Role of DNA Methylation in the Mechanism of Anti- Estrogenic Action of Tamoxifen.
Annual rept. 1 Sep 96-31 Aug 97
GEORGETOWN UNIV WASHINGTON DC
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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 labeled the Not-I ends with alpha-32P-CTP and alpha-32P-GTP by a sequenase reaction, 2 resolution of such landmarked, high molecular weight DNA 40-10 Mb on 0.8 agarose tube gels 3 in-gel digestion of agarose bound Not-I-landmarked DNA fragments with methylase-sensitiveinsensitive enzymes to completion followed by electrophoresis in 5 polyacrylamide slab gels to reveal RLGS patterns. Further studies are in progress to compare and contrast the effects of tamoxifen treatment on DNA methylation and ER expression.
- Medicine and Medical Research