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Role of Nuclear Matrix in Estrogen Regulated Gene Expression in Human Breast Cancer Cells
Annual rept. 15 Jul 97-15 Jul 98
MANITOBA UNIV WINNIPEG
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The goal of this research is to study association of the estrogen receptor ER with the nuclear matrix, and to identify nuclear matrix acceptor sites for ER. We constructed an ER fusion protein GFP-ER with a His6 tag, HA tag, and the green fluorescent protein. Using fluorescent imaging techniques we studied the cellular localization of GFP-ER in the presence of various ER ligands in human breast cancer cell lines transiently transfected to express the GFP-ER protein. Striking differences in the patterns of distribution were observed. Isolation of GFP-ER associated proteins requires a large number of cells expressing GFP-ER. Therefore, three stable cell lines expressing GFP-ER under the control of an inducible promoter were constructed. Characterization of GFP-ER expression in one of the stable cell lines demonstrated that we can control levels of GFP-ER expression. We characterized the effect of expressing different levels of GFP-ER on regulation of the endogenous c-myc gene, to determine what levels of expression caused aberrant effects. We demonstrated that GFP-ER association with nuclear matrix increases with the length of exposure to estrogen, and that ER associated to the nuclear matrix is also binding to DNA, suggesting that nuclear matrix bound ER is transcriptionally active.
APPROVED FOR PUBLIC RELEASE