A ONE MINUTE PULSE OF ESTRADIOL TO MCF-7 BREAST-CANCER CELLS CHANGES ESTROGEN-RECEPTOR BINDING-PROPERTIES AND COMMITS CELLS TO INDUCE ESTROGENIC RESPONSES

Changes in estradiol (E(2))-binding parameters can be detected within minutes, while the estrogenic responses are manifested after several h ours or days of continuous exposure to the steroid. The goal of this s tudy was to determine the time of commitment for the induction of tran scription-dependent responses in the human breast cancer cell line MCF -7. In cultures grown in steroid-deprived serum, a pulse of 1 nM E(2) as short as 1 min was sufficient to maximally increase the level of th e progesterone receptor, as determined by binding of the progestin [H- 3]ORG.2058 after 2 days, and to partially stimulate cell proliferation for 5 days. From uptake experiments it was calculated that after 1 mi n about 7000 E(2) molecules were bound per cell, enough to occupy 5% o f the approx. 150,000 estrogen receptors per cell. Preincubating cells with unlabelled E(2) for 1 min lead to a loss of [H-3]E(2)-binding ca pacity. 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 E(2)- incubation the level of receptor protein remained constant as determin ed by immunoassay with the anti-ER monoclonal antibodies D547 and H222 . The dissociation kinetics of [H-3]E(2) 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 est rogenic response is thus committed by E(2) within less than 1 min and evoked by the activation of a small fraction of estrogen receptors.