O(6)-methylguanine (O(6)MeG) is a highly critical
DNA adduct induced by methylating
carcinogens and anticancer drugs such as
temozolomide,
streptozotocine,
procarbazine and
dacarbazine. Induction of cell death by O(6)MeG lesions requires mismatch repair (MMR) and cell proliferation and is thought to be dependent on the formation of
DNA double-strand breaks (DSBs) or, according to an alternative hypothesis, direct signaling by the MMR complex. Given a role for DSBs in this process, either homologous recombination (HR) or non-homologous end joining (NHEJ) or both might protect against O(6)MeG. Here, we compared the response of cells mutated in HR and NHEJ
proteins to
temozolomide and
N-methyl-N'-nitro-N-nitrosoguanidine (
MNNG). The data show that cells defective in HR (Xrcc2 and Brca2 mutants) are extremely sensitive to cell death by apoptosis and
chromosomal aberration formation and less sensitive to sister-chromatid exchange (SCE) induction than the corresponding wild-type. Cells defective in NHEJ were not (Ku80 mutant), or only slightly more sensitive (
DNA-PK(cs) mutant) to cell death and showed similar aberration and SCE frequencies than the corresponding wild-type. Transfection of
O(6)-methylguanine-DNA methyltransferase (MGMT) in all of the mutants almost completely abrogated the genotoxic effects in both HR and NHEJ defective cells, indicating the mutant-specific
hypersensitivity was due to O(6)MeG lesions.
MNNG provoked H2AX phosphorylation 24-48h after methylation both in wild-type and HR mutants, which was not found in MGMT transfected cells. The gammaH2AX foci formed in response to O(6)MeG declined later in wild-type but not in HR-defective cells. The data support a model where DSBs are formed in response to O(6)MeG in the post-treatment cell cycle, which are repaired by HR, but not NHEJ, in a process that leads to SCEs. Therefore, HR can be considered as a mechanism that causes tolerance of O(6)MeG adducts. The data implicate that down-regulation or inhibition of HR might be a powerful strategy in improving
cancer therapy with methylating agents.