Subcellular compartmentalization of estrogen receptors in human breast cancer cells

Abstract

In MCF-7 human breast cancer cells about 75% of the cell's total estrogen receptor sites were unexpectedly found in nuclei not bound with estrogens [13]. These findings were based on studies with crude nuclei and it was originally proposed that these unfilled nuclear estrogen receptors might be biologically active in the absence of estrogen [23]. In order to examine the subcellular distribution of receptor more critically, we have prepared highly purified MCF-7 nuclei, assessed their purity by electron microscopy RNA DNA and DNA protein ratios, and compared their estrogen receptor content with less pure subcellular fractions. In estrogen-withdrawn cells the percentage of the total unfilled receptors found in nuclear fractions (Rn) decreased dramatically from 30% (1.81 pmol/mg DNA) in the crudest preparation to only 3% (0.19 pmol/mg DNA) in highly purified nuclei. (This decrease is not due to an irreversible loss of receptor sites, as receptors lost from nuclei were recovered in the cytosol.) By contrast, after 1 h incubation in vivo with estradiol, the number of estrogen-filled receptor sites (RnE) found in the crudest nuclear preparation (3.28 pmol/mg DNA) and in the most highly purified nuclei (2.66 pmol/mg DNA) were nearly the same. It is very likely that the high level of unfilled receptors found in crude nuclei is a contamination of the preparation with cytoplasmic receptor. 1. 1. Electron microscopy shows gross contamination of crude nuclei with whole cells and cytoplasmic attachments. Purification of nuclei removes attaching cytoplasm which is parallelled by a dramatic decrease in unfilled receptor sites contained in the nuclear preparations. 2. 2. Multiple extractions with Tris buffer (without KCl) releases as much as 72% of the receptor contained in crude nuclei and treatment with 0.2% Triton X-100 extracts more than half. 3. 3. Tris-extractable receptor from crude nuclei sediments identically with cytosol receptor as a single 8S peak on low salt sucrose gradients. 4. 4. Unfilled receptor does not bind in vitro to purified nuclei. Although we cannot entirely exclude the possibility that unfilled receptors are nuclear in origin but may leak from nuclei during cell fractionation, these results are consistent with the localization of unfilled receptor largely in the cytoplasm of MCF-7 cells, as found in normal target tissues such as the rat uterus.