| In an effort to improve the efficacy of cancer chemotherapy by intervening into the cellular responses to chemotherapeutic change, we have used adenoviral overexpression of N-methylpurine DNA glycosylase (MPG) in breast cancer cells, to study its ability to imbalance base excision repair (BER) and sensitize cells to alkylating agents. Our results demonstrate that MPG overexpressing cells are significantly more sensitive to the alkylating agents methylmethane sulfonate (MMS), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), methyl nitrosourea (MNU), dimethyl sulfate (DMS) and the clinical chemotherapeutic temozolomide (TMZ). We found that this sensitivity was associated with a significant increase in the number of single strand breaks and/or AP sites in overexpressing cells, confirming an MPG-driven accumulation of toxic BER intermediates. Enhanced MPG expression also results in a dramatic induction of double strand breaks shortly after drug treatment and prior to the detection of apoptosis and eventual cell death. This evidence argues in favor of a role for AP sites and/or single strand breaks as well as double strand breaks in the cytotoxic sensitivity to alkylation caused by MPG overexpression.;Importantly, MPG overexpressing cells showed no increased sensitivity to treatment with a 3-MeA-specific alkylating agent, McOSO2(CH 2)2-lexitropsin (Me-lex). We conclude that 3-MeA is not the lesion responsible for MPG-induced sensitivity, as its excision, even at an increased rate replaces a toxic adduct with a similarly toxic AP site or strand break. In contrast, increased removal of 7-MeG by MPG replaces innocuous lesions with toxic repair intermediates, and would be expected to increase the cytotoxicity of alkylation. Dramatically increased removal of 7-meG was demonstrated in MPG overexpressing cells by two independent techniques, and this differential conversion of harmless base changes to toxic genetic interruptions is likely to be responsible for MPG-induced sensitivity to alkylation. This represents a mechanism by which a benign DNA modification can be made toxic through the targeted overexpression of a tolerated gene product. The successful application of this research would eliminate peripheral toxicity and improve the efficacy of chemotherapy, potentially offering cancer patients a cure. |
