Evidence for the origin of the unoccupied oestrogen receptor in nuclei of a human breast-cancer cell line (MCF-7)

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Abstract
The origin of the unoccupied nuclear estrogen receptor (Rn) was studied. Three working hypotheses were investigated: (a) Rn is a dissociation product of the estrogen occupied nuclear receptor (ERn); (b) ERn is only partially occupied, so that additional binding may occur at 0.degree. C (the temperature at which estradiol saturates unoccupied sites); and (c) Rn is derived from the penetration of unoccupied cytoplasmic receptor (Rc) into the nucleus. The MCF-7 cell line was used as a model in the present investigation. The amount of unoccupied receptors was measured by saturation with 7.5 nM-[3H]estradiol at 0.degree. C, whereas the occupied receptors were measured by exchange at 30.degree. C. The cells at preconfluency were exposed to a medium fortified with 10 nM-[3H]estradiol for 1 h, washed and cultured up to 5 days in fresh growth medium. The distribution of estradiol receptors was determined before exposure and during the following 5 days. After 1 h exposure only ERn was found in the nuclear fraction. Thereafter ERn declined continuously so that on day 5 it approached 15% of its value measured 1 h after exposure. Although after 3 days .apprx. 80% of ERn disappeared no Rn appeared, which contradicts hypotheses (a) and (b). On day 4 Rn and Rc appeared simultaneously. The appearance of Rn and Rc was not prevented by culturing the cells in an estrogen-free medium, supporting hypothesis (c). Exposure of cells to increasing concentration of [3H]estradiol (0.1-10 nM) for 1 h resulted in a parallel increase in ERn without increasing the amount of unoccupied binding sites, which contradicts hypothesis (b). The present study supports hypothesis (c), i.e., Rc may also penetrate the nucleus without binding to estradiol.