A one minute pulse of estradiol to MCF-7 breast cancer cells changes estrogen receptor binding properties and commits cells to induce estrogenic responses.

Abstract

Changes in estradiol (E2)-binding parameters can be detected within minutes, while the estrogenic responses are manifested after several hours or days of continuous exposure to the steroid. The goal of this study was to determine the time of commitment for the induction of transcription-dependent responses in the human breast cancer cell line MCF-7. In cultures grown in steroid-deprived serum, a pulse of 1 nM E2 as short as 1 min was sufficient to maximally increase the level of the progesterone receptor, as determined by binding of the progestin [3H]ORG.2058 after 2 days, and to partially stimulate cell proliferation for 5 days. From uptake experiments it was calculated that after 1 min about 7000 E2 molecules were bound per cell, enough to occupy 5% of the approx. 150,000 estrogen receptors per cell. Preincubating cells with unlabelled E2 for 1 min lead to a loss of [3H]E2-binding capacity. As analysed by Scatchard plot, this loss was due to a decrease in the number of exchangeable binding sites and, to a lesser extent, to an increase in the dissociation constant. For up to 30 min of E2-incubation the level of receptor protein remained constant as determined by immunoassay with the anti-ER monoclonal antibodies D547 and H222. The dissociation kinetics of [3H]E2 bound by MCF-7 cells after a 5 min pulse were biphasic, with the slower phase having a rate of 2.3 x 10(-3) min. This rate is characteristic of the activated ER. The estrogenic response is thus committed by E2 within less than 1 min and evoked by the activation of a small fraction of estrogen receptors.