Production of homocysteine in serum-starved apoptotic PC12 cells depends on the activation and modification of Ras

PC12 pheochromocytoma cells expressing a dominant inhibitory mutant of Ha-Ras (M-M17-26) and PC12 cells transfected with normal c-RasH (M-CR3B) have been used to investigate the role of nitrosylation and farnesylation of Ras on the production of homocysteine and the activities of the redox-sensitive transcription factors NF-kappaB and c-Fos. We found that under serum and nerve growth factor withdrawal conditions undifferentiated apoptotic M-CR3B cells accumulated more homocysteine than M-M17-26 cells, and the production of homocysteine decreased in the presence of manumycin and increased in the presence of l-NAME. Furthermore, we have shown that manumycin increased the activity of c-Fos in the M-CR3B cells and decreased the activity of NF-kappaB, while l-NAME decreased the activities of both transcription factors, and accelerated apoptosis of M-CR3B cells. In contrast, in M-M17-26 cells manumycin did not change the activity of c-Fos, nor the activity of NF-kappaB. We conclude that trophic factor withdrawal stimulates Ras, which apparently through the Rac/NADPH oxidase system induces permanent oxidative stress, modulates the activities of NF-kappaB and c-Fos, induces production of homocysteine and accelerates apoptosis. Nitrosylation of Ras is necessary for maintaining the survival of PC12 cells, while farnesylation of Ras stimulates apoptosis under withdrawal conditions.