Aberrant DNA methylation may be involved in human adrenocortical tumorigenesis, which is often accompanied by abnormal hormone production. In this study, we aimed to clarify the effects of DNA methylation on steroidogenesis using the human adrenocortical NCI-H295R cell line as a model. Treatment with the DNA methylation inhibitor 5-aza-28-deoxycytidine (Azad; 10 µM for 7 days) decreased the proliferation rate to approximately 20% and the cell number to 60% of the control, with a simultaneous increase in the expression of the cyclin-dependent kinase inhibitor p57 KIP2 gene. In addition, Azad treatment increased cortisol secretion dose and time dependently, whereas dehydroepiandrosterone sulfate secretion was not affected. Azad treatment decreased basal and (Bu) 2 cAMP-induced expression of low-and high-density lipoprotein receptor, steroidogenic acute regulatory protein (StAR), cholesterol side-chain cleavage enzyme, steroid 17 -hydroxylase/17,20-lyase and steroid 21-hydroxylase mRNA, as well as the StAR protein level. In contrast, Azad treatment increased the basal expression of steroid 11 -hydroxylase and 3 -hydroxysteroid dehydrogenase/ 5 -4 -isomerase genes, although it inhibited the (Bu) 2 cAMP-induced expression of these two genes. The expression of steroidogenic factor-1 (SF-1) and DAX-1 (dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X-chromosome 1) genes (both harboring putative CpG islands in their promoters) and the methylation degree of the HpaII recognition site(s) in the SF-1 gene promoter region were reduced by Azad treatment. The immunostaining pattern of the methyl-CpG-binding protein MeCP2 was also modified by Azad treatment. These results suggest that DNA methylation may be implicated in the regulation of cell proliferation and steroidogenesis in human 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-(TNF ) and the PKA pathway in adrenocortical cell proliferation and apoptosis. The PKA activator Dibutyryl cAMP ((Bu) 2 cAMP) strongly induced differentiation and inhibited proliferation in the human adrenocortical cell line NCI-H295R (H295R). TNF induced apoptosis of H295R cells. Interestingly, (Bu) 2 cAMP treatment clearly enhanced TNF -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) 2 cAMP-induced glucocorticoid secretion since dexamethasone had no significant effect on the TNF -induced apoptosis. (Bu) 2 cAMP 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 TNF -induced apoptosis in adrenocortical H295R cells, and these effects were associated with increased c-myc expression.