The Capacity of the Antiestrogen CI-628 to Activate the Estrogen Receptor in Vitro

The interactions of the antiestrogen CI-628 (α-[4-pyrrolidinoethoxy]phenyl-4-methoxy-α′- nitrostibene) with the cytoplasmic estrogen receptors from rat and calf uteri have been examined. The capacity of CI-628 to activate the estrogen receptor in vitro has been studied using three criteria previously established with the estradiol-receptor complex: 1) the biphasic dissociation kinetics of the estrogen-receptor complex, 2) the temperature- and hormone-dependent transformation of the receptor from a 4S to 5S sedimenting form, and 3) the translocation of the estrogen receptor from the cytoplasmic to the nuclear fraction of uterine tissue. The dissociation of [³H]CI-628 from the cytosol receptor at 0 C (t1/2 = 54 min) is 250-fold faster than the dissociation of estradiol and shows monophasic kinetics. Only a 2- to 3-fold decrease in the rate of the monophasic [³H]- CI-628 dissociation occurs after incubation under conditions sufficient to activate the estradiol-receptor complex. The rapid dissociation of [³H]CI-628 from the receptor causes a redistribution of label during sucrose gradient centrifugation. The true sedimentation profile of the receptor was determined by sedimenting the CI-628-receptor complex through a gradient containing unlabeled CI-628, followed by exchange of the receptorbound CI-628 for [³H]estradiol in the individual gradient fractions. Using this method, the cytoplasmic CI-628-receptor complex sediments in low salt gradients as an 8S species, as does the estradiol-receptor complex. In high salt gradients, the nonactivated CI-628-receptor complex sediments as a 4S species; warming to effect transformation does not produce the 5S (activated) receptor. High concentrations of CI-628 (5-10 µM), compared to 20 nM estradiol, effect only a moderate translocation of the estrogen receptor to the nucleus of uterine tissue incubated in vitro. The nuclear CI-628-receptor complex sedimentation rate was 4S, indicative of the nonactivated form of the receptor. We suggest that the rapid rate of CI-628 dissociation from the receptor results in an exceedingly small quantity and short lifetime of the activated CI-628-receptor complex. Thus, in vitro, in the absence of metabolism, CI-628 occupies cytoplasmic receptor sites, yet is ineffective in inducing receptor activation; this may, in part, account for its antagonistic character.