Role of DNA Methylation in the Mechanism of Anti-Estrogenic Action of Tamoxifen.
Final rept. 1 Sep 96-31 Dec 98,
GEORGETOWN UNIV WASHINGTON DC MEDICAL CENTER
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The expression of estrogen receptor ER is regulated by hypermethylation of CpG islands in ER- breast tumor cells. Hypomethylation with 5-azacytidine was able to restore ER expression in ER- cells. To investigate the possible role of DNA methylation as one of the outcomes of antiestrogen action in ER breast tumor cells, the present study is aimed at detecting methylated CpG sites in breast tumor cells exposed to tamoxifen. The experimental approach is to employ restriction landmark genome scanning RLGS coupled with methylase-sensitiveinsensitive restriction enzyme digestion of genomic DNA. During the first year of the project, we optimized the performance of RLGS using the Iso-Dalt equipment. Toward this end, we achieved 1 landmark digestion of genomic DNA from breast tumor cells, with restriction enzymes Not-I-EcoRV and labeling the Not-I ends with alpha-P-32-dGTP and alpha-P-32-dGTP by a sequenase reaction, 2 resolution of such land marked, high molecular weight DNA 40-10 kb on 0.8 agarose tube gels 3 in-gel digestion of agarose bound Not-I DNA fragments with restriction enzymes followed by electrophoresis in 5 polyacrylamide slab gels to reveal RLGS patterns. At the end of second year of the project, we conclude that it is only practical to work with DNA of less than 4-5 kb rather than genomic DNA to achieve in-gel digestion.
- Medicine and Medical Research