Loss of ATM sensitizes against O6-methylguanine triggered apoptosis, SCEs and chromosomal aberrations

A critical pre-cytotoxic and -apoptotic DNA lesion induced by methylating carcinogens and chemotherapeutic drugs is o(6)-methylguanine (O(6)MeG). The mechanism by which O(6)MeG causes cell death via apoptosis is only partially understood. The current model ascribes a role to DNA replication and mismatch repair, which converts O(6)MeG into a critical distal lesion (presumably a DNA double-strand break) that is finally responsible for genotoxicity and apoptosis. Here we analysed whether the PI3-like kinase ATM is involved in this process. ATM is a major player in recognizing and signaling DNA breaks, but most reports are limited to ionizing radiation. Comparing mouse ATM knockout fibroblasts (ATM-/-) with the corresponding wild-type (ATM+/+) we show that ATM-/- cells are hypersensitive to the cytotoxic and apoptosis-inducing effect of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Inhibition of O-6-methylguanine-DNA methyltransferase (MGMT) activity by O-6-benzylguanine enhanced cell killing whereas the increase of MGMT activity by transfection with an expression vector provoked MNNG resistance. This was more pronounced in ATM-/- than in ATM+/+ cells, suggesting that O(6)MeG is responsible, at least in part, for increased MNNG sensitivity of ATM-/- cells. Cytogenetic studies showed that MNNG-induced sister-chromatid exchange frequencies were the same in ATM-/- and ATM+/+ cells in the first mitoses following treatment, but higher in ATM-/- cells than in the wild-type in the second post-treatment mitoses, when MGMT was depleted. Also, a significant higher frequency of MNNG-induced chromosomal aberrations was observed in ATM-/- than in ATM+/+ cells when analysed at a late recovery time, which is consistent with O(6)MeG being the inducing lesion. In summary, we conclude that ATM is not only involved in resistance to ionizing radiation but also to methylatingagents, playing a role in the repair of secondary DNA damage generated from O(6)MeG lesions. The data also show that ATM is not required for activating the apoptotic pathway in response to O(6)MeG since ATM-/- cells are able to undergo apoptosis with high frequency. (C) 2004 Elsevier B.V. All rights reserved.