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STUDIES ON CHEMOTHERAPEUTIC DEOXYRIBONUCLEOSIDES

化疗脱氧核糖核苷的研究

基本信息

批准号：
3164560
负责人：
GEORGE ACS
金额：
$ 6.36万
依托单位：
MOUNT SINAI SCHOOL OF MEDICINE OF CUNY
依托单位国家：
美国
项目类别：
财政年份：
1977
资助国家：
美国
起止时间：
1977-08-01 至 1985-11-30
项目状态：
已结题

项目摘要

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.

包括我们自己在内的文献中的几份报告都指出了 DNA甲基化模式的改变与细胞内遗传变化之间的关系表型我们的目的是研究改变的可能性， DNA甲基转移酶的活性与随后的修饰，表型它将被确定1）是否致癌物质结合到和/或损伤DNA可以影响DNA甲基转移酶之间的相互作用和它的基质。直接接触致癌物质会改变DNA 在体外或间接通过在它们存在下培养细胞。修饰DNA 将测定它们结合和抑制酶的能力。我们已经表明，一种致癌诱变剂，5-azaCR，在体内外均能抑制DNA甲基转移酶。（二） DNA甲基转移酶的水平是否在细胞或组织中发生变化接触致癌物质和肿瘤促进剂。酶的水平，培养细胞或小鼠皮肤中的细胞毒性将通过直接测定或通过放射免疫测定。 3)是否甲基化的模式在特定的限制位点在癌变过程中发生改变。的HPLC分析来自限制性内切酶切割的3'和5'末端的32 P标记核苷酸来自限制性内切酶切割的DNA的3'和5'末端的DNA核苷酸将用于确定限制性内切酶中C残基甲基化的程度，正常和肿瘤细胞的DNA位点。 4)无论是DNA 可以分离不同位点特异性的甲基转移酶，它们的活性和致癌作用之间的关系是否可以确立了习为此，DNA甲基转移酶将通过以下方法分离：亲和层析及其对半甲基化（基因组）和完全未甲基化的（pBR 322）底物。这些研究可能阐明致癌物的另一种作用机制，确定DNA甲基化在建立正常和异常细胞中基因表达的遗传模式。