Role of Nuclear Matrix in Estrogen Regulated Gene Expression in Human BreastCancer Cells

摘要

The goal of this research is to study the association of the estrogen receptorwith the nuclear matrix, and to identify the nuclear matrix acceptor sites for the estrogen receptor. We have constructed an ER fusion protein (GFP-ER) with a His(6) tag, HA tag, and the green fluorescent protein. In transiently transfected ER negative cells GFP-ER causes the ligand dependent transactivation of an ERE-reporter construct. Using confocal fluorescent microscopy we observed the cellular localization of GFP-ER in living cells. GFP-ER is localized to the nuclei, with exclusion from the nucleoli, both in absence of ligand, and when 17 beta-estradiol, or 4-hydroxytamoxifen are added. In MCF7 cells (ER+), the nuclear distribution has a speckled or punctate pauern which does not change with the addition of ligand. In MDA MB 231 cells (ER-), the distribution of GFP-ER in cells without ligand added is fuzzy or diffuse, with the addition of ligand the nuclear distribution becomes more localized or granular. The pattern of distribution of GFP-ER appears the same on nuclear matrices isolated on slides as in living cells. We corroborated this by extracting nuclear matrix in solution and detecting the presence of GFP-ER by Western blotting with HA-antibody.