HeLa S3 cells contain high affinity, saturable receptors specific for glucocorticoids (congruent to 20,000 per cell). Growth of HeLa S3 cells in media containing dexamethasone (10(-6) M) results in a pronounced (congruent to 70%) reduction in the total cellular level of nuclear or cytoplasmic dexamethasone receptor number without any alteration in steroid-receptor dissociation constant (Kd congruent to 1 X 10(-9) M). This reduction in receptor number is not the result of simple receptor occupation, since this effect also occurs when receptor number is modulated by the direct addition of [3H]dexamethasone. Both control and dexamethasone-treated cells attain equilibrium states of receptor binding by 120 min at 0 C and by 60 min at 37 C, enabling estimation of receptor number and affinity by Scatchard analysis of saturation curves. Down-regulation of glucocorticoid receptor occurs at steroid concentrations as low as 10(-9) M (40% reduction). This alteration in receptor occurs after 24 h of hormone but not after either 2 or 6 h. Only the active glucocorticoids, (dexamethasone, cortisol) down-regulate glucocorticoid receptors. Cortexolone, estradiol, and 5 alpha-dihydrotestosterone have no apparent effect on this process, whereas progesterone (10(-7) M) is partially effective. Subcellular distribution studies indicate that glucocorticoids affect only that population of receptors capable of nuclear translocation at 37 C. We conclude that glucocorticoids can regulate the metabolism of their own receptors, via undefined mechanisms.

HeLa S3 cells contain high affinity, saturable protein receptor molecules which steriospecifically bind active glucocorticoids. The number of glucocorticoid receptors per cell changes as cells in culture proceed through the cell cycle (1). HeLa S3 cells brought to the GI/S boundary by the double thymidine block procedure undergo a rapid synchronous round of DNA synthesis when released into thymidine-free medium. Analysis of glucocorticoid receptor binding indicates a rapid rise in cellular receptor number when measurements are made in whole cells at either 3 C (cytoplasmic) or 37 C (nuclear). Cytoplasmic receptors are maintained at levels about 150% above late GI values throughout S and GII phases until mitosis occurs, whereas the nuclear binding of hormone is dramatically reduced during GII and remains low during mitosis and early GI. Similar cell cycle-dependent alterations in receptor number occur in synchronized cell populations obtained by unit gravity sedimentation. Sucrose density gradient analysis and Sephacryl S-200 gel filtration of cytoplasmic receptors during the cell cycle indicate a rapid increase in approximately 7-8S dexamethasone-binding component during early S phase, when receptors accumulate in cells. The role of RNA, protein, and DNA syntheses in mediating this S phase increase in receptor number were next examined. Both cycloheximide and puromycin blocked the increased receptors accumulation that occurs during the S phase, whereas delayed addition of these inhibitors led to partial increases in receptor binding. Several inhibitors of RNA synthesis [actinomycin D, L-521,818-00E10 (Merck), and alpha-amanitin] all effectively inhibited the S phase accumulation of receptor. Increased receptor binding during the S phase was not dependent on DNA replication. These data suggest that the increase in glucocorticoid receptor number during the S phase results from alterations in RNA and protein synthetic processes.

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