Interactions of estrogen-receptor and antiestrogen-receptor complexes with nuclei in vitro

Interactions of the estrogen-receptor complex (ER) with nuclei in vitro were shown to be dose, time, temperature, and tissue dependent. Specificity was demonstrated by the ability of ER charged with unlabeled 17 beta-estradiol to compete with [3H]ER for the nuclear acceptor sites. The ionic environment affected [3H]ER nuclear interactions; [3H]ER binding varied inversely with ionic strength, and apparent nuclear saturation was observed in the presence of 0.1 M KCl. Nuclear interactions of estrogen receptor charged with 17 beta-estradiol (ER) or monohydroxytamoxifen (AER) were compared. While both ER and AER (nonradiolabeled) were efficient competitors for [3H]ER nuclear binding, differences were observed when 3H-labeled ligands were used for saturation analysis of the nuclear acceptor sites. Scatchard analysis of the data revealed similar apparent Kd values for [3H]ER and [3H]AER binding to the nuclear sites (mean +/- SD, 1.2 +/- 0.5 X 10(-9) and 2.6 +/- 0.2 X 10(-9) M, respectively). However, the relative number of nuclear binding events consistently differed, with 28,000 +/- 7,300 sites/nucleus (mean +/- SD) for ER vs. 17,800 +/- 6,300 sites/nucleus for AER. Treatment of nuclei with 0-300 unit-min/ml DNAase I before incubation with receptor complexes resulted in a parallel percent decrease in the number of ER and AER nuclear binding sites. Saturation analysis performed with nuclei previously digested with 0-30 unit-min/ml DNAase I demonstrated that the apparent affinities of the receptor complexes for nuclear sites remained unchanged. Therefore, we suggest that both AER and ER bind to acceptor sites in the small portion of the chromatin hypersensitive to DNase I, but that fewer AER than ER can bind at least some of the ER nuclear acceptor sites. The lower binding capacity for AER may result in a pattern of gene expression that produces the agonist/antagonist effects observed with antiestrogens.