We. Rainey et al., REGULATION OF HUMAN ADRENAL CARCINOMA CELL (NCI-H295) PRODUCTION OF C19 STEROIDS, The Journal of clinical endocrinology and metabolism, 77(3), 1993, pp. 731-737
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.