TY - JOUR
T1 - Overexpression of Bcl-2 protects human hepatoma cells from Fas-antibody- mediated apoptosis
AU - Takahashi, Masahiko
AU - Saito, Hidetsugu
AU - Okuyama, Torayuki
AU - Miyashita, Toshiyuki
AU - Kosuga, Motomichi
AU - Sumisa, Futoshi
AU - Yamada, Masao
AU - Ebinuma, Hirotoshi
AU - Ishii, Hiromasa
N1 - Funding Information:
The authors thank Dr. Y. Tsujimoto, Osaka University for providing us with pB4, and Dr. Jayanta Roy Chowdhury, Department of Medicine, Division of Gastroenterology and Liver Diseases, Albert Einstein College of Medicine, New York, for stimulating discussions and help with preparing the manuscript. The authors also thank Naoko Funeshima for expert technical assistance. This work was supported by the Japanese Ministry of Health and Welfare, the Japanese Ministry of Education, Science and Culture, Mitsukoshi Fund of Medicine 1997 and Keio University.
PY - 1999/8
Y1 - 1999/8
N2 - 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.
AB - 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.
KW - Antisense oligonucleotide
KW - Gene transfer
KW - Human hepatoma cell line
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U2 - 10.1016/S0168-8278(99)80230-X
DO - 10.1016/S0168-8278(99)80230-X
M3 - Article
C2 - 10453946
AN - SCOPUS:0032775972
SN - 0168-8278
VL - 31
SP - 315
EP - 322
JO - Journal of Hepatology
JF - Journal of Hepatology
IS - 2
ER -