Endocrine Abstracts

The human adrenal cortex consists of three layers in which specific steroid hormones are produced. Human adrenal NCI-H295A (A) and NCI-H295R (R) cells, originate from the same adrenocortical tumor and express all genes essential for steroidogenesis. Therefore they often serve as a suitable model to study human steroidogenesis. No data are available comparing steroidogenesis of A vs. R cells. Assuming no difference, research data from these two cell lines are directly compared. To characterize A and R cells, we investigated steroidogenesis of both cell lines. We found differences in the steroid profile of A and R cells. A cells converted [³H]-pregnenolone predominantly to aldosterone and cortisol while only traces of androgens were produced. R cells converted [³H]-pregnenolone to aldosterone, cortisol and androgens. The observed differences may be either due to differences in gene expression and /or posttranslational modifications which may lead to different activities of specific enzymes. Having found a profound difference in androgen synthesis, we compared HSD3B2 and CYP17 gene expression performing RT and real time PCR. We observed higher HSD3B2 expression in A cells compared to R cells while no difference in the expression of CYP17 was found. Functional studies were performed for P450c17 and 3betaHSDII enzymes. To study the activities of P450c17 (17alpha-hydroxylase and 17, 20 lyase), cells were treated with trilostane (3betaHSD inhibitor) prior to [³H]-pregnenolone or [³H]-17alpha-hydroxypregnenolone incubations. R cells showed higher 17, 20 lyase activity. To study 3betaHSDII activity, cells were incubated with [³H]-DHEA. Interestingly, lower 3betaHSDII activity was detected in R cells. In summary, we show that A and R cells differ in their steroid profile. R cells produce significantly more androgens. Further comparative studies of A vs. R cells may help to understand mechanism/s regulating human androgen production in health and disease.