Dissociation of [3H]estradiol from perfused monolayer cultures of estrogen receptor-positive MCF-7 human breast cancer cells is described. A kinetic model is proposed to which loss of estradiol from the cells occurs from an intracellular compartment of free estradiol that is in dynamic equilibrium with estradiol bound to both specific (receptor) and nonspecific cellular binding sites. We found that the estrogen receptor in MCF-7 cells has little effect on the dissociation rate constant of estradiol from MCF-7 cells by comparing the dissocation of estradiol from MCF-7 cells with estrogen receptor-negative MDA-MB-231 cells. The major determinant of the rate constant of estradiol dissociation from both MCF-7 and MDA-MB-231 cells is the equilibrium between free intracellular estradiol and estradiol bound to slowly dissociating nonspecific binding sites. This result indicates that slow dissocation of estradiol from nonspecific cellular binding sites may prolong the occupancy of specific receptor sites. Thus, the equilibrium between estradiol bound to specific receptor-binding sites and nonspecific binding sites may play a role in determining estrogenic activity. The antiestrogens, tamoxifen [trans-1-(p-β-diethylaminoethoxyphenyl)1,2-diphenyl but-1-ene], clomiphene [1-(p-diethylaminoethoxyphenyl)1,2-diphenyl-2-chloroethylene citrate], and nafoxidine [1-(2-[p-(3,4-dihydro-6-methoxy-2-phenyl-1-naphthyl)phenoxy]ethyl pyrrolidine hydrochloride] accelerate the loss of estradiol from receptor-binding sites in perfused MCF-7 cells. One possible explanation for this effect is that antiestrogens inhibit the binding of free intracellular estradiol to the receptor and thereby greatly reduce the influence of the slowly dissociating, nonspecifically bound pool of estradiol on receptor occupancy. This effect of antiestrogens may contribute to their antagonism of estrogenic effects if temporal requirements for estrogen-receptor complexes exist. Evidence which suggests that estrogen-induced proliferation of rat uterine tissue has specific temporal requirements for nuclear estrogen-receptor complexes is the relatively rapid loss of nuclear estriol-receptor complexes and the relatively weak ability of estriol to induce growth compared to estradiol. We find that estriol-receptor complexes disappear from perfused MCF-7 cells about 4 times faster than do estradiol-receptor complexes. The rate of loss of estradiol-receptor complexes from MCF-7 cells perfused with antiestrogens is nearly identical to the rate of loss of estriol-receptor complexes from MCF-7 cells. Thus, acceleration of the loss of estradiol-receptor complexes from MCF-7 cells by antiestrogens may be sufficient to block or attenuate certain estrogen-induced responses. Dissociation of estradiol from specific binding sites in MCF-7 cells is first order after incubation with low estradiol concentrations (0.03 or 0.06 nM) and sequential first order after incubation of MCF-7 cells with higher estradiol concentrations (0.5, 4, or 30 nM). This difference in the dissociation kinetics of specifically bound estradiol after incubation with high or low estradiol concentrations suggests that there may be two types of competible or specific binding sites in the MCF-7 cells. A slow phase of estradiol dissociation is observed after incubation with all estradiol concentrations. By several criteria, this phase of dissociation appears to be the loss of estradiol from approximately 20,000 high affinity estrogen receptor-binding sites/cell. The nature of the more rapidly dissociating type of specific binding observed after incubating cells with 0.5, 4, or 30 nM estradiol is uncertain but may be distinct from the lower affinity type II estrogen receptors identified in rat uterine tissue.
ASJC Scopus subject areas