Androgen influences transforming growth factor-beta1 gene expression in human adrenocortical cells

Sex steroid hormones have been shown to affect adrenocortical function and trophism, yet little is known about androgen action in human adrenocortical gland. In this study we examined the effects of androgens on transforming growth factor-beta1 (TGF/beta1) production by the human adrenocortical cell line, NCI-H295, which we recently demonstrated to express androgen receptor and whose growth is significantly reduced by dihydrotestosterone (DHT) treatment. TGFbeta1 is an important regulator of human adrenal development, with marked effects on steroid-producing cell function, and the production of distinct TGFbeta subtypes has been suggested to be regulated by steroid hormones in several tissues. To address potential TGFbeta1 induction by DHT, quantitative PCR and enzyme-linked immunoadsorbent assay were performed in NCI-H295 cells treated with DHT (from 10(-12)-10(-9) mol/L). DHT led to a significant dose-dependent increase in TGFbeta1 messenger ribonucleic acid expression and in biologically active TGFbeta1 protein levels in the conditioned media of NCI-H295 cells, demonstrating that androgen can induce TGFbeta1 expression and production. TGFbeta1 (10(-7)-10(-6) mol/L) was capable of significantly reducing cell proliferation (P < 0.05) after 24 h of treatment, as assessed by measuring [3H]thymidine incorporation in NCI-H295 cells. The addition of TGFbeta1-neutralizing antibody to cell cultures treated with different DHT concentrations (10(-9) and 10(-10) mol/L) blocked the inhibitory effect of TGF/beta1 on adrenocortical cell proliferation. These findings suggest that TGFbeta1 exerts an inhibitory action on adrenocortical cell proliferation. Therefore, it might be reasonable to suppose that DHT could also influence human adrenocortical cell growth by involving TGFbeta1.     

Tumour-derived human adrenocortical cells express beta-adrenergic receptors: steroidogenic effects of beta-adrenergic input

It is well established that catecholamines have potent actions on adrenocortical function and steroidogenesis in different species. The effect of these substances on steroid production of the human adrenal cell line H295R is the subject of this study. H295R cells were cultured in the presence of the synthetic catecholamine, isoproterenol for four hours. Aldosterone, cortisol, and DHEA secretion was measured using direct radioimmunoassays. Administration of 10(-11)-10(-7) mol/L isoproterenol produced a dose-dependent increase in secretion of aldosterone, cortisol, and DHEA by H295R cells resulting in 3-fold, 2.5-fold, and 2-fold stimulation respectively, relative to basal values. Analysis of mRNA using nested PCR revealed the presence of all three types of beta-adrenergic receptors namely beta1, beta2, and beta3 in H295R cells. Isoproterenol had no effect on the proliferation rate of H295R cells as determined by 3H-incorporation assay and the colorimetric WST-1 cell proliferation assay.     

The effects of ACTH on steroid metabolomic profiles in human adrenal cells

The adrenal glands are the primary source of mineralocorticoids, glucocorticoids, and the so-called adrenal androgens. Under physiological conditions, cortisol and adrenal androgen synthesis are controlled primarily by ACTH. Although it is well established that ACTH can stimulate steroidogenesis in the human adrenal gland, the effect of ACTH on overall production of different classes of steroid hormones has not been defined. In this study, we examined the effect of ACTH on the production of 23 steroid hormones in adult adrenal primary cultures and 20 steroids in the adrenal cell line, H295R. Liquid chromatography/tandem mass spectrometry analysis revealed that, in primary adrenal cell cultures, cortisol and corticosterone were the two most abundant steroid hormones produced with or without ACTH treatment (48 h). Cortisol production responded the most to ACTH treatment, with a 64-fold increase. Interestingly, the production of two androgens, androstenedione and 11β-hydroxyandrostenedione (11OHA), that were also produced in large amounts under basal conditions significantly increased after ACTH incubation. In H295R cells, 11-deoxycortisol and androstenedione were the major products under basal conditions. Treatment with forskolin increased the percentage of 11β-hydroxylated products, including cortisol and 11OHA. This study illustrates that adrenal cells respond to ACTH through the secretion of a variety of steroid hormones, thus supporting the role of adrenal cells as a source of both corticosteroids and androgens.     

Effects of steroid hormones on spermatogenesis and GnRH release in male Leopard frogs, Rana pipiens

Several lines of evidence suggest reproduction in the ranid frogs is potently regulated by the gonadal steroids, in particular 5alpha-dihydrotestosterone (DHT) and 17beta-estradiol (E(2)), and a non-gonadal steroid, the stress hormone corticosterone (Cort). Little is known about how these steroid hormones act upon the GnRH system to regulate the downstream reproductive events. We address these gaps in our knowledge by investigating the effects of Cort, E(2), and DHT administration on the in vitro release of GnRH and on the spermatogenesis of adult male leopard frog, Rana pipiens. R. pipiens were implanted for 20 days with silastic capsules containing cholesterol (Ch; control), Cort, E(2), or DHT. Upon sacrifice, acute hypothalamic explants were cultured and measured for GnRH release, and testes processed for histological analysis. Although only E(2) implant significantly reduced the gonadosomatic index, all three steroid hormones altered spermatogenesis. Cort modestly but significantly reduced the presence of spermatids. The effects of E(2) and DHT were both stimulatory and inhibitory, depending on the stage of spermatogenesis. None of the steroid hormones altered baseline GnRH release. Interestingly, only E(2) significantly stimulated veratridine-induced GnRH release, suggesting E(2) treatment increased the releasable pool of GnRH and/or enhance the excitability of GnRH neurons. In sum, this is the first study to report the direct measurement of GnRH secretion in a poikilothermic tetrapod. Our results revealed potent but sometimes paradoxical effects of steroid hormones, especially E(2), on the reproductive regulation of the male R. pipiens.     

Effect of leptin on ACTH-stimulated secretion of cortisol in rhesus macaques and on human adrenal carcinoma cells

OBJECTIVE: Because glucocorticoids stimulate leptin release and, at least in vitro, leptin inhibits cortisol secretion, a feedback system between glucocorticoids and leptin has been proposed. However, in humans and non-human primates there are no in vivo studies to support any role for leptin in the control of the hypothalamic-pituitary-adrenal axis. In this study, we investigated the effect of leptin on (i) ACTH-stimulated secretion of cortisol in six male rhesus monkeys and (ii) basal and forskolin (FSK)-stimulated cortisol secretion by the human adrenal carcinoma cell H295R in vitro. DESIGN AND METHODS: In vivo studies: after suppression of endogenous ACTH with either dexamethasone (n=6) or a corticotropin-releasing factor (CRF) antagonist (d-Phe CRF(12-41)) (n=3), 1 microg bolus of human ACTH(1-24) was administered to stimulate adrenal cortisol release. Blood samples were collected every 15 min for 3 h. Leptin (1 mg) was infused over 4 h, starting 1 h before ACTH bolus. In vitro studies: NCI-H295R cells were incubated for 6, 12, 24 and 48 h in the absence or presence of 20 micromol/l FSK in combination with leptin (100 ng/ml medium). Cortisol levels in serum and medium were measured by solid phase radioimmunoassay. RESULTS: Acute leptin infusion to rhesus monkeys did not change basal cortisol levels, peak cortisol levels after ACTH(1-24) or the area under the curve when compared with studies in which leptin was not given. FSK increased cortisol levels in medium at 24 and 48 h, but leptin did not change cortisol release in either control or FSK-stimulated cells. CONCLUSIONS: Short-term leptin infusion affected neither the cortisol response to ACTH in non-human primates in vivo nor cortisol release (basal or FSK stimulated) by H295R cells, in vitro. These data suggest that leptin may not be an acute regulator of primate adrenal cortisol secretion.     

Characterization of adrenal autonomy in Cushing's syndrome: a comparison between in vivo and in vitro responsiveness of the adrenal gland

We measured cortisol and precursor steroid production in response to ACTH, cholera toxin, and forskolin by the dispersed adrenocortical cells prepared from the adrenal glands of 10 patients with different forms of Cushing's syndrome. The cells prepared from the hyperplastic adrenal glands from 4 patients with Cushing's disease responded in a dose-dependent manner to ACTH, cholera toxin, and forskolin. The adrenal cells prepared from 4 encapsulated adrenal adenomas showed no (n = 2), a lowered (n = 1), or a clear (n = 1) response of cortisol release to ACTH. The cells prepared from the adrenal glands of 1 patient with dysplastic micronodular adrenal glands showed a limited response to ACTH, while the cells from an adrenocortical carcinoma, which secreted very little cortisol per cell, were unresponsive to ACTH, cholera toxin, and forskolin. The reaction of the dispersed adrenal cells from these 10 patients to ACTH, cholera toxin, and forskolin showed a close correlation (P less than 0.001 in all instances). This suggests that the defect in autonomous glands is not located at the level of the ACTH receptor, but, rather, involves the adenylate cyclase complex as a whole or its coupling to cAMP-dependent protein kinase. The release into the medium of the cortisol precursors deoxycortisol, 17-hydroxyprogesterone, and progesterone showed that the four autonomous nodules were characterized by a significantly higher deoxycortisol/cortisol ratio in the medium (P less than 0.01), suggesting a relative blockade of 11 beta-hydroxylase in these adrenal adenomas. This was further substantiated in cells from several adrenals by a significant increase in the release of these precursors in response to ACTH in the absence of a cortisol response. We conclude the following. 1) Adrenal adenoma formation in patients with Cushing's syndrome is accompanied by a parallel decrease in the stimulation of the release of steroid hormones in response to ACTH, cholera toxin, and forskolin. This points to a defect in the adenoma cells beyond the ACTH receptor. 2) Adrenal adenoma formation in patients with Cushing's syndrome is accompanied by a relative blockade of 11 beta-hydroxylase activity. 3) By comparing the preoperative dynamic tests of the pituitary-adrenal axis, the plasma ACTH concentration, the morphology of the adrenal glands, and their in vitro responsiveness, a gradual transition from pituitary to (partial) adrenal autonomy could be recognized in several patients.     

Antiestrogens upregulate estrogen receptor beta expression and inhibit adrenocortical H295R cell proliferation

The molecular mechanisms involved in adrenocortical tumorigenesis are still not completely understood. In this study, using the H295R cell line as a model system, we investigated the role of estrogens and estrogen receptor (ER) alpha and ER beta in the growth regulation of adrenocortical tumors. We demonstrated that H295R cells are able to convert androgens to estrogens by a constitutive expression of active cytochrome P450 aromatase protein and express ER beta to a greater extent than ER alpha. Moreover, physiological concentrations of 17beta-estradiol (E2) determined an increase of thymidine incorporation, suggesting the presence of an autocrine mechanism in maintaining H295R cell proliferation. Evaluating the response to ER antagonists like 4-hydroxytamoxifen (OHT) and ICI 182 780 (ICI), we observed an up-regulation of ER beta and a dose-dependent inhibition of H295R cell proliferation. Whereas ICI determined the growth arrest of H295R cells, OHT induced morphological changes that were characteristic of apoptosis. According to the above-mentioned observations, OHT but not ICI clearly induced a marked expression of FasL and the cleavage of both caspase-8 and caspase-3. Interestingly, the apoptotic effects of OHT in H295R cells may be consequent to the enhanced levels of ER beta which stimulate the expression of FasL interacting with activating protein (AP)-1 sites located within its promoter sequence. In conclusion, we have demonstrated that H295R cells are able to transform androgens to estrogens that activate an autocrine mechanism, mediated by their own receptors, and contribute to regulate the proliferation of these cells. Moreover, this study points towards a role for ER beta as an important mediator of the repressive effects exerted by antiestrogens on H295R cells; however, further studies are needed to clarify its role in the control of adrenocortical cell proliferation and on the potential benefits of antiestrogens for treatment of adrenocortical cancer.     

Effect of long-term androgen treatment on the function of hypothalamo-hypophysial and -adrenal axes in transsexual agonadal women

The study was aimed at assessing the influence of prolonged (18-24 months) androgen treatment of 11 agonadal transsexual women on basal concentrations of LH, FSH, testosterone (T), estradiol (E2), progesterone (P) and SHBG, on the hypothalamic-hypophysial (GH, PRL, ACTH) and hypothalamic-adrenal (cortisol) response in insulin test and TSH and PRL response after TRH administration. Fifteen healthy women in follicular phase of menstrual cycle and 15 men served as controls. In TS women basal concentrations of E2 were comparable with those in healthy women in follicular phase and the lowered T value showed negative correlation with SHBG. In most TS women the stimulated secretion of GH, PRL, ACTH and cortisol in insulin test was diminished. Basal values of these hormones oscillated within normal range except ACTH levels which were higher as compared with control values. In most cases the PRL response to TRH was diminished, but in three patients excessive secretion of PRL was found. Long-term priming with androgen was found to produce a dramatic change in the patterns of hormonal response to post-insulin hypoglycaemia and TRH in female-to-male transsexuals. It was concluded that in prolonged treatment of agonadal transsexual women the doses of testosterone preparations should be adjusted to individual patients in order to monitor steroid and gonadotropin hormone values, as well as the response of pituitary hormones, particularly that of PRL, to stimuli.     

Direct effects of sex steroids on prolactin release at the anterior pituitary level: interactions with dopamine, thyrotropin-releasing hormone, and isobutylmethylxanthine

When present alone for 4 or 8 days, 5 alpha-dihydrotestosterone (DHT) or the pure progestin R5020 (17,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione) inhibits spontaneous PRL release by 33--50% in rat anterior pituitary cells in primary culture. This inhibitory effect of DHT and R5020 can only be partially reversed by 17 beta-estradiol (E alpha). DHT and R5020 inhibit spontaneous PRL release in E2-primed cells at ED50 values of 0.5 and 3 nM, respectively. While E2 diminishes by 30--60% the maximal inhibitory effect of dopamine on PRL release and increases by 10-fold the ED50 value of dopamine action, DHT and R5020 can prevent by 30--60% the action of E2 and thus increase the potency of dopamine to inhibit PRL release. The inhibitory action of DHT and R5020 as well as the stimulatory action of E2 on spontaneous PRL release are similarly expressed on TRH- and 3-isobutyl-1-methylxanthine-induced PRL release, thus suggesting that at least part of the highly effective modulatory effects of sex steroids are exerted at a step after cAMP formation.     

Hormonal regulation of H19 gene expression in prostate epithelial cells

The H19 gene is transcribed in an mRNA-like noncoding RNA. When tumors of various organs or cell types are considered, H19 oncogene or tumor-suppressor status remains controversial. To address the potential regulation of H19 gene expression by an androgen steroid hormone (DHT: dihydrotestosterone) or by a peptidic hormone (PRL: prolactin), we performed experiments in rats systemically treated with chemical mediators. This range of in vivo experiments demonstrated that chronic hyperprolactinemia upregulated the H19 expression in epithelial and stromal cells whereas DHT downregulated the gene. PRL and DHT appeared to be opposite mediators in the H19 RNA synthesis. We investigated these hormonal effects in three human prostate epithelial cell lines. In LNCaP cancer cells, the opposite effect of PRL and DHT was corroborated. However, in normal cells (PNT1A), H19 remained insensitive to the hormones in fetal calf serum (FCS) medium but became responsive in a serum-stripped medium. In the DU-145 cancer cell line, tested for its androgen-independence and aggressiveness, the hormones had no effect on H19 expression whatever the culture conditions. Finally, we demonstrated that PRL upregulated the H19 expression in LNCaP cells by the JAK2-STAT5 transduction pathway. We conclude that H19 expression is regulated by both a peptidic and a male steroid hormone.     

cAMP-dependent protein kinase activation inhibits proliferation and enhances apoptotic effect of tumor necrosis factor-alpha in NCI-H295R adrenocortical cells

Adrenocorticotropin is the major regulator of adrenocortical development and function. It acts mainly through the cAMP-dependent protein kinase A (PKA) pathway. Our aim was to study the interaction of tumor necrosis factor-alpha (TNFalpha) and the PKA pathway in adrenocortical cell proliferation and apoptosis. The PKA activator Dibutyryl cAMP ((Bu)2cAMP) strongly induced differentiation and inhibited proliferation in the human adrenocortical cell line NCI-H295R (H295R). TNFalpha induced apoptosis of H295R cells. Interestingly, (Bu)2cAMP treatment clearly enhanced TNFalpha-induced apoptosis in H295R cells, but not in another human adrenocortical cell line SW-13, the mouse adrenocortical Y-1 cell line or the human HeLa cell line. This synergistic effect was not due to the (Bu)2cAMP-induced glucocorticoid secretion since dexamethasone had no significant effect on the TNFalpha-induced apoptosis. (Bu)2cAMP treatment rapidly increased the expression of the proto-oncogene c-myc in H295R cells, but not in SW-13, Y-1 or HeLa cells. In transient c-myc transfection assay, c-myc expression associated with decreased expression of the proliferation marker Ki-67 in H295R cells. In conclusion, cAMP-dependent protein kinase activation reduced proliferation and augmented TNFalpha-induced apoptosis in adrenocortical H295R cells, and these effects were associated with increased c-myc expression.     

Human proopiomelanocortin-(79-96), a proposed cortical androgen-stimulating hormone, does not affect steroidogenesis in cultured human adult adrenal cells.

The existence of a cortical androgen-stimulating hormone (CASH), distinct from ACTH, regulating the secretion of human adrenal androgens has long been postulated. Recently, it has been reported that an 18-amino acid peptide, corresponding to the first part of the joining peptide of proopiomelanocortin [POMC-(79-96)], was able to stimulate the secretion of dehydroepiandrosterone from cultured human adult adrenocortical cells, but had no effect on cortisol production. We have studied the acute and long term effects of ACTH (10(-11) and 10(-9) M), CASH-18 (10(-8) M), or both on cortisol and dehydroepiandrosterone sulfate by human adult adrenocortical cells. Although ACTH increased steroid secretion and enhanced the steroidogenic responsiveness to further ACTH stimulation, CASH-18 alone or together with ACTH (10(-11) or 10(-9) M) had no effect. In addition, we were unable to demonstrate any specific binding of [125I]CASH-18 to human adrenocortical cells, although [125I] ACTH-(1-39) binds specifically to the same cell preparation.     

Acute administration of melatonin at two opposite circadian stages does not change responses to gonadotropin releasing hormone, thyrotropin releasing hormone and ACTH in healthy adult males

We evaluated the effect of a single oral administration of 100 mg melatonin (MT) vs placebo (PL) on the pituitary release of LH, FSH, TSH and prolactin (PRL) after GnRH + TRH and on the adrenocortical release of cortisol, aldosterone and progesterone after ACTH in healthy adult males. We carried out a double blind study in 6 volunteers in winter, at two opposite stages of the circadian cycle: 08:00 and 20:00 h. Injection of GnRH (100 micrograms), TRH (200 micrograms) and ACTH (10 micrograms of the synthetic ACTH 1-17 analogue, Alsactide) was performed one h after MT or PL ingestion. Plasma MT levels were 200-4,000-fold higher after MT than PL thus confirming the effective gastrointestinal absorption of the pineal hormone. The hormonal patterns were superimposable after MT and PL. A higher response of PRL, FSH and cortisol was observed in the evening vs morning protocols independently of previous MT or PL. Our data demonstrate that the acute oral administration of a pharmacological dose of MT at two opposite circadian stages is ineffective to change a variety of pituitary and adrenocortical responses in human male subjects. The circadian chronosusceptibility of pituitary and adrenocortical cells to specific stimuli deserves interest to future investigation.     

The effects of suramin on human adrenocortical cells in vitro: suramin inhibits cortisol secretion and adrenocortical cell growth.

Suramin, a polycyclic and polyanionic drug, has been successfully used in the therapy of inoperable adrenocortical cancer. The present study was undertaken to investigate the effects of suramin on normal human adrenocortical cells in primary monolayer cultures. The proliferation and the basal, as well as the adrenocorticotropin (ACTH)-stimulated, cortisol secretion of these cells were studied. The data show that suramin decreases basal, as well as ACTH-stimulated, cortisol secretion in a dose-dependent manner (P less than .05 from 300 mumol/L upward). At a suramin concentration of 3 mmol/L, cortisol secretion was inhibited by 70% +/- 4% in ACTH-stimulated cells and by 42% +/- 6% in unstimulated cells. The proliferation of adrenocortical cells in response to fetal calf serum was also inhibited by suramin at concentrations from 300 mumol/L upward, maximal suppression (71% +/- 6%, P less than .01) being observed at a concentration of 10 mmol/L. Both inhibition of cortisol secretion and inhibition of adrenocortical cell proliferation were not due to toxicity of the compound, as could be shown by restimulation of cortisol secretion in suramin-treated cells with ACTH. Our results indicate that suramin exerts an inhibitory influence on the cortisol secretion and on the proliferation of normal human adrenocortical cells. Suramin may not only be useful in the treatment of adrenocortical cancer, but may also have an ameliorative effect on other malignant conditions with augmented steroid hormone production, resistant to conventional forms of therapy.     

Evidence for a role of testosterone-androgen receptor interactions in mediating masculine sexual behavior in male rats

The purpose of this study was 2-fold: 1) to use gonadal steroid hormone exposures in the physiological range to assess the relative roles of testosterone (T), estradiol (E2), and dihydrotestosterone (DHT) in the expression of male sexual behavior, and 2) to determine whether androgen receptor (AR) or estrogen receptor (E2R) occupation is increased after exposure to these various gonadal steroid hormones. Sexually experienced, castrated male rats implanted sc with Silastic capsules containing T, 10% E2, DHT, 10% E2 plus DHT, or blanks provided hormone levels in the physiological range. Copulatory behavior was measured on days 2-4, 5-7, 10-12, and 14-16 of steroid treatment. Although T, E2, and E2 plus DHT treatments all activated mounting, only T was effective in restoring ejaculation in 100% of the males. DHT alone had no effect on any aspect of male sexual behavior. Brains of males given these various hormone treatments were assayed for both cell nuclear AR and cell nuclear E2R binding in the hypothalamus, preoptic area, amygdala, and septum. Results indicate that when hormone levels in the physiological range were employed, T and DHT bind primarily to AR, whereas E2 binds to E2R. In a second experiment, 0.5% E2 plus DHT was found to yield AR and E2R levels comparable to those in rats receiving T capsules. Male rats bearing these capsules showed virtually no sexual behavior, demonstrating that elevation of AR and E2R levels comparable to those generated by T is not sufficient to induce male sexual behavior. We then measured intact AR and E2R levels and determined that in intact males E2R levels were higher than in T-treated males. These E2R levels could be replicated using 1.0% E2. Males exposed to 1.0% E2 plus DHT failed to display male sexual behavior. These data suggest that 1) relatively high and prolonged levels of E2R occupation are required for estrogen activation of male sexual behavior, 2) high levels of AR occupation induced by DHT are not sufficient to activate male sexual behavior, and 3) in intact male rats T, acting via androgen receptors, plays a primary role in mediating the expression of masculine sexual behavior.     

Human adrenal corticocarcinoma NCI-H295R cells produce more androgens than NCI-H295A cells and differ in 3beta-hydroxysteroid dehydrogenase type 2 and 17,20 lyase activities

The human adrenal cortex produces mineralocorticoids, glucocorticoids, and androgens in a species-specific, hormonally regulated, zone-specific, and developmentally characteristic fashion. Most molecular studies of adrenal steroidogenesis use human adrenocortical NCI-H295A and NCI-H295R cells as a model because appropriate animal models do not exist. NCI-H295A and NCI-H295R cells originate from the same adrenocortical carcinoma which produced predominantly androgens but also smaller amounts of mineralocorticoids and glucocorticoids. Research data obtained from either NCI-H295A or NCI-H295R cells are generally compared, although for the same experiments no direct comparison between the two cell lines has been performed. Therefore, we compared the steroid profile and the expression pattern of important genes involved in steroidogenesis in both cell lines. We found that steroidogenesis differs profoundly. NCI-H295A cells produce more mineralocorticoids, whereas NCI-H295R cells produce more androgens. Expression of the 3beta-hydroxysteroid dehydrogenase (HSD3B2), cytochrome b5, and sulfonyltransferase genes is higher in NCI-H295A cells, whereas expression of the cytochrome P450c17 (CYP17), 21-hydroxylase (CYP21), and P450 oxidoreductase genes does not differ between the cell lines. We found lower 3beta-hydroxysteroid dehydrogenase type 2 but higher 17,20-lyase activity in NCI-H295R cells explaining the 'androgenic' steroid profile for these cells and resembling the zona reticularis of the human adrenal cortex. Both cell lines were found to express the ACTH receptor at low levels consistent with low stimulation by ACTH. By contrast, both cell lines were readily stimulated by 8Br-cAMP. The angiotensin type 1 receptor was highly expressed in NCI-H295R than NCI-H295A cells and angiotensin II stimulated steroidogenesis in NCI-H295R but not NCI-H295A cells. Our data suggest that comparative studies between NCI-H295A and NCI-H295R cells may help find important regulators of mineralocorticoid or androgen biosynthesis.