TY - JOUR
T1 - Expression of 17α-hydroxylase and 3β-hydroxysteroid dehydrogenase in fetal human adrenocortical cells transfected with SV40 T antigen
AU - Cheng, C. Y.
AU - Flasch, M. V.
AU - Hornsby, P. J.
N1 - Copyright:
Copyright 2018 Elsevier B.V., All rights reserved.
PY - 1992
Y1 - 1992
N2 - Primary fetal human adrenocortical cells of definitive zone origin were transfected by electroporation with pSV3neo, a plasmid coding for SV40 T antigen and neo, which confers resistance to the antibiotic G418. The clones obtained proliferated for 30 to 40 population doublings after isolation when grown under standard medium conditions, and then entered 'crisis'. When early-passage clones were incubated with cyclic AMP (1:1 N6-monobutyryl and 8-bromo analogues), cell rounding was observed, as in primary cultures of human adrenocortical cells. As previously shown in bovine adrenocortical cells, rounding was inhibited with a monoclonal antibody against urokinase plasminogen activator but not with a monoclonal antibody against tissue plasminogen activator. The regulation of the steroidogenic pathway in clones was investigated. The effects of cyclic AMP and activation of protein kinase C were examined in cells maintained in defined medium or in the presence of serum. 17α-Hydroxylase was strongly induced by cyclic AMP, as evidenced by Northern blotting and by the conversion of progesterone or 25-hydroxy-[1,2-3H]cholesterol, this induction being blocked by low concentrations of 12-O-tetradecanoylphorbol-13-acetate (TPA). Cholesterol side-chain cleavage enzyme was strongly induced by cyclic AMP, and clones also showed low activities of 21-hydroxylase and 11β-hydroxylase. Under all circumstances levels of 3β-hydroxysteroid dehydrogenase (3β-HSD), as assessed by Northern blotting or by conversion of 25-hydroxycholesterol, were very low. 3β-HSD was not induced by cyclic AMP or TPA alone, but was induced by the combination of the two agents. The regulation of 17α-hydroxylase and 3β-HSD resembles that previously described in primary cultures of human fetal adrenocortical cells. Thus, transfection with SV40 T antigen resulted in the production of clones which preserve the unique characteristics of the human adrenal cortex.
AB - Primary fetal human adrenocortical cells of definitive zone origin were transfected by electroporation with pSV3neo, a plasmid coding for SV40 T antigen and neo, which confers resistance to the antibiotic G418. The clones obtained proliferated for 30 to 40 population doublings after isolation when grown under standard medium conditions, and then entered 'crisis'. When early-passage clones were incubated with cyclic AMP (1:1 N6-monobutyryl and 8-bromo analogues), cell rounding was observed, as in primary cultures of human adrenocortical cells. As previously shown in bovine adrenocortical cells, rounding was inhibited with a monoclonal antibody against urokinase plasminogen activator but not with a monoclonal antibody against tissue plasminogen activator. The regulation of the steroidogenic pathway in clones was investigated. The effects of cyclic AMP and activation of protein kinase C were examined in cells maintained in defined medium or in the presence of serum. 17α-Hydroxylase was strongly induced by cyclic AMP, as evidenced by Northern blotting and by the conversion of progesterone or 25-hydroxy-[1,2-3H]cholesterol, this induction being blocked by low concentrations of 12-O-tetradecanoylphorbol-13-acetate (TPA). Cholesterol side-chain cleavage enzyme was strongly induced by cyclic AMP, and clones also showed low activities of 21-hydroxylase and 11β-hydroxylase. Under all circumstances levels of 3β-hydroxysteroid dehydrogenase (3β-HSD), as assessed by Northern blotting or by conversion of 25-hydroxycholesterol, were very low. 3β-HSD was not induced by cyclic AMP or TPA alone, but was induced by the combination of the two agents. The regulation of 17α-hydroxylase and 3β-HSD resembles that previously described in primary cultures of human fetal adrenocortical cells. Thus, transfection with SV40 T antigen resulted in the production of clones which preserve the unique characteristics of the human adrenal cortex.
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U2 - 10.1677/jme.0.0090007
DO - 10.1677/jme.0.0090007
M3 - Article
C2 - 1325152
AN - SCOPUS:0026769660
VL - 9
SP - 7
EP - 17
JO - Journal of Molecular Endocrinology
JF - Journal of Molecular Endocrinology
SN - 0952-5041
IS - 1
ER -