STUDIES ON CHEMOTHERAPEUTIC DEOXYRIBONUCLEOSIDES

点击翻译按钮获取中文摘要

Several reports in the literature, including our own, point to a link between altered patterns of DNA methylation and heritable changes in cell phenotype. It is our aim to examine the possibility that alterations in the activity of DNA methyl-transferase with subsequent modification in the phenotype. It will be determined 1) Whether carcinogens that bind to and/or damage DNA can affect the interaction between DNA methyltransferase and its substrate. DNA will be modified by direct exposure to carcinogens in vitro or indirectly bu culturing cells in their presence. Modified DNAs will be assayed for their ability to bind and inactivate the enzyme. We have already shown that one carcinogen-mutagen, 5-azaCR, when invorporated into DNA can inhibit DNA methyltransferase in vivo and in vitro. 2) Whether the levels of DNA methyltransferase change in cells or tissues exposed to carcinogens and tumor promoters. The levels of enzyme in cultured cells or in mouse skin will be determined either by direct assay or by radioimmune assay. 3) Whether the pattern of methylation at specific restriction sites is altered during carcinogenesis. HPLC analysis of 32P-labeled nucleotides from 3' and 5' ends of restriction enzyme cleaved DNA nucleotides from 3' and 5' ends of restriction enzyme cleaved DNA will be used to determine the extent of methylation of C residues in restriction sites of DNA from normal and tumor cells. 4) Whether DNA methyltransferases of differing site specificity can be isolated and whether a relationship between their activity and carcinogenesis can be established. For this purpose, DNA methyltransferases will be isolated by affinity chromatography and its activity assayed on hemimethylated (genomic) and completely unmethylated (pBR322) substrates. These studies may elucidate an alternative mechanism of action for carcinogens and have the potential to define the role of DNA methylation in establishing heritable patterns of gene expression in both normal and abnormal cells.