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-ras(Val12) 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-β-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 p34(cdc2) kinase was decreased, suggesting that p34(cdc2) 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 ras(Asn17) or dominant-negative raf-1(C4B) 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.