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

Masahiko Takahashi, Hidetsugu Saito, Torayuki Okuyama, Toshiyuki Miyashita, Motomichi Kosuga, Futoshi Sumisa, Masao Yamada, Hirotoshi Ebinuma, Hiromasa Ishii

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Abstract

Background/Aims: 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 β-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 μg/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 β-galactosidase-positive by X-gal staining and Bcl-2-positive. In cells surviving after anti-Fas treatment, the proportion of β-galactosidase-positive cells increased to 50% and the β- 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. Conclusion: Bcl-2 confers protection to human hepatoma cells against Fas-mediated apoptosis, and is essential for survival of some, but not all, hepatoma cells.

Original languageEnglish
Pages (from-to)315-322
Number of pages8
JournalJournal of Hepatology
Volume31
Issue number2
DOIs
Publication statusPublished - 1999 Aug

Keywords

  • Antisense oligonucleotide
  • Gene transfer
  • Human hepatoma cell line

ASJC Scopus subject areas

  • Hepatology

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    Takahashi, M., Saito, H., Okuyama, T., Miyashita, T., Kosuga, M., Sumisa, F., Yamada, M., Ebinuma, H., & Ishii, H. (1999). Overexpression of Bcl-2 protects human hepatoma cells from Fas-antibody- mediated apoptosis. Journal of Hepatology, 31(2), 315-322. https://doi.org/10.1016/S0168-8278(99)80230-X