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

Abstract

While it is well known that MNU induces thymic lymphomas in the mouse, it remains unclear which pre-mutagenic lesions are responsible for lymphomagenic transformation. One lesion thought to play a critical role is O⁶methylguanine [O⁶mG] which initiates G : C to A : T transition mutations in K-ras and other oncogenes. O⁶alkylguanine-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 lacI 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 lacI mice. Sixty-two per cent of the lymphomas had a GGT to GAT activating mutation in codon 12 of K-ras consistent with O⁶mG adduct-mediated point mutagenesis. LacI mutation frequency in thymus of MNU treated Big Blue^TM mice was 45-fold above background whereas it was 11-fold above background in MNU treated MGMT+/Big Blue^TM mice. Most lacI mutations were G : C to A : T transitions, implicating O⁶mG even in the MGMT+ mice. No mutations were attributable to chromosomal aberrations or rearrangements. Thus, O⁶mG adducts account for the carcinogenic effect of MNU and MGMT overexpression is selectively able to reduce O⁶methylguanine adducts below a carcinogenic threshold. Other adducts are mutagenic but appear to contribute much less to malignant transformation or oncogene activation.