Apoptosis induced by DNA damage O6-methylguanine is Bcl-2 and caspase-9/3 regulated and Fas/caspase-8 independent.
In the therapy of various kinds of tumors, methylating agents generating O6-methylguanine (O6MeG) in DNA are used. We studied the molecular mechanism of cell death induced by these agents by comparing isogenic cell lines proficient (MGMT+) and deficient (MGMT-) for the DNA repair protein alkyltransferase and exhibiting the tolerance phenotype. Hypersensitivity to methylation-induced cell killing of MGMT- cells is attributable to the potent induction 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 cytochrome c release and hypophosphorylation of Bad. Other members of the Bcl-2 family (Bag-1, Bak, Bax, and Bcl-xL) were not altered in expression. Transfection of MGMT- cells with bcl-2 protected against methylation-induced apoptosis, 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 of caspase-8 was ineffective in modifying the apoptotic response, whereas inhibition of caspase-3 and caspase-9 blocked apoptosis. Tolerant cells that are unable to repair O6MeG and are impaired in mismatch repair were less sensitive regarding the induction of apoptosis and Bcl-2 decline, supporting the view that O6MeG-induced apoptosis requires mismatch repair. The ultimate O6MeG-derived lesions triggering the apoptotic pathway are likely to 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 O6MeG induces apoptosis via secondary lesions that trigger Bcl-2 decline, cytochrome c release, and caspase-9 and caspase-3 activation independently of Fas/Fas ligand and p53, for which the cells are mutated.
Ochs
K
K
Division of Applied Toxicology, Institute of Toxicology, University of Mainz, Germany.
Kaina
B
B
D000818:Animals; D017209:Apoptosis / Q000235:genetics*; D016466:CHO Cells / Q000201:enzymology; D002352:Carrier Proteins / Q000378:metabolism; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism / Q000502:physiology*; D006224:Cricetinae; D003574:Cytochrome c Group / Q000378:metabolism; D004249:DNA Damage / Q000502:physiology*; D004789:Enzyme Activation; D053222:Fas Ligand Protein; D006147:Guanine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000502:physiology*; D008562:Membrane Glycoproteins / Q000096:biosynthesis; D008769:Methylnitronitrosoguanidine; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011480:Protease Inhibitors / Q000494:pharmacology; D011506:Proteins / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000096:biosynthesis / Q000378:metabolism / Q000502:physiology*; D051025:bcl-Associated Death Protein; D019014:fas Receptor / Q000096:biosynthesis / Q000502:physiology*
D002352:Carrier Proteins; D061945:Caspase Inhibitors; D003574:Cytochrome c Group; D053222:Fas Ligand Protein; D008562:Membrane Glycoproteins; D011480:Protease Inhibitors; D011506:Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051025:bcl-Associated Death Protein; D019014:fas Receptor; D008769:Methylnitronitrosoguanidine; D006147:Guanine; C008449:O-(6)-methylguanine; D019853:O(6)-Methylguanine-DNA Methyltransferase; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases