TY - JOUR
T1 - Oncogene-transformed granulosa cells as a model system for the study of steroidogenic processes
AU - Amsterdam, A.
AU - Hanukoglu, I.
AU - Suh, B. S.
AU - Keren-Tal, I.
AU - Plehn-Dujowich, D.
AU - Sprengel, R.
AU - Rennert, H.
AU - Strauss, J. F.
N1 - Funding Information:
Acknowledgements--We thank Drs A. Ben Ze'ev and J. T. Billheimer for fruitful collaboration, Dr F. Kohen for providing the antibodies for RIA of progesterone and 20,t-hydroxy-4-pregnen-3-one and Mrs M. Kopelowitz for excellent secretarial assistance. A. Amsterdam is the Joyce and Ben B. Eisenberg Professor of Molecular Endocrinology and Cancer Research. I. Hanukoglu is the incumbent of the Delta Research Career Development Chair. Our research has been supported by the Minerva Foundation, Munchen, Germany, the Leo and Julia Forseheimer Center for Molecular Genetics and the Josef Cohn Center for Biomembrane Research at the Weizmann Institute of Science.
PY - 1992/12
Y1 - 1992/12
N2 - Highly steroidogenic granulosa cell lines were established by transfection of primary granulosa cells from preovulatory follicles with SV40 DNA and Ha-ras oncogene. Progesterone production in these cells was enhanced to levels comparable to normal steroidogenic cells, by prolonged (> 12 h) stimulation with 8-Br-cAMP, forskolin and cholera toxin, which elevate intracellular cAMP. The steroidogenic capacity of individual lines correlated with the expression of the ras oncogene product (p21) and the morphology of the cells. Formation of the steroid hormones was associated with de novo synthesis of the mitochondrial cytochrome P450scc system proteins. Since cholesterol import into mitochondria is essential for steroidogenesis, the expression of the peripheral benzodiazepine receptor (PBR) and the sterol carrier protein 2 was characterized in these cells. The induction of the expression of the genes coding for both proteins appeared to be mediated, at least in part, by cAMP. Stimulation of the PBR by specific agonists enhanced progesterone production in these cells. The phorbol ester 12-O-tetradecanoyl-phorbol 13-acetate (TPA) dramatically suppressed the cAMP-induced steroidogenesis, in spite of enhanced intracellular cAMP levels, suggesting that TPA can modify the effects of cAMP. cAMP stimulation suppressed growth of transformed cells concomitantly with induction of steroidogenesis. The transformed cells lacked receptors for the native stimulants, the gonadotropic hormones. After transfection of the cells with a lutropin (LH) receptor expression plasmid, the LH and hCG response was reconstituted. In these newly established cell lines gonadotropins were able to stimulate the formation of cAMP and progesterone in a dose-dependent manner with an ED50 characteristic of the native receptor. High doses caused desensitization to gonadotropins as observed in normal cells. These newly established oncogene-transformed granulosa cell lines can serve as a useful model to study inducible steroidogenesis and the effect of oncogene expression on this process.
AB - Highly steroidogenic granulosa cell lines were established by transfection of primary granulosa cells from preovulatory follicles with SV40 DNA and Ha-ras oncogene. Progesterone production in these cells was enhanced to levels comparable to normal steroidogenic cells, by prolonged (> 12 h) stimulation with 8-Br-cAMP, forskolin and cholera toxin, which elevate intracellular cAMP. The steroidogenic capacity of individual lines correlated with the expression of the ras oncogene product (p21) and the morphology of the cells. Formation of the steroid hormones was associated with de novo synthesis of the mitochondrial cytochrome P450scc system proteins. Since cholesterol import into mitochondria is essential for steroidogenesis, the expression of the peripheral benzodiazepine receptor (PBR) and the sterol carrier protein 2 was characterized in these cells. The induction of the expression of the genes coding for both proteins appeared to be mediated, at least in part, by cAMP. Stimulation of the PBR by specific agonists enhanced progesterone production in these cells. The phorbol ester 12-O-tetradecanoyl-phorbol 13-acetate (TPA) dramatically suppressed the cAMP-induced steroidogenesis, in spite of enhanced intracellular cAMP levels, suggesting that TPA can modify the effects of cAMP. cAMP stimulation suppressed growth of transformed cells concomitantly with induction of steroidogenesis. The transformed cells lacked receptors for the native stimulants, the gonadotropic hormones. After transfection of the cells with a lutropin (LH) receptor expression plasmid, the LH and hCG response was reconstituted. In these newly established cell lines gonadotropins were able to stimulate the formation of cAMP and progesterone in a dose-dependent manner with an ED50 characteristic of the native receptor. High doses caused desensitization to gonadotropins as observed in normal cells. These newly established oncogene-transformed granulosa cell lines can serve as a useful model to study inducible steroidogenesis and the effect of oncogene expression on this process.
UR - http://www.scopus.com/inward/record.url?scp=0027056803&partnerID=8YFLogxK
U2 - 10.1016/0960-0760(92)90315-A
DO - 10.1016/0960-0760(92)90315-A
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C2 - 22217832
AN - SCOPUS:0027056803
SN - 0960-0760
VL - 43
SP - 875
EP - 884
JO - Journal of Steroid Biochemistry and Molecular Biology
JF - Journal of Steroid Biochemistry and Molecular Biology
IS - 8
ER -