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M. SALMENPERÄ
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A. I. KAHRI
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A. SAURE
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SUMMARY

The effects of corticosterone in concentrations found in adrenal venous plasma on ACTH-induced changes in cultured cortical cells derived from foetal rat adrenals were studied. Corticosterone at a concentration of 5·7 × 10−5 mol/l completely inhibited mitochondrial differentiation to fasciculate-like morphology. The same cultures revealed significant inhibition of 11β- and 18-hydroxylation compared with cultures treated with ACTH only. This was shown by the reduced formation of corticosterone and 18-OH-deoxycorticosterone (48%, P < 0·001) and simultaneous enhancement of deoxycorticosterone formation (33%, P < 0·05) from added [4-14C]progesterone. Similar inhibition was observed when dibutyryl cyclic AMP replaced ACTH as an inducer of differentiation. Lower concentrations of corticosterone (1·2 × 10−5 and 2·4 × 10−5 mol/l) inhibited ACTH-stimulated formation of corticosterone and 18-OH-deoxycorticosterone from endogenous precursors.

The results demonstrate that corticosterone regulates the stage of differentiation in cultured adrenocortical cells. The possible role of corticosterone in the regulation of growth and steroidogenic capacity of the adrenal cortex is discussed.

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P Heikkilä
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J Arola
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A Salmi
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A I Kahri
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Abstract

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.

Journal of Endocrinology (1995) 145, 379–385

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J Liu
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R Voutilainen
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A I Kahri
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P Heikkilä
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Abstract

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.

Journal of Endocrinology (1996) 148, 523–529

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J. Arola
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P. Heikkilä
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R. Voutilainen
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A. I. Kahri
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ABSTRACT

ACTH has a biphasic effect on the proliferation of fetal rat adrenocortical cells in primary culture. Dramatic changes occurred during the first 72 h of ACTH stimulation, when incorporation of bromodeoxyuridine was used as an indicator of proliferation. The primary effect of ACTH was the inhibition of proliferation during the first 24 h, which was followed by an intense stimulatory phase during the third day of ACTH treatment. Cycloheximide (a protein synthesis inhibitor) prevented both the inhibitory and the stimulatory effects of ACTH, but did not affect the basal proliferation of unstimulated zona glomerulosa-like cells. Although adrenocortical cells stimulated with cyclic AMP (cAMP) derivatives, 8-bromo cAMP (8-Br cAMP) or dibutyryl cAMP ((Bu)2cAMP), differentiated morphologically into fasciculata-like cells, and secreted corticosterone and 18-OH-deoxycorticosterone, as did ACTH-stimulated cells, neither of the derivatives inhibited proliferation during the first 24 h of treatment. In contrast to ACTH, (Bu)2cAMP had a stimulatory effect on bromodeoxyuridine incorporation during the first 24 h of treatment. 8-Br cAMP did not change proliferation during the 24 h of treatment, but had a stimulatory effect after 72 h, which was not seen with (Bu)2cAMP.

Thus, these results suggest that (1) differentiation, steroid hormone synthesis and the mitogenic effect of ACTH are transduced through the cAMP-mediated system, (2) the antimitogenic effect of ACTH is transduced via a cAMP-independent pathway and (3) both antimitogenic and mitogenic effects of ACTH are dependent on protein synthesis.

Journal of Endocrinology (1993) 139, 451–461

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J Arola
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P Heikkilä
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R Voutilainen
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A I Kahri
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Abstract

ACTH exerts a biphasic effect on the growth of fetal rat adrenocortical cells in primary culture when bromodeoxyuridine (BrdU) incorporation is used as an indicator of proliferation. The immediate inhibitory effect during the first 24 h of ACTH stimulation is not dependent on cyclic AMP (cAMP). Protein kinase C (PKC) inhibitors H-7 and staurosporine blocked this inhibitory effect of ACTH, whereas 12-0-tetradecanoyl phorbol-13-acetate (TPA; a PKC activator) mimicked the ACTH-induced antimitogenic effect. The stimulatory growth effect of ACTH appears after 72 h of treatment. A similar mitogenic effect is also achieved with cAMP derivative 8-bromo cAMP (8-Br cAMP). However, both ACTH- and 8-Br cAMP-induced proliferations could be reduced with H-7.

ACTH-induced corticosterone secretion was inhibited 50% with H-7 after 24 h, but 8-Br cAMP-induced secretion was unaffected. However, if the treatments were continued for 72 h, H-7 no longer reduced the steroid secretions. Reduction (50–75%) of cholesterol side-chain cleavage enzyme (P450scc) mRNA expression was also noted with H-7 in ACTH-treated cultures after 6 and 24 h. In contrast, TPA doubled the corticosterone secretion induced by 8-Br cAMP, but did not further increase the ACTH-induced secretion after 24 h. TPA alone, however, was not able to induce steroid secretion or P450scc mRNA expression. The morphological differentiation of fetal rat adrenocortical cells with ACTH or 8-Br cAMP from zona glomerulosa-like cells into zona fasciculata-like cells was not disturbed by H-7 nor was it induced by TPA alone.

These results therefore suggest that PKC- and cAMP-dependent signal transductions are involved in the ACTH-induced biphasic growth effect of fetal rat adrenocortical cells. PKC plays a role in the inhibitory growth effect, and both PKC and cAMP are involved in the stimulatory growth phase of ACTH. Both PKC and cAMP are also involved in the steroid secretion of zona glomerulosa-type cells, but differentiation into zona fasciculata-type cells and their steroid production is transduced through cAMP.

Journal of Endocrinology (1994) 141, 285–293

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J Liu
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R Voutilainen
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A I Kahri
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P Heikkilä
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Abstract

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.

Journal of Endocrinology (1997) 152, 175–181

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J Liu
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P Heikkilä
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A I Kahri
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R Voutilainen
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Abstract

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, adrenocorticotropin (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.

Journal of Endocrinology (1996) 150, 43–50

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J Liu
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A I Kahri
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P Heikkilä
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W F Blum
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R Voutilainen
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Abstract

Human phaeochromocytomas abundantly express insulin-like growth factor-II (IGF-II), but its regulation and biological role in these neoplasms is not known at present. To clarify the regulation of IGF-II gene expression in phaeochromocytomas, we studied the effects of glucocorticoids, nerve growth factor (NGF), and protein kinase A and C regulators in primary cultures of human phaeochromocytoma cells. Cytoplasmic RNA was extracted and analysed by Northern and dot blotting with a 32P-labelled cDNA probe for IGF-II. Dexamethasone treatment (500 ng/ml) for 3 and 7 days resulted in a 260% and 515% increase in the accumulation of IGF-II mRNA respectively. The stimulatory effect of dexamethasone was time-and dose-dependent. The increases in the 6·0 and 2·2 kb species of IGF-II mRNAs were the most apparent. Cortisol (1 μg/ml) increased the amount of IGF-II mRNA by threefold compared with the control. NGF (200 ng/ml), dibutyryl cyclic AMP (1 mm) and 12-O-tetradecanoyl phorbol-13-acetate (a protein kinase C activator; 100 ng/ml) had no significant effect on IGF-II mRNA levels. These data suggest that IGF-II gene expression in human phaeochromocytomas may be regulated by microenvironmental glucocorticoids.

Journal of Endocrinology (1994) 142, 29–35

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