Characteristics of binding to estrogen, androgen, progestin, and glucocorticoid receptors in 7,12-dimethylbenz(a)anthracene-induced mammary tumors and their hormonal control.

Abstract In order to perform simultaneous measurement of binding of four classes of steroids and facilitate study of their mechanism of action in 7,12-dimethylbenz( a )anthracene-induced mammary tumors in the rat, we have investigated in detail the binding characteristics of 17β-[2,4,6,7,16,17- 3 H]estradiol (17β-[ 3 H]estradiol), 17,21-[6,7- 3 H]dimethyl-19-norpregna-4,9-diene-3,20-dione ([ 3 H]R5020), [ 3 H]dexamethasone (DEX), and 5α-[ 3 H]dihydrotestosterone (DHT) in cytosol prepared from these tumors. Following assessment of optimal buffer composition, separation of bound and free steroids was achieved with dextran-coated charcoal, protamine sulfate, or hydroxylapatite. Using the same buffer and the optimal method of separation for each tracer, we have found that the time course of binding of each labeled steroid was fast, the slowest rate of association being obtained with [ 3 H]DEX ( t ⋍ 100 to 150 min) at 4°. In addition, the level of specific binding of each tracer was stable for a period of at least 21 hr at 4° but decreased markedly at 23° (except with 17β-[ 3 H]estradiol). Complete exchange of the tracer from the binding component was achieved at 4° for [ 3 H]R5020 and at 23° for 17β-[ 3 H]estradiol. Although [ 3 H]DEX dissociated from its binding component at 4°, the receptor complex was unstable during long-term incubations necessary to achieve complete exchange. No significant exchange of [ 3 H]DHT binding occurred at 4°. Using six increasing concentrations of a large series of unlabeled steroids, each tracer showed a high degree of binding specificity. Sucrose gradient analysis performed in low-ionic-strength buffer revealed specific and protamine sulfate-precipitable steroid-binding components migrating in the 8 to 9S area for 17β-[ 3 H]estradiol, 7S and 4 to 5S for [ 3 H]R5020, and 7 to 8S for [ 3 H]DEX and [ 3 H]DHT. Sucrose gradient analysis in a high-ionic-strength buffer (0.4 m KCl) gave only one labeled area (3 to 5S) for each tracer. Scatchard analysis revealed K d 9s of 0.14 nm for 17β-[ 3 H]estradiol, 2.9 nm for [ 3 H]R5020, 13 nm for [ 3 H]DEX, and 0.45 nm for [ 3 H]DHT binding. In a second series of experiments, while adrenalectomy had no effect on the binding of any of the four classes of steroids, 17β-[ 3 H]estradiol, [ 3 H]R5020, and [ 3 H]DHT binding levels were decreased in 7,12-dimethylbenz( a )anthracene-induced mammary tumors after ovariectomy plus adrenalectomy or hypophysectomy, while the binding of [ 3 H]DEX remained unchanged. The present data describe the optimal conditions for simultaneous and specific measurement of binding of four classes of steroids in cytosol from 7,12-dimethylbenz( a )anthracene-induced mammary tumors in the rat. Moreover, as revealed by hypophysectomy, adrenalectomy, and ovariectomy, the levels of the progesterone and glucocorticoid receptors are under different hormonal control.