Isolation of Estrogen-Responsive Genes in Human Breast Cancer Cells
Annual rept. 1 Sep 2001-1 Sep 2002
MANITOBA UNIV WINNIPEG
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The purpose of this proposal is to isolate and identify estrogen-responsive genes in human breast cancer cells using the chromatin immunoprecipitation ChIP protocol. Estrogen receptor ER -bound DNA has been isolated using ChIP from human breast cancer NCF-7 T5 cells ER positive and hormone dependent. Southern blotting analysis shows that ER-DNA contains ER-responsive sequences ER, PR, P52 and c-myc. PCR analysis demonstrated that ER-DNA contains the p52 gene promoter, which is expressed in ER positive cells. Recent evidence shows that ER and associated coactivators are recruited to the estrogen inducible promoters in a cyclical manner. It is reasonable to believe that ER interaction with DNA is dynamically altered during estrogen induction. ER-bound DNA from MCF-7 T5 cells treated with estradiol E2 for 30, 60 and 120 min were isolated and analyzed by POR. A library of ER-bound DNA from 30 min E2 treated MCF-7 T5 cells has been constructed. ChIP-ER-DNA fragments were cloned, sequenced, and blast searched in GenBank. Approximately 900 positive colonies were collected from this library, and approximately 100 cloned DNA fragments were sequenced. A - 12ERE-Sp1 sequence has been identified in PACE4 pro-protein convertase and EWS Ewings sarcoma genes. Studies to determine whether these genes respond to estrogen is currently being determined.
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