calcimycin and Adrenal-Cortex-Neoplasms

We have studied the hormonal regulation of type 1 angiotensin-II receptor (AT1-R) mRNA expression and [125I]angiotensin-II ([125I]AII) binding in human adrenocortical carcinoma H295 cells, which exhibit predominantly AT1-subtype receptors. Activation of the cAMP signaling pathway with forskolin or (Bu)2cAMP caused a rapid decrease in AT1-R mRNA levels (decreased 65% within 3 h). This preceded a time-dependent (maximal, 70% within 12 h) and dose-dependent (IC50, 2 microM forskolin) loss of [125I]AII binding together with decreased phosphoinositidase-C activation (72% decrease) on subsequent AII challenge. Thus, the decreases in AT1-R mRNA levels and functional receptor expression parallel each other in response to activation of protein kinase-A. AII treatment also caused a rapid loss in AT1-R mRNA (maximal, 80% decrease within 3 h), but 48-h treatment caused both [125I]AII binding and the subsequent phosphoinositidase-C response to decrease by only 6% (P < 0.05) and 22% (P < 0.05), respectively. The effect of AII on AT1-R mRNA levels was fully reproduced by the combination of calcium ionophore (A23187) and phorbol ester (12-O-tetradecanoylphorbol 13-acetate), suggesting that AII action was through protein kinase-C and possibly other Ca(2+)-sensitive protein kinases. The effect of AII, but not forskolin, was reversed by treatment in the presence of cycloheximide. In conclusion, control of AT1-R expression is differentially regulated by adenylate cyclase and phosphoinositidase-C signaling pathways, which act at multiple levels in human adrenocortical cells.

Topics: Adrenal Cortex Neoplasms; Angiotensin II; Base Sequence; Bucladesine; Calcimycin; Colforsin; Cyclic AMP; Cycloheximide; Gene Expression Regulation, Neoplastic; Humans; Kinetics; Molecular Sequence Data; Phosphoric Diester Hydrolases; Receptors, Angiotensin; RNA, Messenger; Signal Transduction; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

NCI-H295 is a recently described human adrenocortical carcinoma cell line that makes a variety of steroid hormones. We sought to determine if steroidogenesis in these cells employs the same enzymes as those used in normal adrenal steroidogenesis, and if the genes encoding those enzymes exhibit characteristic responsiveness to activators of the protein kinase-A and -C pathways of intracellular second messengers. Northern blots show that NCI-H295 cells contain abundant mRNAs for three key steroidogenic enzymes, cytochrome P450scc, cytochrome P450c17, and cytochrome P450c21. These mRNAs accumulated in a time- and dose-dependent fashion in response to 8-bromo-cAMP (8Br-cAMP), forskolin, cholera toxin, and 3-isobutyl-1-methylxanthine, all activators of the protein kinase-A pathway. Nuclear run-on assays and actinomycin-D transcriptional inhibition experiments show that cAMP regulates the expression of all three genes primarily at the transcriptional level. Inhibition of protein synthesis with cycloheximide did not prevent the cAMP-induced accumulation of P450scc or P450c17 mRNAs, but did inhibit accumulation of P450c21 mRNA, suggesting that cAMP is acting through a mechanism dependent on protein synthesis to promote accumulation of P450c21 mRNA. Stimulation of the protein kinase-C pathway with phorbol ester decreased P450scc and P450c17 mRNAs, but stimulated the accumulation of P450c21 mRNA. RNase protection experiments, Northern blot hybridizations, and reverse transcription-polymerase chain reaction show that NCI-H295 cells express both the 11 beta-hydroxylase (P450c11 beta) encoded by the P450c11B1 gene and the aldosterone synthetase (P450c11AS) encoded by the P450c11B2 gene. 8Br-cAMP increased the abundance of both of these mRNAs with similar kinetics, with maximal accumulation of both after about 24 h. NCI-H295 cells also contain the mRNAs for aromatase and insulin-like growth factor-II. 8Br-cAMP increased the abundance of aromatase mRNA and decreased the abundance of IGF-II mRNA. These studies show that NCI-H295 cells express most of the enzymes needed for human adrenal steroidogenesis, and that the genes encoding these enzymes respond to stimulation of second messenger pathways in a manner similar to that of human adrenals. NCI-H295 cells appear to be a good model for studying the molecular regulation of human adrenal steroidogenesis.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Adrenal Cortex; Adrenal Cortex Hormones; Adrenal Cortex Neoplasms; Adrenocorticotropic Hormone; Angiotensin II; Aromatase; Base Sequence; Calcimycin; Carcinoma; Cycloheximide; Cytochromes; Enzymes; Gene Expression Regulation, Enzymologic; Glyceraldehyde-3-Phosphate Dehydrogenases; Humans; Insulin-Like Growth Factor II; Molecular Sequence Data; RNA, Messenger; Second Messenger Systems; Tetradecanoylphorbol Acetate; Transcription, Genetic; Tumor Cells, Cultured; Zona Glomerulosa

The conversion of cholesterol to pregnenolone, the rate-limiting step in steroid hormone synthesis, occurs on mitochondrial cytochrome P450scc, which catalyzes this reaction by receiving electrons from NADPH via a flavoprotein [adrenodoxin reductase (AdRed)] and an iron sulfur protein [adrenodoxin (Adx)]. The behavior of the genes and mRNAs encoding these proteins has been studied in several systems, but little is known about the behavior of the human proteins. Using cloned cDNAs for human P450scc and AdRed, we constructed bacterial expression vectors to make milligram quantities of the corresponding proteins. These, plus purified human Adx similarly prepared by Dr. L. Vickery, were injected into rabbits to raise antiserum to each of the proteins. Each antiserum was highly specific and did not cross-react with other mitochondrial proteins detectable by Western blotting. Human JEG-3 choriocarcinoma cells and mouse Y-1 adrenocortical carcinoma cells were then incubated for 0-24 h with 1 mM 8-bromo-cAMP (8Br-cAMP) or 30 nM phorbol 12-myristate 13-acetate (PMA; phorbol ester) plus 1 microM A23187 (calcium ionophore) to activate the protein kinase-A and -C pathways, respectively. In JEG-3 cells, 8Br-cAMP increased and PMA/A23187 slightly decreased the abundance of P450scc and Adx, but neither treatment had a detectable effect on AdRed. The production of pregnenolone by these cells increased 3-fold in response to 8Br-cAMP and fell to one third in response to PMA/A23187. In Y-1 cells, 8Br-cAMP increased the abundance of all three proteins, while PMA/A23187 decreased the abundance of P450scc and Adx. The production of pregnenolone by these cells increased 9-fold in response to 8Br-cAMP and was unaffected by TPA/A23187. These studies show that the three proteins of the cholesterol side-chain cleavage system behave in response to 8Br-cAMP and PMA/A23187 as predicted from the study of their genes and mRNAs, indicating that the chronic regulation of steroidogenesis in these cell systems is regulated principally at the level of mRNA abundance.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Adrenal Cortex Neoplasms; Adrenodoxin; Animals; Calcimycin; Cholesterol Side-Chain Cleavage Enzyme; Choriocarcinoma; Cloning, Molecular; Escherichia coli; Ferredoxin-NADP Reductase; Genetic Vectors; Humans; Kinetics; Mice; Mitochondria; Plasmids; Pregnenolone; Protein Kinase C; Protein Kinases; Recombinant Proteins; Restriction Mapping; RNA, Messenger; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

Very little has been known of the biochemical function of a human adrenocortical carcinoma cell line, SW-13. In this study, the production of several adrenal steroids and 3', 5'-cyclic adenosine monophosphate (cAMP) were investigated in this cell line. The cells were incubated in L-15 medium containing 0.1% bovine serum albumin with several reagents in an atmosphere of 5% CO2 and 95% air for 2 hours at 37 degrees C. Aldosterone (Ald), corticosterone (B), cortisol (F), dehydroepiandrosterone sulfate (DHEA-S) and cAMP were simultaneously assayed by specific radioimmunoassays in the medium and cells. Significant increases in cAMP production were observed by cholera toxin (10 ng/ml) and forskolin (10 nM), both direct stimulators of adenylate cyclase, in the cAMP concentration without an increase in the steroids. The DHEA-S concentration in the medium was significantly increased by angiotensin-II (10(-7)M), noradrenalin (3 X 10(-5) M), adrenalin (3 X 10(-5) M) or alpha-melanocyte-stimulating hormone (alpha-MSH, 10(-7) M), none of which was associated with cAMP production. Neither adrenocorticotropin (10(-10) M) nor human chorionic gonadotropin (500 mIU/ml) stimulated the release of the steroids or cAMP production. A calcium ionophore, A23187 (10(-7) M), and 12-O-tetradecanoylphorbol-13-acetate (10(-8) M), a direct stimulator of protein kinase C, stimulated the release of DHEA-S, but not those of Ald, B and F. The results suggest that SW-13 retains functioning adenylate cyclase which, however, is not linked with steroidogenesis and that DHEA-S is produced probably by the mechanisms which involve protein kinase C system or calcium ion. This report provides the first demonstration of cAMP and DHEA-S production in SW-13 and suggests that this cell line is potentially useful for investigating the mechanisms of steroidogenesis in the human adrenal cortex.

Topics: Adrenal Cortex Neoplasms; Adrenocorticotropic Hormone; alpha-MSH; Angiotensin II; Calcimycin; Carcinoma, Small Cell; Cell Line; Cholera Toxin; Chorionic Gonadotropin; Colforsin; Cyclic AMP; Dehydroepiandrosterone; Dehydroepiandrosterone Sulfate; Epinephrine; Humans; Norepinephrine; Tumor Cells, Cultured

Topics: Adrenal Cortex Neoplasms; Adrenocorticotropic Hormone; Bucladesine; Calcimycin; Calcium; Cell Line; Corticosterone; Humans; Time Factors