AMINOGLUTETHIMIDE SUPPRESSES ADRENOCORTICOTROPIN RECEPTOR EXPRESSION IN THE NCI-H295 ADRENOCORTICAL TUMOR-CELL LINE

The adrenostatic compound aminoglutethimide (AG), a potent inhibitor o f the P450 side chain cleavage enzyme, is used in the treatment of ACT H-dependent or adrenal Gushing's syndrome. Recently, AG has been shown to inhibit ACTH receptor (ACTH-R) mRNA expression in ovine adrenocort ical cells in a time-dependent fashion. To investigate whether ACTH-R down-regulation will also be induced in tumor cells, we studied the ef fect of AG on ACTH-R expression in the human NCI-h295 adrenocortical c arcinoma cell line, which expresses functional ACTH receptors and prod uces steroids of the glucocorticoid, mineralocorticoid and androgen pa thway. The cells were incubated in triplicate with increasing doses of AG (3, 30, 300 mu M) which suppressed steroid secretion dose-dependen tly. After 48 h, cells were harvested, and total RNA was extracted, el ectrophoresed, blotted and hybridized with a human ACTH-R cDNA probe. In parallel experiments, after preincubation with AG the cells were st imulated with ACTH (10 nM) for 10 min and the intracellular cAMP accum ulation was determined by RIA. AG significantly suppressed the baselin e ACTH-R mRNA expression in a dose-dependent fashion (300 mu M AGI 5 /- 1%; 30 mu M AG, 64 +/- 1%; 3 mu M AG, 108 +/- 19% compared with con trol cells, 100 +/- 11%). The reduced ACTH-R mRNA expression was paral leled by low ACTH-induced cAMP accumulation indicating reduced express ion of the ACTH-R protein. The adrenostatic compound metyrapone, an in hibitor of 11 beta-hydroxylase activity, also suppressed ACTH-R mRNA e xpression in a similar fashion. Stimulation of the protein kinase A pa thway by simultaneous incubation of ACTH (10 nM) or forskolin (10 mu M ) together with AG was not able to overcome the steroid biosynthesis b lockade, but reversed the inhibitory effects of AG on the ACTH-R mRNA expression. Also, cortisol (12 mu M) reversed the AG induced ACTH-R mR NA expression. We conclude that AG induces profound ACTH-R down-regula tion in the NCI-h295 cell line either by affecting the gene expression or by decreasing transcript accumulation via an effect on RNA stabili ty. This novel action of AG can be reversed by stimulation of the cAMP pathway and of the glucocorticoid-mediated signal transduction cascad e. As the down-regulation occurs in vitro at concentrations which are reached during treatment with AG in humans it may contribute to its th erapeutic activity in adrenal disease.