Overexpression of Bcl-2 protects human hepatoma cells from Fas-antibody-mediated apoptosis

Background/Aims: Fas is a cell surface antigen, that triggers apoptosis upo n specific ligand or antibody binding. The proto-oncogene bcl-2 prevents ap optosis 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. Cel l viability and apoptotic change were examined after anti-Fas- and antisens e oligodeoxynucleotide treatments. Apoptotic cells were detected by nick-en d labelling and the TUNEL method. To test if Bcl-2 expression can protect H epG2 cells from Fas-mediated apoptosis, the cells were transduced using ret roviral vector, LZBC, designed to coexpress E. coli beta-galactosidase and human Bcl-2. To further confirm the protective effect of Bcl-2 expression a gainst 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 tre atment with an agonist anti-Fas antibody (1 mu g/ml for 3 days), whereas 33 % of the HepG2 cells were killed by this treatment. Inhibition of Bcl-2 exp ression by transfection of antisense oligodeoxynucleotides caused spontaneo us apoptosis in HCC-T, but not in HepG2 cells, suggesting that Bcl-2 is ess ential for survival of HCC-T cells, whereas other proteins may substitute f or it in HepG2 cells. Following LZBC infection, 10% HepG2 cells were beta-g alactosidase-positive by X-gal staining and Bcl-2-positive. In cells surviv ing after anti-Fas treatment, the proportion of beta-galactosidase-positive cells increased to 50% and the beta-galactosidase activity increased 6-fol d, indicating that Bcl-2 expression protected the cells from Fas-mediated a poptosis. In the cloned HepG2 cells stably expressing Bcl-2, the extent of Fas-mediated apoptosis was inversely related to the level of Bcl-2 expressi on. Conclusion: Bcl-2 confers protection to human hepatoma cells against Fas-me diated apoptosis, and is essential for survival of some, but not all, hepat oma cells.