Inhibin/activin betaB-subunit expression in pheochromocytomas favors benign diagnosis.

Malignancy of pheochromocytomas is difficult to estimate on the basis of histopathological features. Good prognostic markers are not available. In our search for new markers to differentiate malignant pheochromocytomas from benign ones we tested the value of inhibin/activin subunit expression. Inhibins are heterodimeric glycoproteins consisting of an alpha-subunit and either a betaA- or a betaB-subunit. Activins are composed of beta-subunits only. Immunohistochemically inhibin/activin betaB-subunit was strongly positive in the normal adrenal medulla, but the cortex was negative. A striking difference was found in inhibin/activin betaB expression between benign and malignant pheochromocytomas. The majority of benign adrenal tumors (27 of 30) showed strong or moderate immunoreactivity, whereas all seven malignant tumors were negative or only weakly positive for inhibin/activin betaB-subunit. The percentage of positively staining cells varied greatly in extraadrenal pheochromocytomas and in those benign tumors that showed over 5 mitoses/10 high power fields, necrosis, or capsular or vascular invasion, here called borderline tumors. Inhibin/activin betaB messenger ribonucleic acid was also found in pheochromocytomas. However, no significant differences in messenger ribonucleic acid levels were found in various types of tumors. Weak immunohistochemical positivity for inhibin/activin betaA-subunit was detected in the adrenal cortex, but the medulla and most of the pheochromocytomas were negative. Our data show that inhibin/activin betaB-subunit is expressed in normal adrenal medullary cells. Strong staining is found in most benign adrenal pheochromocytomas, whereas malignant tumors are almost negative. This suggests that loss of inhibin/activin betaB-subunit expression in pheochromocytomas may be used as an indicator of malignant potential.

Expression of low and high density lipoprotein receptor genes in human adrenals.

Corticosteroids are synthesized from cholesterol which may arise from de novo synthesis or from the uptake of low or high density lipoproteins (LDL or HDL). In the present study, we compared the expression and regulation patterns of LDL receptor and CLA-1 (CD36 and LIMPII Analogous-1, an HDL receptor) genes in adult human adrenocortical tissues to shed more light on the relative contribution of LDL and HDL in human adrenal steroidogenesis. By screening 64 normal and pathological adrenal samples by Northern blotting, we found a positive correlation between LDL receptor and CLA-1 mRNA expression in the adrenal tissues (r=0.547; spearman rank correlation test P<0.01). Adrenal tissues adjacent to Cushing's adenomas contained consistently less LDL receptor and CLA-1 mRNA than normal adrenals (Mann-Whitney P<0.05). In primary cultures of normal adrenal cells, accumulation of both LDL receptor and CLA-1 mRNAs was upregulated by ACTH in a dose- and time-dependent manner, with an earlier induction of LDL receptor than CLA-1 mRNA expression. (Bu)(2)cAMP also increased the levels of these two mRNAs. Addition of LDL, but not HDL, into the culture medium increased cortisol production in untreated adrenocortical cells. Both LDL and HDL enhanced ACTH-induced cortisol production, with the effect of LDL much stronger than that of HDL. Our data show that LDL receptor and CLA-1's expression is ACTH-dependent and occurs in parallel in human adrenal tissues. LDL rather than HDL may be used as the preferential source of cholesterol for steroidogenesis in human adult adrenocortical cells.

Expression of inhibin alpha in adrenocortical tumours reflects the hormonal status of the neoplasm.

Inhibins are gonadal glycoprotein hormones whose main endocrine function is to inhibit pituitary FSH secretion. In addition to testes and ovaries, other steroid-producing organs are sites of inhibin alpha subunit expression. To study the role of inhibins in human adrenal gland, we screened a panel of 150 adrenals (10 normal adrenals, 25 adrenocortical hyperplasias, 65 adrenocortical adenomas, 30 adrenocortical carcinomas and 20 phaeochromocytomas) for inhibin alpha expression. mRNA levels of inhibin alpha subunit were studied in 57 samples and all tissues were stained immunohistochemically with an inhibin alpha subunit-specific antibody. Inhibin alpha mRNA was detected in all adrenocortical tissues. Virilizing adenomas possessed a 10-fold higher median inhibin alpha mRNA expression than did normal adrenals. Bilaterally and nodularly hyperplastic adrenals and other than virilizing adrenocortical tumours had their median inhibin alpha mRNA levels close to those of normal adrenals. Immunohistochemically, inhibin alpha subunit was detectable in all normal and hyperplastic adrenals, as well as in 73% of the adrenocortical tumours. However, the percentage of inhibin alpha-positive cells varied greatly in different tumour types. The median percentage of positive cells was 10 in non-functional and Conn's adenomas, 30 in Cushing's adenomas and 75 in virilizing adenomas. In malignant adrenocortical tumours the median percentage of inhibin alpha-immunopositive cells was 20 in non-functional carcinomas, 30 in Conn's carcinomas, 65 in Cushing's carcinomas and 75 in virilizing carcinomas. All phaeochromocytomas were negative for inhibin alpha subunit both at the mRNA level and immunohistochemically. Our data show that inhibin alpha subunit is highly expressed in both normal and neoplastic androgen-producing adrenocortical cells, with less expression in cortisol-producing and hardly any in aldosterone-producing cells. This suggests a specific role for inhibins in the regulation of adrenal androgen production. We did not find any significant difference in inhibin alpha expression between benign and malignant adrenocortical tumours. Thus inhibin alpha gene does not seem to have a tumour suppressor role in human adrenal cortex.

Expression of activin A and its receptors in human pheochromocytomas.

Activin A (a homodimer of two activin betaA subunits) has been shown to induce the neuronal differentiation of rat pheochromocytoma PC12 cells. We studied activin A and its receptor gene expression in human pheochromocytomas in vivo and in vitro to clarify the potential involvement of activin A in the pathophysiology of these tumors. We first screened 20 pheochromocytomas and nine normal adrenal tissues for activin betaA mRNA expression. Northern blots hybridized with specific oligonucleotide probes detected weak signals for activin betaA transcripts in pheochromocytomas. Both type I and type II activin receptor (ActR-I, ActR-IB and ActR-II) mRNA expression was also detectable in the pheochromocytoma tissues. In primary cultures of pheochromocytoma cells, expression of activin betaA mRNA was readily detectable by Northern blotting, and secretion of activin A into the conditioned medium was confirmed by an enzyme-linked immunosorbent assay. The expression of activin betaA mRNA and secretion of activin A were induced by (Bu)(2)cAMP after 1 and 3 days of treatment (all P<0.05). A protein kinase inhibitor, staurosporine, inhibited the basal and (Bu)(2)cAMP-induced accumulation of activin betaA mRNA (P<0.05). In addition, induction of chromaffin phenotype by dexamethasone also inhibited the basal and (Bu)(2)cAMP-induced expression of activin A at both mRNA and protein levels (all P<0.05). In contrast, the expression of ActR-I and ActR-IB mRNAs was not affected by these agents in cultured pheochromocytoma cells. In summary, activin betaA subunit and activin receptors are expressed in human pheochromocytomas. Production of activin A in cultured pheochromocytoma cells is induced through the protein kinase A pathway, but reduced during chromaffin differentiation. Therefore, activin A may function as a local neurotrophic factor via an auto/paracrine manner in human pheochromocytomas.

p53 and Ki67 in adrenocortical tumors.

The p53 tumor-supressor gene has been reported as the most frequent genetic abnormality seen in human malignancies. Here we studied immunohistochemically the expression of p53 in a large series of adrenocortical tumors. The proliferative activity was assessed by the expression of Ki67. Tumor material consisted of 60 adrenocortical adenomas and 27 adrenocortical carcinomas. A tumor was scored as positive for p53 if more than 10% of the cells showed nuclear staining. All adrenocortical adenomas were negative for p53 and the percentage of Ki67 positive cells was mostly 1-2% but never exceeded 5%. Hormonal activity did not reflect the proliferation index. Adrenocortical carcinomas, however, behaved differently depending on hormonal activity. 10/13 of non-functional , 0/3 Conn's, 3/7 Cushing's and 3/4 virilizing carcinomas were positive for p53. The proliferative activity was also higher in non-fuctional carcinomas compared with hormonally active tumors. Our data show that majority of adrenocortical carcinomas are positive for p53, whereas all adenomas are negative. Hormonal activity of carcinomas reflects both p53 status and proliferation index. Thus, immunohistochemical levels of p53 and Ki67 are higher in hormonally inactive adrenocortical carcinomas.

Expression of vascular endothelial growth factor in human adrenals.

Angiogenesis is an important component in many biological processes and also in pathologic conditions including neoplastic diseases. Vascular endothelial growth factor (VEGF) is a secreted endothelial cell-specific growth factor, which is induced by tissue hypoxia and is angiogenic in vivo. Adrenal gland is a well-vascularized organ, and the roles of VEGF in normal adrenal and in adrenal tumorigenesis is not well characterized. We therefore investigated VEGF mRNA expression in normal human adrenals and in cultured adrenocortical cells. VEGF mRNA was constantly expressed in normal adrenals as well as in cultured adrenocortical cells. The mRNA levels were increased after 24h stimulation with either ACTH or cAMP. The effect of cAMP was dose-dependent. This suggests that ACTH-induced VEGF mRNA expression is mediated via protein kinase A dependent pathway.

Regulation of neuropeptide Y mRNA expression in cultured human pheochromocytoma cells.

The expression of the neuropeptide Y (NPY) gene varies considerably in human pheochromocytomas, but the mechanisms for this variation have not been clarified. To investigate the regulation pattern of the NPY gene in human pheochromocytomas, we screened 16 pheochromocytomas and 9 normal adrenal tissues with Northern blots. The expression level of NPY mRNA in normal adrenal medulla was low and relatively constant, while the pheochromocytomas showed a very wide variation in NPY mRNA levels in both malignant and benign tumors. This indicates that NPY gene expression is not correlated with malignancy in pheochromocytomas. In primary cultures of human pheochromocytoma cells, nerve growth factor treatment (causing neuronal differentiation) increased NPY mRNA accumulation 2- to 5-fold (P < 0.05). NPY mRNA levels were also induced by protein kinase modulators (Bu)(2)cAMP and staurosporine in the cultures (P < 0.05). In contrast, treatment with dexamethasone and IGF-II (causing or linked with chromaffin differentiation) reduced NPY mRNA accumulation (P < 0.05). These data show that the regulation pattern of NPY mRNA expression in cultured human pheochromocytoma cells is different from that previously described in rat pheochromocytoma PC12 cells. Regulation of NPY mRNA expression in primary cultures by these differentiating factors suggests that the expression of NPY mRNA in pheochromocytoma tissues may be associated with the neuronal differentiation of the tumor cells affected by multiple factors.

pG2 gene expression and its regulation in human adrenocortical and medullary tumors.

The cDNA clone pG2 was originally isolated from a human pheochromocytoma. The respective gene was found to be strongly expressed in normal adrenal zona glomerulosa and medulla, as well as in Conn's adenomas and pheochromocytomas. To shed more light on the expression and regulation of the pG2 gene, we investigated its expression in a wide variety of different adrenal neoplasms and cultured adrenal cells. Northern blot analysis was used to determine the steady state level of pG2 mRNA. Besides normal adrenals, Conn's adenomas and pheochromocytomas, we found abundant expression of pG2 mRNA in Cushing's, virilizing and nonfunctional adrenocortical adenomas and carcinomas, as well as in hyperplastic adrenals. The relative levels of pG2 mRNA in various adrenocortical tumors were not significantly different from those in normal adrenals and pheochromocytomas. In primary cultures of normal adrenal cells, treatment with adrenocorticotropin induced a 3- to 15-fold increase in the expression of pG2 mRNA (P<0.01), and this effect was reproduced by incubation with (Bu)2cAMP. In cultured pheochromocytoma cells, treatment with (Bu)2cAMP and a protein kinase inhibitor, staurosporine, increased pG2 mRNA accumulation (2- to 4-fold over the control level, P<0.01, and 3- to 8-fold, P<0.01, respectively). These results indicate that pG2 is widely expressed in normal and pathological adrenal tissues from both cortical and medullary origin, which eliminates its usefulness as a specific marker for zona glomerulosa or medullary adrenal tumors. Accumulation of pG2 mRNA is regulated by multiple differentiating factors through different pathways in primary cultures of normal adrenal and pheochromocytoma cells.

Expression of insulin-like growth factor binding protein 1-6 genes in adrenocortical tumors and pheochromocytomas.

The insulin-like growth factor (IGF) system appears to be important in the regulation of adrenal growth and hormone synthesis. As IGF-binding proteins (IGFBPs) modify IGF bioactivity, we investigated the expression of IGFBP 1-6 genes in different adrenal tumors and hyperplasias to further clarify the role of the IGF system in adrenal pathophysiology. IGFBP-1 mRNA levels were too low to be detected by Northern blot analysis, but could be found by RT-PCR in some tumors and hyperplastic adrenals. Other IGFBPs were detected by Northern blotting. IGFBP-3 mRNA levels were very low in normal adrenals. In adrenal tumors and hyperplastic adrenals, IGFBP-3 mRNA expression was usually higher than in normal adrenals. In hormonally active adrenocortical carcinomas, IGFBP-2, -4, -5 and -6 mRNA levels were lower than in nonfunctional carcinomas and normal adrenals. The low IGFBP mRNA expression in the hormone-producing carcinomas was associated with high IGF-II mRNA content. In adrenocortical adenomas from patients with Cushing's or Conn's syndrome, mean IGFBP mRNA levels were higher than in normal adrenals or in hormonally inactive adenomas. In nodular and bilateral hyperplasias, IGFBP-2, -3 and -4 mRNA expression was on average higher than in normal adrenals but varied substantially, as did IGFBP mRNA levels in pheochromocytomas. In comparison to normal adrenals, pheochromocytomas expressed on average higher levels of IGFBP-2 and -4 but less IGFBP-5 and -6 mRNAs. Our data show that the six IGFBPs 1-6 are expressed at variable level in adrenal tumors and hyperplasias. The low level of IGFBP mRNAs in hormonally active adrenocortical carcinomas was of particular interest.

ACTH regulates LDL receptor and CLA-1 mRNA in the rat adrenal cortex.

Rat adrenocortical cells utilize both low density lipoprotein (LDL) and high density lipoprotein (HDL) cholesterol for steroid hormone production. In addition to exogenous lipoprotein-derived cholesterol, cells produce cholesterol de novo. Adrenocorticotropin (ACTH) increases both steroid hormone secretion and uptake of LDL and HDL. We studied the expression of LDL receptor mRNA and CLA-1 (a putative HDL receptor) mRNA in cultured rat adrenocortical cells. ACTH increased the amounts of LDL receptor mRNA during 2 to 48 h of stimulation, the highest levels being detected after 2-4 h. Similar results were obtained with cyclic AMP (cAMP) derivatives, 8-bromo cAMP (8-Br cAMP) or dibutyryl cAMP. ACTH increased CLA-1 mRNA during 2 to 24 h of stimulation, the highest levels being detected after 4 h. In conclusion, ACTH up regulates both LDL and HDL receptor mRNA in rat adrenocortical cells.

Ribonucleic acid expression of the CLA-1 gene, a human homolog to mouse high density lipoprotein receptor SR-BI, in human adrenal tumors and cultured adrenal cells.

Human CLA-1 is homologous to the mouse SR-BI gene, which was recently identified as a high density lipoprotein receptor involved in selective cholesterol uptake in rodent adrenal cells. We screened 42 normal and pathological adrenal samples by Northern blotting and found abundant expression of CLA-1 messenger ribonucleic acid (mRNA) in normal adult and fetal adrenals, adrenocortical adenomas, and hyperplasias. Adrenocortical carcinomas and the adrenals adjacent to Cushing's adenomas contained less CLA-1 mRNA than normal adrenals. CLA-1 mRNA was also highly expressed in a Leydig cell tumor, but much less in liver, kidney, and pheochromocytomas. The accumulation of CLA-1 mRNA in primary cultures of normal adrenocortical cells was up-regulated by ACTH in a dose- and time-dependent manner. Both dibutyryl cAMP and staurosporine increased the basal expression of CLA-1 mRNA. Although there was no additive effect of ACTH and dibutyryl cAMP, staurosporine slightly enhanced the stimulatory effect of ACTH on the expression of CLA-1 mRNA. The abundant expression of CLA-1 mRNA and its regulation by the physiological hormone ACTH in human adrenal cells suggest that CLA-1 has a role in adrenal steroidogenesis, probably as a lipoprotein receptor mediating the selective cholesterol uptake in these cells.

Ribonucleic acid expression of the clustered imprinted genes, p57KIP2, insulin-like growth factor II, and H19, in adrenal tumors and cultured adrenal cells.

The recently cloned cyclin-dependent kinase inhibitor gene p57KIP2 is genomically imprinted and located on human chromosome 11p15.5. This region contains two other imprinted genes, insulin-like growth factor II (IGF-II) and H19, both of which seem to be implicated in adrenal neoplasms. We analyzed the expression of the putative tumor suppressor p57KIP2 gene by Northern blotting in normal and hyperplastic adrenals, adrenocortical tumors, and pheochromocytomas. The expression of p57KIP2 messenger ribonucleic acid (mRNA) correlated positively with H19 and negatively with IGF-II RNA in adrenocortical tissues. p57KIP2 mRNA (and H19 RNA) was abundantly expressed in normal human adrenals, adrenocortical adenomas from patients with Cushing's or Conn's syndrome or without clinical evidence of hormone overproduction, hyperplastic adrenals, and tumor-adjacent adrenal tissues, in which IGF-II mRNA expression was low. In most adrenocortical carcinomas and virilizing adrenal adenomas, very low levels of both p57KIP2 and H19 RNAs were observed, whereas IGF-II was highly expressed. In pheochromocytomas, p57KIP2 and H19 RNA expression was highly variable, but on the average it was about 45% and 27%, respectively, of that in normal and tumor-adjacent adrenals. In cultured adrenocortical cells, ACTH and dibutyryl cAMP treatment slightly reduced the predominant 1.7-kilobase (kb) transcript of p57KIP2 gene, but induced a 2.5-kb transcript with a simultaneous increase in H19 RNA expression. The stimulatory effect of ACTH on the 2.5-kb p57KIP2 and H19 transcript accumulation was enhanced by exogenous IGF-II and IGF-I. Our data show that p57KIP2 and H19 RNAs are expressed usually in parallel in normal and pathological adrenocortical tissues. The decreased expression of both p57KIP2 and H19 RNAs in conjunction with elevated IGF-II mRNA expression in hormonally active adrenocortical carcinomas suggests that the loss of expression of the putative tumor suppressor genes p57KIP2 and H19 may be involved in the pathogenesis of these neoplasms.

Expression patterns of the c-myc gene in adrenocortical tumors and pheochromocytomas.

Abundant c-myc gene expression in neoplasms has been often linked to poor prognosis. As c-myc mRNA is expressed and hormonally regulated in human adrenals, we examined the c-myc gene expression in adrenal tumors by RNA analysis and immunohistochemistry to find out the possible role of c-myc in adrenal neoplasms. The abundant expression of the c-myc gene in normal adrenals was localized to the zona fasciculata and zona reticularis, with much lower expression in the zona glomerulosa and adrenal medulla. In hormonally active adrenocortical carcinomas (n = 6) and in virilizing adenomas (n = 4), c-myc mRNA levels were approximately 10% of those in normal adrenals (n = 11). In contrast, adrenal adenomas from patients with Cushing's (n = 4) and Conn's (n = 9) syndrome, non-functional adenomas (n = 2), adrenocortical hyperplasias (bilateral, n = 5; nodular, n = 4), and non-functional adrenocortical carcinomas (n = 3) expressed c-myc mRNA to the same extent as normal adrenals. The c-myc mRNA abundance in benign adrenal pheochromocytomas (n = 19) was similar to that in normal adrenal medulla. However, in malignant adrenal pheochromocytomas (n = 6), the average c-myc mRNA levels were approximately threefold that in benign adrenal pheochromocytomas. There was a good correlation between c-myc mRNA expression and immunohistochemical reactivity in both normal and pathological adrenal tissues. Southern blot analysis revealed no amplification or rearrangement of the c-myc gene in any of the adrenal tumors. In conclusion, c-myc expression localized to zona fasciculata and reticularis in normal adrenals. Virilizing adenomas and hormonally active adrenocortical carcinomas expressed c-myc mRNA clearly less than the other adrenal neoplasms and normal adrenal tissue. On the other hand, malignant pheochromocytomas contained more c-myc mRNA than benign ones. Further studies are required to clarify the mechanisms and significance for the distinct expression pattern of the c-myc gene in different adrenal neoplasms.

Adrenomedullin gene expression and its different regulation in human adrenocortical and medullary tumors.

Adrenomedullin (ADM) is a polypeptide originally discovered in a human pheochromocytoma and is also present in normal adrenal medulla. It has been proposed that ADM could be involved in the regulation of adrenal steroidogenesis via paracrine mechanisms. Our aim was to find out if ADM gene is expressed in adrenocortical tumors and how ADM gene expression is regulated in adrenal cells. ADM mRNA was detectable by Northern blotting in most normal and hyperplastic adrenals, adenomas and carcinomas. The average concentration of ADM mRNA in the hormonally active adrenocortical adenomas was about 80% and 7% of that in normal adrenal glands and separated adrenal medulla respectively. In adrenocortical carcinomas, the ADM mRNA concentration was very variable, but on average it was about six times greater than that in normal adrenal glands. In pheochromocytomas, ADM mRNA expression was about ten times greater than that in normal adrenals and three times greater than in separated adrenal medulla. In primary cultures of normal adrenal cells, a protein kinase C inhibitor, staurosporine, reduced ADM mRNA accumulation in a dose- and time-dependent fashion (P < 0.01), whereas it simultaneously increased the expression of human cholesterol side-chain cleavage enzyme (P450 scc) gene (a key gene in steroidogenesis). In cultured Cushing's adenoma cells, adrenocorticotropin, dibutyryl cAMP ((Bu)2cAMP) and staurosporine inhibited the accumulation of ADM mRNA by 40, 50 and 70% respectively (P < 0.05), whereas the protein kinase C activator, 12-O-tetradecanoyl phorbol 13-acetate (TPA), increased it by 50% (P < 0.05). In primary cultures of pheochromocytoma cells, treatment with (Bu)2cAMP for 1 and 3 days increased ADM mRNA accumulation two- to threefold (P < 0.05). Our results show that ADM mRNA is present not only in adrenal medulla and pheochromocytomas, but also in adrenocortical neoplasms. Both protein kinase A- and C-dependent mechanisms regulate ADM mRNA expression in adrenocortical and pheochromocytoma cells supporting the suggested role for ADM as an autocrine or paracrine (or both) regulator of adrenal function.

Expression of the steroidogenic acute regulatory protein mRNA in adrenal tumors and cultured adrenal cells.

The steroidogenic acute regulatory protein (StAR) has recently been shown to be a factor necessary for cholesterol transport into adrenal and gonadal mitochondria, which is the regulated, rate-limiting step in steroidogenesis. We show here that StAR mRNA is highly expressed in normal adult adrenals (n = 9), adrenocortical adenomas (n = 16), adrenal hyperplasias (n = 6), adrenocortical carcinomas (n = 6) and adrenals adjacent to tumor tissues (n = 9). There was a good correlation between the expression of StAR and the cholesterol side-chain cleavage enzyme/20,22-desmolase (P450 scc) mRNAs both in normal (r = 0.93; P < 0.01) and in tumor (r = 0.97; P < 0.001) tissues. No StAR mRNA was detected in Northern blots of liver, kidney, breast, parathyroid or phaeochromocytoma RNAs. In cultured adrenocortical cells, adrenocorticortropin (ACTH), (Bu)2cAMP, and cholera toxin increased StAR and P450 scc mRNA accumulation 6- to 18-fold, dose- and time-dependently. StAR (and P450 scc) mRNA increased relatively slowly in response to ACTH treatment, with the maximal increment at 24 h, while the mRNA of the early response gene c-fos peaked within 2 h. The protein kinase inhibitor H-7 inhibited basal and ACTH-induced StAR mRNA expression. Our results show that StAR mRNA is expressed at high levels in normal human adrenals and adrenocortical neoplasms. It is up-regulated in parallel with P450 scc by ACTH in adult adrenocortical cells, which suggests that ACTH is at least one of the key regulators of adrenal StAR expression.

Expression of the c-myc gene in human adrenals: regulation by adrenocorticotropin in primary cultures.

We have analyzed the expression of the c-myc proto-oncogene in human adrenal glands in vivo and in primary cell cultures by Northern blot analysis. c-myc mRNA was consistently expressed in all human adrenals studied. Expression in adult adrenals was found to be approximately 50% of that in fetal adrenals, but much higher than that in adult liver and kidney. Adrenocorticotropin (ACTH) treatment increased c-myc mRNA accumulation dose- and time-dependently up to more than 5-fold (on average), with the maximal effect at 2 h. (Bu)2cAMP and 12-O-tetradecanoyl phorbol 13-acetate (TPA) also induced c-myc gene expression. There was no synergistic effect between the ACTH, (Bu)2cAMP and TPA treatments. The basal level of c-myc expression was reduced by the protein kinase inhibitors H-7 (1-(5-isoquinolinesulfonyl)-2-methyl-piperazine dihydrochloride), staurosporine and HA1004 (N-(2-guanidinoethyl)-5-isoquinolinesulfonamide hydrochloride). H-7 totally abolished ACTH-, TPA- and (Bu)2cAMP-induced c-myc expression, while staurosporine inhibited the stimulatory effects of ACTH and TPA, and HA1004 weakly inhibited the effects of ACTH and (Bu)2cAMP. Incubation with cycloheximide or 10% fetal calf serum increased c-myc mRNA levels 3- and 4-fold respectively. Our data show that the c-myc gene is expressed abundantly in normal human adrenals, and that this expression can be regulated by multiple factors in the primary cultures.

ACTH-induced c-myc proto-oncogene expression precedes antimitogenic effect during differentiation of fetal rat adrenocortical cells.

The regulation of proto-oncogenes has been connected with proliferation and differentiation in various cell types. In the present study, the ACTH-induced expression of c-myc mRNA and proliferation of fetal rat adrenocortical cells have been studied. Low levels of c-myc mRNA were detected in undifferentiated zona glomerulosa-like cells. Stimulation with ACTH for 2 to 6 h transiently increased the c-myc mRNA levels. Both basal and ACTH-induced expression levels were increased by the protein synthesis inhibitor cycloheximide. Treatment with a protein kinase C (PKC) activator 12-0-tetradecanoyl phorbol-13-acetate mimicked the effect of ACTH, whereas c-myc mRNA levels decreased by inhibiting the PKC with H-7. ACTH inhibited proliferation of fetal rat adrenocortical cells during the first 24 h of stimulation. The inhibitory effect began from 6 h, reached its maximum at 12 h and slowly vanished at 24 h. Our data demonstrated that ACTH transiently increased c-myc mRNA expression. Adrenocortical c-myc expression was mediated via PKC. In contrast to previous reports, where c-myc expression precedes proliferation of various cells, ACTH-induced c-myc mRNA expression of cultured fetal rat adrenocortical cells was followed by inhibition of proliferation.

H19 and insulin-like growth factor-II gene expression in adrenal tumors and cultured adrenal cells.

The expression of H19 and insulin-like growth factor-II (IGF-II) genes is important for fetal growth, and the misexpression of these genes may also be involved in the development of some tumors. In human fetal adrenals, H19 and IGF-II expression levels are very high. We show here that H19 is strongly expressed (approximately 50% of the expression in fetal adrenals and 6-fold higher than that in adult liver) in normal adult adrenals (n = 9), adrenocortical adenomas (n = 28), and hyperplastic adrenals (n = 11). In four hormonally active adrenocortical carcinomas, very low levels of H19 ribonucleic acid (RNA) were detected, whereas IGF-II was highly expressed. In cultured adrenocortical cells, ACTH, (Bu)2cAMP, and cholera toxin increased H19 RNA accumulation 2- to 5-fold (P < 0.01), but had no significant effect on IGF-II messenger RNA levels. In pheochromocytomas (n = 22), H19 expression was variable, on the average, about 13% of the expression in the adjacent adrenal cortex. In primary cultures of pheochromocytoma cells, H19 RNA was not detectable via Northern blot analysis. Our data show that H19 expression is maintained at high levels in adult human adrenals and benign neoplasms. H19 RNA is up-regulated by ACTH in adult adrenocortical cells. The very low levels of H19 expression in hormonally active adrenocortical carcinomas suggest that loss of H19 expression may be associated with malignancy in these neoplasms.

Corticosterone regulates cell proliferation and cytochrome P450 cholesterol side-chain cleavage enzyme messenger ribonucleic acid expression in primary cultures of fetal rat adrenals.

Glucocorticoids are known to inhibit growth in many different cell types. Although corticosterone is secreted by the adrenal cortex, its direct effect on the growth of different zones is poorly determined. We studied the effects of corticosterone on cell proliferation and cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc; the rate-limiting step in adrenal steroidogenesis) messenger RNA (mRNA) accumulation in primary cultures of fetal rat adrenals. Adrenocortical cells, grown in the absence of ACTH for 3 weeks, possess typical features of zona glomerulosa cells. These cells differentiate into fasciculata-type cells and undergo biphasic proliferation when stimulated with ACTH. The primary antimitogenic phase of 24 h is followed by rapidly increased bromodeoxyuridine incorporation after 72 h of ACTH treatment. If the treatment is continued, the proliferation decreases again, but remains higher than the proliferation of the untreated cells. Undifferentiated zona glomerulosa-type cells secrete very low amounts of corticosterone. The 10% basal proliferation was not affected if exogenous corticosterone was added. However, if corticosterone was combined with ACTH for 3 days, it blocked the stimulatory growth effect of ACTH dose dependently. Etomidate, an inhibitor of steroidogenic enzymes, completely blocked corticosterone secretion. In our cultures it inhibited 50% of the proliferation of the zona glomerulosa-type cells. However, its effect was totally opposite in long term ACTH-treated cultures; in these fasciculata-type cells, etomidate stimulated the proliferation rate 3-fold. P450scc gene expression was low in undifferentiated zona glomerulosa-like cells. ACTH stimulation increased P450scc mRNA expression 10-fold. Exogenous corticosterone inhibited ACTH-induced P450scc mRNA accumulation by 50%, whereas etomidate doubled it. Our data suggest that a low corticosterone concentration supports the proliferation of undifferentiated zona glomerulosa-type cells, whereas a high corticosterone concentration inhibits the proliferation of differentiated zona fasciculata-type cells. In addition, a high corticosterone concentration may inhibit steroidogenesis by reducing P450scc expression. Thus, corticosterone may be an important modulator of adrenocortical cell proliferation and steroidogenesis in different zones of the adrenal cortex.