O6-methylguanine-DNA methyltransferase (MGMT), a ubiquitous protein in bacteria and higher organisms, is responsible for repair of the carcinogenic and mutagenic adduct O6-alkylguanine induced in DNA by various simple alkylating carcinogens (e.g., N-alkylnitrosamines) and antitumor drugs (Procarbazine and haloethyl-N-nitrosoureas). MGMT, a stoichiometric protein without turnover, is strictly regulated in both bacteria and mammals. The level of mammalian MGMT activity is tissue- and cell type-specific and is modulated during the cell cycle. Some cultured cell lines called Mex-/Mer-, and often derived from tumors, have no detectable MGMT activity even though they appear to have the MGMT gene. In contrast, some drug-resistant tumor xenografts and lines have higher levels of MGMT than their progenitor cells. With the recent availability of cDNAs of human and mouse MGMT genes and of antibodies against the human protein, the broad objective of the project is a comprehensive understanding of the regulation of MGMT gene in mammals and the impact of this regulation on mutagenesis and carcinogenesis induced by alkylating agents and on antitumor activity of alkylating drugs. specifically, the following topics are to be addressed in both individual and collaborative efforts. (1) The organization of human and mouse MGMT genes. (2) Identification of regulatory elements of these genes. (3) Molecular basis of extinction of MGMT in Mex- cells. (4) Induction of MGMT gene and its amplification, particularly in drug-resistant human tumors. (5) Involvement of trans-acting factors in MGMT regulation and their characterization. (6) Construction of expression vectors for large-scale production of human and mouse MGMT for subsequent biochemical studies and X ray crystallographic analysis of the 3-D structure of the MGMT polypeptides. (7) Construction of mammalian expression vectors to be used in the creation of transgenic cells and mice with modulated expression of human MGMT in order to investigate the role of O6-alkylguanine in mutagenesis, carcinogenesis, toxicity, and other biological endpoints. (8) Construction of gene-targeting vectors containing mouse MGMT gene sequences for use in targeted mutagenesis of embryonic stem (ES) cells. (9) Insertion of the ES cells into blastocysts for subsequent production of heterozygous and homozygous mice with null mutation in the MGMT gene in order to study the essentiality of MGMT in development and in the prevention of germ-line mutation and tumorigenesis.
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