Apoptosis induced by DNA damage O-6-methylguanine is Bcl-2 and caspase-9/3regulated and Fas/caspase-8 independent

In the therapy of various kinds of tumors, methylating agents generating O- 6-methylguanine (O(6)MeG) in DNA are used, We studied the molecular mechani sm of cell death induced by these agents by comparing isogenic cell lines p roficient (MGMT+) and deficient (MGMT-) for the DNA repair protein alkyltra nsferase and exhibiting the tolerance phenotype. Hypersensitivity to methyl ation-induced cell killing of MGMT- cells Is attributable to the potent ind uction of apoptosis. We show that apoptosis is a late event occurring >48 h after methylation. It was preceded by decrease in Bcl-2 protein level and accompanied by activation of caspase-9 and caspase-3, We also observed cyto chrome c release and hypophosphorylation of Bad, Other members of the Bcl-2 family (Bag-1, Bak, Bax, and Bcl-x(L)) were not altered in expression, Tra nsfection of MGMT- cells with bcl-2 protected against methylation-induced a poptosis, indicating that Bcl-2 plays a key role in the response. Induction of apoptosis in MGMT- cells was not triggered by Fas and Fas ligand (CD95, Apo-1) because both proteins remained unaltered in expression and receptor -proximal caspase-8 was not activated after methylation. Also, inhibition o f caspase-8 was ineffective in modifying the apoptotic response, whereas in hibition of caspase-3 and caspase-9 blocked apoptosis. Tolerant cells that are unable to repair O(6)MeG and are impaired in mismatch repair were less sensitive regarding the induction of apoptosis and Bcl-2 decline, supportin g the view that O(6)MeG-induced apoptosis requires mismatch repair, The ult imate O(6)MeG-derived lesions triggering the apoptotic pathway are likely t o be DNA double-strand breaks, which were significantly formed in MGMT- but not in MGMT+ and tolerant cells and which preceded apoptosis, Overall, the data indicate that O(6)MeG induces apoptosis via secondary lesions that tr igger Bcl-2 decline, cytochrome c release, and caspase-9 and caspase-3 acti vation independently of Fas/Fas ligand and p53, for which the cells are mut ated.