Chang M Y, Won S J, Yang B C, Jan M S, Liu H S
College of Medicine, National Cheng Kung University, Tainan, Taiwan, Republic of China.
Exp Cell Res. 1999 May 1;248(2):589-98. doi: 10.1006/excr.1999.4436.
This is the first report demonstrating that NIH/3T3 fibroblasts utilize the Raf-1/MAPK pathway to sensitize themselves to tumor necrosis factor-alpha (TNF-alpha) cytotoxicity under Ha-rasVal12 oncogene-overexpressed conditions. This paper clearly shows that the sensitivity of NIH/3T3 cells to TNF-alpha cytotoxicity positively correlated with the expression level of activated Ha-ras transgene, which was manipulated either positively by isopropyl-beta-d-thiogalactoside (IPTG) induction or negatively by a ribozyme or a dominant negative Ras suppression. Further analysis revealed that after TNF-alpha treatment, Ha-ras-overexpressed transformants underwent apoptosis. Overexpression of dominant negative Raf-1, Rac1, or RhoA in the Ha-ras transformants clarified that among these factors, only dominant negative Raf-1 could reverse the cell sensitivity to TNF-alpha, indicating that Raf-1, as a proapoptotic factor, indeed participates in TNF-alpha cytotoxicity. The anti-apoptotic roles of Bcl-2 and PI(3) kinase are also demonstrated by the Ha-ras transformants which became more resistant to TNF-alpha while overexpressing Bcl-2 or the activated p110 catalytic subunit. The analyses of the cell cycle and nuclear transcription factor activities revealed that TNF-alpha treatment caused the Ha-ras overexpressed transformants to shift from S to G0/G1 phase and increased the responses of AP-1, c-fos, and c-myc. Taken together, we suggest that the possible action of Ha-ras overexpression to sensitize TNF-alpha-treated fibroblasts is predominantly through the Ras/Raf-1/MAPK pathway to increase the responses of AP-1, c-fos, and c-myc, which are possibly involved in the aberration of cell cycle machinery, and subsequently to turn on the death program.
这是首份表明NIH/3T3成纤维细胞在Ha-rasVal12癌基因过表达条件下利用Raf-1/丝裂原活化蛋白激酶（MAPK）信号通路使自身对肿瘤坏死因子-α（TNF-α）细胞毒性敏感的报告。本文清楚显示，NIH/3T3细胞对TNF-α细胞毒性的敏感性与活化的Ha-ras转基因表达水平呈正相关，活化的Ha-ras转基因可通过异丙基-β-D-硫代半乳糖苷（IPTG）诱导正向调控，或通过核酶或显性负性Ras抑制负向调控。进一步分析表明，TNF-α处理后，Ha-ras过表达转化体发生凋亡。在Ha-ras转化体中过表达显性负性Raf-1、Rac1或RhoA表明，在这些因子中，只有显性负性Raf-1可逆转细胞对TNF-α的敏感性，这表明Raf-1作为促凋亡因子确实参与了TNF-α细胞毒性作用。Ha-ras转化体在过表达Bcl-2或活化的p110催化亚基时对TNF-α更具抗性，这也证明了Bcl-2和磷脂酰肌醇-3激酶（PI(3)激酶）的抗凋亡作用。细胞周期和核转录因子活性分析表明，TNF-α处理使Ha-ras过表达转化体从S期转变为G0/G1期，并增强了活化蛋白-1（AP-1）、原癌基因c-fos和原癌基因c-myc的反应。综上所述，我们认为Ha-ras过表达使TNF-α处理的成纤维细胞敏感的可能作用主要是通过Ras/Raf-1/MAPK信号通路增强AP-1、c-fos和c-myc的反应，这些反应可能参与细胞周期机制的异常，随后启动死亡程序。
