Liu H S, Chen C Y, Lee C H, Chou Y I
Department of Microbiology and Immunology, College of Medicine, National Cheng Kung University, Tainan, Taiwan, Republic of China.
Br J Cancer. 1998 Jun;77(11):1777-86. doi: 10.1038/bjc.1998.296.
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.
源自小鼠NIH/3T3成纤维细胞的Ha-ras转化体“7-4”被用于研究活化型Ha-ras的过表达与细胞凋亡之间的关系。该细胞系含有一个可诱导的Ha-rasVal12癌基因，其受大肠杆菌（E. coli）乳糖操纵子/阻遏物系统的调控。我们证明，在血清饥饿条件下，通过外源性异丙基-β-D-硫代半乳糖苷（IPTG）过表达活化型Ha-ras癌基因可刺激细胞凋亡。细胞周期分析表明，大多数过表达Ha-ras的7-4细胞积聚在S期，且p34cdc2激酶的表达水平降低，这表明p34cdc2可能参与7-4细胞的凋亡过程。在这些细胞中过表达bcl-2转基因可阻断Ha-ras诱导的凋亡，且这种阻断在Ha-ras基因表达的下游得到证实。放线菌酮以剂量依赖的方式阻断7-4细胞的凋亡，这表明可能通过Ha-ras的过表达合成特定的调节凋亡的蛋白质。在表达显性负性rasAsn17或显性负性raf-1C4B以在不同阶段抑制Ha-ras信号转导的7-4衍生物中，Ha-ras过表达引发的凋亡也受到抑制，这表明活化型Ha-ras的过表达可诱导细胞凋亡，且raf-1信号通路的活性是这一过程所必需的。
