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 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 expression. mRNA levels of inhibin subunit were studied in 57 samples and all tissues were stained immunohistochemically with an inhibin subunit-specific antibody. Inhibin mRNA was detected in all adrenocortical tissues. Virilizing adenomas possessed a 10-fold higher median inhibin mRNA expression than did normal adrenals. Bilaterally and nodularly hyperplastic adrenals and other than virilizing adrenocortical tumours had their median inhibin mRNA levels close to those of normal adrenals. Immunohistochemically, inhibin subunit was detectable in all normal and hyperplastic adrenals, as well as in 73% of the adrenocortical tumours. However Our data show that inhibin subunit is highly expressed in both normal and neoplastic androgenproducing adrenocortical cells, with less expression in cortisol-producing and hardly any in aldosteroneproducing cells. This suggests a specific role for inhibins in the regulation of adrenal androgen production. We did not find any significant difference in inhibin expression between benign and malignant adrenocortical tumours. Thus inhibin gene does not seem to have a tumour suppressor role in human adrenal cortex.

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.

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.

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.

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.