from Fas-mediated apoptosis.
  
  
  METHODS
  Two human cell lines, HCC-T and HepG2 were used. Expression of Fas antigen and Bcl-2 was detected by flow cytometry and Western blotting. Cell viability and apoptotic change were examined after anti-Fas- and antisense oligodeoxynucleotide treatments. Apoptotic cells were detected by nick-end labelling and the TUNEL method. To test if Bcl-2 expression can protect HepG2 cells from Fas-mediated apoptosis, the cells were transduced using retroviral vector, LZBC, designed to coexpress E. coli beta-galactosidase and human Bcl-2. To further confirm the protective effect of Bcl-2 expression against Fas-mediated apoptosis in HepG2, Bcl-2 expressing plasmid vector was produced and a cell line stably expressing Bcl-2 was cloned.
  
  
  RESULTS
  Western blot analysis showed constitutive Bcl-2 expression in HCC-T cells, but not in HepG2 cells. HCC-T was resistant to apoptosis after treatment with an agonist anti-Fas antibody (1 microg/ml for 3 days), whereas 33% of the HepG2 cells were killed by this treatment. Inhibition of Bcl-2 expression by transfection of antisense oligodeoxynucleotides caused spontaneous apoptosis in HCC-T, but not in HepG2 cells, suggesting that Bcl-2 is essential for survival of HCC-T cells, whereas other proteins may substitute for it in HepG2 cells. Following LZBC infection, 10% HepG2 cells were beta-galactosidase-positive by X-gal staining and Bcl-2-positive. In cells surviving after anti-Fas treatment, the proportion of beta-galactosidase-positive cells increased to 50% and the beta-galactosidase activity increased 6-fold, indicating that Bcl-2 expression protected the cells from Fas-mediated apoptosis. In the cloned HepG2 cells stably expressing Bcl-2, the extent of Fas-mediated apoptosis was inversely related to the level of Bcl-2 expression.
  
  
  CONCLUSIONS
  Bcl-2 confers protection to human hepatoma cells against Fas-mediated apoptosis, and is essential for survival of some, but not all, hepatoma cells.
authors.affiliation: Department of Internal Medicine, School of Medicine, Keio University, Tokyo, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Takahashi
authors.forename: H
authors.initials: H
authors.lastname: Saito
authors.forename: T
authors.initials: T
authors.lastname: Okuyama
authors.forename: T
authors.initials: T
authors.lastname: Miyashita
authors.forename: M
authors.initials: M
authors.lastname: Kosuga
authors.forename: F
authors.initials: F
authors.lastname: Sumisa
authors.forename: M
authors.initials: M
authors.lastname: Yamada
authors.forename: H
authors.initials: H
authors.lastname: Ebinuma
authors.forename: H
authors.initials: H
authors.lastname: Ishii
mesh_terms: D017209:Apoptosis; D015153:Blotting, Western; D006528:Carcinoma, Hepatocellular / Q000378:metabolism* / Q000648:ultrastructure*; D005434:Flow Cytometry; D015972:Gene Expression Regulation, Neoplastic; D019254:Genes, bcl-2 / Q000235:genetics; D006801:Humans; D020287:In Situ Nick-End Labeling; D066298:In Vitro Techniques; D008113:Liver Neoplasms / Q000378:metabolism* / Q000648:ultrastructure*; D016376:Oligonucleotides, Antisense / Q000378:metabolism; D000090063:Proto-Oncogene Mas; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014407:Tumor Cells, Cultured; D015854:Up-Regulation; D019014:fas Receptor / Q000235:genetics / Q000378:metabolism*
chemical_list: C000715470:MAS1 protein, human; D016376:Oligonucleotides, Antisense; D000090063:Proto-Oncogene Mas; D019253:Proto-Oncogene Proteins c-bcl-2; D019014:fas Receptor