| Background and object DNA methylation is catalyzed by specific DNA methyltransferases (DNMTs), with S-adenosyl-L-methionine (SAM) as the methyl donor. In mammals, the major target for DNA methylation is the cytosine located to a guanine (5'-CpG-3'). These targets of methylation are not equally distributed in the genome, but found in CpG islands. CpG islands are sequences longer than 200bp with a GC content of over 50% (in contrast to a genome-wide average of about 40%) and an observed over expected ratio of 0.6 or greater of CpG. In mammals cells, approximately, sixty percent to ninety percent of CpG island are methylated. Unmethylated CpG islands are found mainly in the 5'-regions of housekeeping genes as well as some other tissues specifically expressed genes and usually extend from the promoter region into the first exon and sometimes into intron 1. Increased expression of DNMTs has been reported in human cancer, Nonetheless, DNMT1 has been more prominently implicated in cancer development than other DNMTs. Glutathione S-transferases p1 (GSTP1) is an autosomal gene located at chromosome 11q13 within 30kb, including 6 introns and 7 exons, and encoding 210 amino acid. GSTP1 belongs to a family of GSTs isoenzymes, that defend cells against damage mediated by oxidant and electrophilic carcinogens. It catalyzes the conjugation of glutathione with electrophilic compounds, including carcinogens and exogenous drugs, resulting in less toxic and more readily excreted metabolites. AS a Tumor suppressor gene, the CpG island encompassing the GSTP1 becomes hypermethylated associated with pathogenesis of many human cancers. During the pathogenesis of human hepatocellular carcinoma (HCC), 90% HCC tissues fail to express GSTP1 mRNA or GSTP1 polypeptides, 85% HCC tissues in which the CpG island encompassing the GSTP1 becomes hypermethylated. HepG2,Hep3B cells, the human HCC lines, have been shown to contain densely hypermethylated GSTP1 CpG island sequences and to be devoid of GSTP1 mRNh. Treatment with DNMT inhibitor has been reported to trigger the reactivation of genes carrying somatic CpG island hypermethylation.Therefore, restoration of "silenced" gene expression via therapeutic reversal of CpG island hypermethylation may be considered for use as cancer chemoprevention drugs as well as cancer treatment drugs.In previous study of our laboratory work, inhibition of growth of cancer cells derived from different tissues by CDP has been demonstrated in a dose, time-dependent mode. On this basis, further study on reactivation of hypermtehylated GSTP1 Promoter activity in MCF-7 Cells by CDP and explanation of mechanism was carried on in present work. Methods Recombinant plasmid of pGL3-GSTPlpro~m that contains hypermethylated GSTP1 promoter were constructed and then transiently transfected into human breast cancer cell line MCF-7 cell. After treated with CDP and 5-aza-C, The luciferase activity in lysates were assayed.Results The GSTP1 promoter DNA fragment was modified with the CpG methylase, M. Sssâ… , identified with the Hpaâ…¡, an sensitive restriction enzyme to methylation. The methylated GSTP1 promoter DNA fragment was not cleaved. It suggested that The recombinant plasmid pGL3-GSTPlpro~m with methylated GSTP1 promoter was successfully constructed. Low promoter activities were found in hypermethylated GSTP1 promoter. The promoter activities were reactivated and in a dose-dependent mode.Conclusion CDP has the ability to reactivate the hypermethylated GSTP1 gene promoter activity.
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