Steroid derivatives for electrophilic affinity labelling of glucocorticoid binding sites: interaction with the glucocorticoid receptor and biological activity.

To investigate the possible use of electrophilic affinity labelling for the characterization of glucocorticoid receptors, different chemically reactive derivatives of deoxycorticosterone (deoxycorticosterone 21-mesylate and deoxycorticosterone 21-(1-imidazole) carboxylate), dexamethasone (dexamethasone 21-mesylate, dexamethasone 21-iodoacetate and dexamethasone 21-bromoacetate) and progesterone (21-chloro progesterone) were tested for their ability to bind irreversibly to the glucocorticoid receptor from goat lactating mammary gland. Using partially purified receptor, only one of the steroids tested, dexamethasone 21-mesylate (DXM-M) was found more effective than dexamethasone (DXM) in preventing exchange of radioactive dexamethasone in the receptor binding site. The affinity of DXM-M for the glucocorticoid receptor, measured by competitive binding assay, was 1/15 that of DXM. Polyacrylamide gel electrophoresis in sodium dodecyl sulphate of the [3H]-DXM-M labeled glucocorticoid receptor revealed a specific covalently radiolabeled fraction corresponding to an apparent molecular weight of 75,000 to 80,000. The biological activity of DXM-M was studied in RPMI 3460-clone 6 Syrian hamster melanoma cells, a cell line which is sensitive to growth inhibition by glucocorticoids. Like DXM, DXM-M inhibits the growth of RPMI 3460-clone 6 cells and it acts as a slowly reversible glucocorticoid agonist at concentrations which correlate with the affinity of DXM-M for the glucocorticoid receptor in vitro.

Growth inhibition by glucocorticoids in RPMI 3460 melanoma cells.

We have shown previously that dexamethasone inhibits the growth of RPMI 3460 melanoma cells and that cytosols of these cells contain a dexamethasone-binding macromolecule which has properties expected for a glucocorticoid receptor. In this paper, we show that two other glucocorticoids, triamcinolone acetonide and hydrocortisone, also cause growth inhibition in RPMI 3460 cells and that progesterone can block this response. The biological effect of these steroids correlates well with their previously reported ability to bind receptor, a result consistent with the concept that the glucocorticoid-induced growth inhibition is a receptor-mediated event. We have also investigated the nature of the growth response and shown that glucocorticoids inhibit growth in these melanoma cells by increasing the population-doubling time rather than by cytolytic effects. Moreover, a limited exposure to dexamethasone fails to trigger the growth inhibition, suggesting that the continued presence of steroid is necessary for growth inhibition to occur. Since serum-free medium and medium conditioned by exposure to cells do not affect the glucocorticoid-induced growth inhibition, we have obtained no evidence that either the interaction of glucocorticoids with serum factors or cell-induced changes in medium components are involved in the response. In addition to the effect on growth, we have also described morphological alterations which occur in the presence of dexamethasone.