Almost all living organisms have evolved DNA alkyltransferases to repair lethal and mutagenic adducts induced at the O6-position of guanine in DNA by alkylating agents such as methylnitrosourea. These O6-alkyguanine-DNA alkyltransferases (ATases) can also repair precursors of cytotoxic DNA cross-links induced by anticancer chloroethylating drugs (e.g., BCNU). Whereas all normal human tissues and cultured cell lines (Mer+) constitutively express ATase, termed MGMT, 20-30% of tumor cell lines (Mer-) completely lack this repair activity and are hypersensitive to the cytotoxic and mutagenic effects of simple methylating agents as well as to the therapeutic effects of chloroethylating agents. MGMT appears to be constitutively regulated at levels characteristic for a given tissue or cell type; however, there is considerable variability among individuals, and a wide range of levels in malignant tumors. The major goal of this proposal is to determine the mechanisms that regulate MGMT expression in Mer+ cells and that result in its total suppression in Mer- cells. Because regulation appears to be at the transcriptional level, the specific aims focus primarily on the promoter region of the gene. We propose that cytosine methylation in the promoter region determines complete suppression of MGMT transcription in Mer- cells (Aim 1), and that binding of proteins to enhancer or suppressor elements determines constitutive levels of MGMT in Mer+ cells (Aims 2 and 3). Transcription factors that bind such DNA elements in a methylation-dependent manner will be sought (Aim 2). An enhancer element recently identified at the first exon/intron boundary will be characterized with respect to its specific binding protein and the effect of methylation (Aim 4). Hypermethylated CpG islands in the body of the MGMT gene will be located and the levels of specific binding proteins that correlate with MGMT expression will be determined (Aim 5). These studies will employ DNA band-shift and footprinting techniques to identify and characterize DNA binding proteins, and site-directed mutagenesis of binding sequences in reporter gene constructs to establish functional significance. Elucidation of critical regulatory elements in the MGMT gene, and the role of CpG methylation and regulatory binding proteins in MGMT expression, will provide new markers of potential susceptibility to environmental carcinogens and of potential resistance to cancer chemotherapy. The information gained will also suggest new avenues for intervention in these processes.
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