Detection of Genetic Lesions in Breast Cancer
Abstract
The purpose of this proposal is to develop a "next generation" representational difference analysis that uses transcribing nuclear DNA. Organomercury and Sulfolink Coupling gel chromatography could isolate soluble transcribed chromatin from breast cancer cells. However, most transcribed chromatin is insoluble. Thus, we used ChIPs (chromatin immunoprecipitation) technique to isolate active chromatin that is bound to highly acetylated histones. Before applying Chips, we studied the dynamics of histone acetylation in breast cancer cells, and the effect of estradiol on these processes. The kinetics of histone acetylation in hormone dependent (T47D5) and hormone independent (MDA MB 231) breast cancer cells were determined. Estradiol increased hi stone acetylation by decreasing the rate of histone deacetylation in T47D5 cells. Estradiol had no effect on histone acetylation in hormone independent cells. The mechanism by which estradiol decreased the rate of histone deacetylation was explored. Estradiol affected the sub nuclear trafficking of the estrogen receptor (ER) and ER associated coactivators (SRC- 1 and SRC-3, which have hi stone acetyltransferase activity) which become tightly bound to the nuclear matrix. However, estradiol did not affect the activity or subnuclear distribution of histone deacetylases. Finally, the Chips protocol was successfully used to isolate transcriptionally active DNA from T47D5 and MDA MB 231 cells.
Document Details
- Document Type
- Technical Report
- Publication Date
- Oct 01, 2000
- Accession Number
- ADA387879
Entities
People
- James R. Davie
Organizations
- University of Manitoba