REGULATION AND ENZYMOLOGY OF DNA METHYLASE IN CANCER

最近的实验表明，酶促DNA甲基化参与

Recent experiments indicate that enzymatic DNA methylation participates in the control of gene expression in higher eukaryotes. Presently, the mechanics of regulation and specificity of the enzyme, DNA methyltransferase (DMase) to methylate DNA are unclear. Studies have demonstrated aberrations in tissue-specific patterns of genomic methylation during carcinogenesis, tumor hypomethylation, and recently carcinogen interference in the transmethylation reaction catalyzed by DMase. Since cancer is charaterized by abnormalities in gene expression and persistant DNA replication, changes in genomic methylation by carcinogen binding may be causally related to carcinogenic initiation and/or malignant evolution. The proposed research focuses on regulation and enzymology of DMase in normal liver and during hepatocarcinogenesis; the elucidation of factors for faithful transmission of the methylation of specific sequences, and the mechanics by which carcinogens may alter this process at the template level of by direct interaction with the enzyme. In this renewal, one goal will be to produce monoclonal antibodies against the major DMase specie(s). These will be used to simplify the purification scheme as well as detect active and inactive DMase during carcinogenesis, and to test for the possible presence of multiple enzyme species. These antibodies will also be used to identify DMase polypeptides in in vitro translation assays and colony lysates so that DMase cDNA can be generated, clone and detected in either expression (pUC) cDNA libraries or in nonexpression vectors (pBR). The purpose of obtaining these probes is to eventually isolate the DMase gene. Because our understanding of the role of methylation is hampered by the inability to preserve the methylation pattern on molecular cloning, one of our goals in cloning full-length DMase in an expression vector is to be able to genetically engineer the expression in a bacterial host for this purpose. In this way, it will be easier to test the function(s) of DNA methylation as controlled by DMase and to determine whether it is an obligate component in the development of cancer.