Selective activation of Ha-ras(val12) oncogene increases susceptibilityof NIH/3T3 cells to TNF-alpha.
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.
Chang
M Y
MY
College of Medicine, National Cheng Kung University, Tainan, Taiwan, Republic of China.
Won
S J
SJ
Yang
B C
BC
Jan
M S
MS
Liu
H S
HS
D017209:Apoptosis; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005786:Gene Expression Regulation; D011905:Genes, ras; D006801:Humans; D020128:Inhibitory Concentration 50; D016760:Proto-Oncogene Proteins c-fos / Q000096:biosynthesis; D016271:Proto-Oncogene Proteins c-myc / Q000096:biosynthesis; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism; D015398:Signal Transduction; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014170:Transformation, Genetic; D019076:Transgenes; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D018631:ras Proteins / Q000378:metabolism*
D016760:Proto-Oncogene Proteins c-fos; D016271:Proto-Oncogene Proteins c-myc; D018808:Transcription Factor AP-1; D014409:Tumor Necrosis Factor-alpha; D019908:Proto-Oncogene Proteins c-raf; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D018631:ras Proteins