Selective activation of Ha-rasval12 oncogene increases susceptibility of NIH/3T3 cells to TNF-α

This is the first report demonstrating that NIH/3T3 fibroblasts utilize the Ras/Raf-1/MAPK pathway to sensitize themselves to tumor necrosis factor-alpha (TNF-alpha) cytotoxicity under Ha-ras(Va112) 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, Rad, or RhoA in the Haras 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 pro-apoptotic 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 G(0)/G(1) 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. (C) 1999 Academic Press.