Apoptosis Induced by DNA Damage O-Methylguanine Is Bcl-2 and Caspase-9/3 Regulated and Fas/Caspase-8 Independent

K Ochs, B Kaina - Cancer research, 2000 - AACR
K Ochs, B Kaina
Cancer research, 2000AACR
In the therapy of various kinds of tumors, methylating agents generating O 6-methylguanine
(O 6MeG) 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 …
Abstract
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 crelease, and caspase-9 and caspase-3 activation independently of Fas/Fas ligand and p53, for which the cells are mutated.
AACR