Two sex steroid receptors in SC-115 mammary tumor cells

Abstract

The growth of the SC-115 mammary carcinoma in mice is androgen dependent. Estrogens antagonize the androgen effect. The high affinity binding of androgens and estrogens has been studied in soluble extracts of the tumor, of primary culture cells and clone MI 1 cells. Results indicate that two distinct specific steroid hormone-binding sites (termed ‘receptors’) are found in all cytosol fractions. The androgen-receptor (A) binds testosterone, androstanolone, cyproterone (an anti-androgen), progesterone and estradiol, but only very weakly non-steroidal diethylstilbestrol. The estrogen-receptor (E) binds estrogenic substances such as estradiol and diethylstilbestrol, but no androgen. The apparent K D, eq for A and E receptors of [ 3H]androstanolone and [ 3H]estradiol respectively, is identical (-0.5-1 nM at 4 °C). The affinity of estradiol for the A-receptor, when measured against [ 3H]androstanolone binding, indicates a K i = 17.5 nM. The concentration of binding sites is of the order of 0.1 pmole/mg protein (somewhat higher for A than for E receptor) in MI 1 cell cytosol. Studies by ultracentrifugation through glycerol-Tris gradients (low salt medium) reveal the macromolecular nature of the cytosol A and E receptors (7–7.5 S). Evidence is presented of the transfer of the A and the E receptors to nuclei after incubation of tumor slices as well as of clone MI 1 cells with the corresponding hormones. Experiments suggest that the two different binding sites are present on two separated macromolecular moieties. After incubation at 37 °C of tumor slices with 10–20 nM [ 3H]testosterone, or with 10 nM [ 3H]estradiol, the corresponding radioactive hormone-receptor complexes are, as expected, found in the nuclear KCl extracts. In parallel experiments, where slices are incubated with non-radioactive hormones at the same concentration and the nuclear KCl extracts subsequently treated by radioactive steroids, no available androgen-binding sites are found in the nuclei after exposure to estradiol, nor estrogen-binding sites after exposure to testosterone. Therefore, in the same cell, two receptors are present which bind androgens and estrogens with high affinity, and one given hormone (estradiol) can be specifically bound (with different affinities) by two different receptors which, however, discriminate a synthetic analog (diethylstilbestrol). The data may give some molecular background for interpreting responses to the same hormone which may differ at various concentrations, for studying effects of analogs, and for analysing the control of tumor growth by antagonistic steroids.