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

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