Selective activation of oncogenic Ha-ras-induced apoptosis in NIH/3T3 cells.
A Ha-ras transformant '7-4', derived from mouse NIH/3T3 fibroblasts, was used to study the relationship between overexpression of activated Ha-ras and cell apoptosis. This cell line contains an inducible Ha-rasVal12 oncogene, which was under the regulation of the Escherichia coli (E. coli) lac operator/repressor system. We demonstrate that overexpression of activated Ha-ras oncogene by exogenous isopropyl-beta-D-thiogalactoside (IPTG) under serum-depleted conditions can stimulate cell apoptosis. Cell cycle analysis showed that most of the 7-4 cells with Ha-ras overexpression accumulated at S-phase and that the expression level of p34cdc2 kinase was decreased, suggesting that p34cdc2 may be involved in 7-4 cell apoptosis. Overexpression of bcl-2 transgene in these cells blocked Ha-ras-induced apoptosis, and this blockage was confirmed downstream of Ha-ras gene expression. Cycloheximide blocked the apoptosis of 7-4 cells in a dose-dependent manner, indicating that specific protein regulating apoptosis may be synthesized through Ha-ras overexpression. Ha-ras overexpression-triggered apoptosis was also prevented in the 7-4 derivatives that express either dominant-negative rasAsn17 or dominant-negative raf-1C4B to suppress Ha-ras signal transduction at different stages, indicating that overexpression of activated Ha-ras can induce cell apoptosis and that raf-1 pathway activity is required for this process.
Department of Microbiology and Immunology, College of Medicine, National Cheng Kung University, Tainan, Taiwan, Republic of China.
H S
HS
Liu
C Y
CY
Chen
C H
CH
Lee
Y I
YI
Chou
D016475:3T3 Cells; D000818:Animals; D017209:Apoptosis; D016203:CDC2 Protein Kinase / Q000032:analysis; D002461:Cell Line, Transformed; D003513:Cycloheximide / Q000494:pharmacology; D011905:Genes, ras; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D051379:Mice; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000502:physiology; D019908:Proto-Oncogene Proteins c-raf / Q000502:physiology; D015398:Signal Transduction; D019076:Transgenes
D019253:Proto-Oncogene Proteins c-bcl-2; D007544:Isopropyl Thiogalactoside; D003513:Cycloheximide; D019908:Proto-Oncogene Proteins c-raf; D016203:CDC2 Protein Kinase