The adrenostatic compound aminoglutethimide (AG), a potent inhibitor of the P450 side chain cleavage enzyme, is used in the treatment of ACTH-dependent or adrenal Cushing's syndrome. Recently, AG has been shown to inhibit ACTH receptor (ACTH-R) mRNA expression in ovine adrenocortical cells in a time-dependent fashion. To investigate whether ACTH-R down-regulation will also be induced in tumor cells, we studied the effect of AG on ACTH-R expression in the human NCI-h295 adrenocortical carcinoma cell line, which expresses functional ACTH receptors and produces steroids of the glucocorticoid, mineralocorticoid and androgen pathway. The cells were incubated in triplicate with increasing doses of AG (3, 30, 300 microM) which suppressed steroid secretion dose-dependently. After 48 h, cells were harvested, and total RNA was extracted, electrophoresed, blotted and hybridized with a human ACTH-R cDNA probe. In parallel experiments, after preincubation with AG the cells were stimulated with ACTH (10 nM) for 10 min and the intracellular cAMP accumulation was determined by RIA. AG significantly suppressed the baseline ACTH-R mRNA expression in a dose-dependent fashion (300 microM AG, 5+/-1%; 30 microM AG, 64+/-1%; 3 microM AG, 108+/-19% compared with control cells, 100+/-11%). The reduced ACTH-R mRNA expression was paralleled by low ACTH-induced cAMP accumulation indicating reduced expression of the ACTH-R protein. The adrenostatic compound metyrapone, an inhibitor of 11beta-hydroxylase activity, also suppressed ACTH-R mRNA expression in a similar fashion. Stimulation of the protein kinase A pathway by simultaneous incubation of ACTH (10 nM) or forskolin (10 microM) together with AG was not able to overcome the steroid biosynthesis blockade, but reversed the inhibitory effects of AG on the ACTH-R mRNA expression. Also, cortisol (12 microM) reversed the AG-induced ACTH-R mRNA expression. We conclude that AG induces profound ACTH-R down-regulation in the NCI-h295 cell line either by affecting the gene expression or by decreasing transcript accumulation via an effect on RNA stability. This novel action of AG can be reversed by stimulation of the cAMP pathway and of the glucocorticoid-mediated signal transduction cascade. As the down-regulation occurs in vitro at concentrations which are reached during treatment with AG in humans it may contribute to its therapeutic activity in adrenal disease.

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