Endogenous therapeutic and environmental methylating agents form O6- methylguanine [06mG] DNA adducts, which have been implicated as human carcinogens. During the previous funding period for this grant, the investigators have shown the importance of O6mG DNA adducts in the induction of lymphoma in mice treated with MNU. Transgenic expression of the DNA repair protein O6-alkylguanine DNA alkyltransferase which removes O6mG DNA adducts markedly reduce the risk of lymphomagenesis, indicating the O6mG is the dominant carcinogenic adduct formed by MNU. In this proposal, the role of O6mG DNA adducts in carcinogenesis will be reassessed by studying the involvement of the mismatch repair system in the carcinogenic process. Mismatch repair recognition of O6-mG:T mispairs leads to single strand breaks, chromosomal rearrangements and aberrations. Previously, however, carcinogenesis of O6-mG has been thought to be entirely due to G to A point mutations because the O6-mG preferentially mispairs with thymine during DNA synthesis. The hypothesis to be tested is that in mismatch repair competent mice, chromosomal rearrangements are important in carcinogenesis whereas in mismatch repair defective mice, carcinogenesis precedes through G to A point mutations. In the first Specific Aim, transgenic mice defective in one of two mismatch repair proteins, PMS2 or MSH2 will be treated with MNU and followed for induction of tumors. Since a hallmark of mismatch repair mutation is the lack of cytotoxicity of methylating agents, more cells will survive with persistent O6-mG DNA adducts/ resulting in G to A point mutations. Thus, an increased incidence of MNU induced tumors is expected. These tumors will be analyzed to determine whether mismatch repair defects influence the type and incidence of tumors, the presence of chromosomal aberrations and G to A point mutations in K-ras.
In Specific Aim 2, MNU treatment of RAG2 knockout mice will be utilized. RAG2 mice are incapable of undergoing V(D)J joint formation and are unable to produce mature T-cells with rearranged T-cell receptors. Surprisingly, however, preliminary data indicates that RAG2 mice are susceptible to MNU induction of lymphomas and these contain T-cell receptor rearrangements. Thus, Specific Aim 2 will define the ability of MNU to induce T-cell receptor rearrangements in RAG2 knockout mice leading to lymphomagenesis. The investigators propose that the RAG2 defect can be bypassed by O6mG adduct mediated activation of the mismatch repair system leading to chromosomal rearrangements at the T-cell receptor locus. The overall goal of these studies is to understand the complex carcinogenic pathways of methylating agents and the impact of DNA repair on the process.
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