Mice over-expressing human O6alkylguanine-DNA alkyltransferase selectively reduce O6methylguanine mediated carcinogenic mutations to threshold levels after N-methyl-N-nitrosourea

While it is well known that MNU induces thymic lymphomas in the mouse, it remains unclear which premutagenic lesions are responsible for lymphomagenic transformation. One lesion thought to play a critical role is O(6)methylguanine [O(6)mG] which initiates G:C to A:T transition mutations in K-ras and other oncogenes, O(6)alkylguanine-DNA alkyltransferase (AGT), encoded by the methylguanine methyltransferase gene [MGMT], removes the methyl group thereby preventing the mutation from occurring, When overexpressed in the thymus, MGMT protects mice from MNU-induced thymic lymphomas, To determine whether MGMT overexpression reduced G:C to A:T mutation frequency after MNU, Big Blue(TM) IacI and MGMT+/Big Blue(TM) mice were treated with MNU and analysed for mutations in the lacI and K-ras genes. The incidence of MNU-induced lymphomas was 84% in Big Blue(TM) lacI mice compared to 14% in MGMT+Big Blue(TM) lad mice. Sixty-two per cent of the lymphomas had a GGT to GAT activating mutation in codon 12 of K-I ns consistent with O(6)mG adduct-mediated paint mutagenesis, LacI mutation frequency in thymus of MNU treated Big Blue(TM) mice was 45-fold above background whereas it was ii-fold above background in MNU treated MGMT+/Big Blue(TM) mice, Most IacI mutations were G:C to A:T transitions, implicating O(6)mG even in the MGMT+ mice. No mutations were attributable to chromosomal aberrations or rearrangements, Thus, O(6)mG adducts account for the carcinogenic effect of MNU and MGMT overexpression is selectively able to reduce O(6)methylguanine adducts below a carcinogenic threshold. Other adducts are mutagenic but appear to contribute much less to malignant transformation or oncogene activation.