REGULATION OF HUMAN ADRENAL CARCINOMA CELL (NCI-H295) PRODUCTION OF C19 STEROIDS

The regulation of biosynthesis of the adrenal C19 steroids (the so-cal led adrenal androgens) remains unclear. Understanding adrenal producti on of C19 steroids is important when the benefits of these steroids ar e considered on processes and diseases associated with aging. In vitro studies defining the mechanisms that regulate the production of human adrenal C19 steroids have been limited because of the difficulties in obtaining adrenal tissue. A cell line that retains differentiated adr enal functions would greatly facilitate research in this area. Herein, we describe the use of the human adrenocortical tumor H295 cell line as a model to evaluate mechanisms controlling C19 and C21 steroid prod uction. The cells were characterized with regard to ACTH, forskolin, a nd dibutyryl cAMP (dbcAMP) responsiveness, as measured by increased cA MP production, synthesis of steroids, and induction of 17alpha-hydroxy lase cytochrome P450 (P450c17). Forskolin and dbcAMP, which were more effective than ACTH, enhanced the production of cortisol, dehydroepian drosterone (DHEA), DHEA sulfate (DHEAS), and androstenedione over a 48 -h treatment period. Comparison of the relative amounts of measured st eroid secreted under forskolin treatment indicated that the primary pr oduct was cortisol (70%), followed by androstenedione, (14%), DHEA (9% ), and DHEAS (7%). Cortisol was also demonstrated to be the major ster oid product by examination of UV-detectable steroids after high perfor mance liquid chromatographic, separation. The increases in steroid pro duction caused by ACTH, forskolin, and dbcAMP occurred in a concentrat ion- and time-dependent manner. A key enzyme in the production of C19 steroids is P450c17. ACTH, forskolin, and dbcAMP increased the activit y of 17alpha-hydroxylase by approximately 2.5-, 10-, and 10-fold, resp ectively. These effects on enzyme activity occurred in a concentration -dependent manner and coincided with increased levels of P450c17 mRNA. In summary, H295 cells should provide a much-needed model to study me chanisms controlling the secretion of glucocorticoids and C19 steroids , because steroid production in these cells is hormonally controlled a nd associated with the induction of P450c17.