Establishment of highly steroidogenic granulosa cell lines by cotransfection with SV40 and Ha-ras oncogene: induction of steroidogenesis by cyclic adenosine 3'-5'-monophosphate and its suppression by phorbol ester.
We have established new cell lines from preovulatory follicles of PMSG-treated immature rats by cotransfection of primary cells with SV40 DNA and Ha-ras oncogene. These cell lines secrete progesterone and 20 alpha-hydroxy-4-pregnen-3-one in response to stimulation with 8-bromo-cAMP, forskolin, or cholera toxin at levels similar to primary cultures of granulosa cells (500 ng/10(6) cells/48 h). 12-O-Tetradecanoylphorbol 13-acetate inhibits progesterone production induced by 8-bromo-cAMP (67%, P less than 0.001), forskolin (88%, P less than 0.001), and cholera toxin (75%, P less than 0.001), in spite of enhancing cAMP accumulation (200%, P less than 0.001) in response to forskolin or cholera toxin. LH, FSH, prostaglandins E1 and E2, and PRL do not stimulate progesterone production. The beta-adrenergic agonist, isoproterenol, stimulates significantly both cAMP accumulation (80%, P less than 0.05) and progesterone and 20 alpha-hydroxy-4-pregnen-3-one secretion (70%, P less than 0.05). In contrast to primary cultured cells in which progesterone secretion increases within 90 min of stimulation, in the cell lines progesterone secretion becomes evident only after 12-24 h of stimulation. The stimulation of these lines by forskolin produced a maximum rise in intracellular cAMP levels at 45 min which declined to 50% (P less than 0.001) of this value within 12 h. It was found that cAMP suppressed growth concomitantly with induction of steroidogenesis when cotransfected cells were examined for growth by total cell protein and [3H]thymidine incorporation to DNA. These granulosa cell lines are uniquely suited for further studies of cAMP induction and the role of oncogenes in steroidogenesis.
Suh
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Department of Hormone Research, Weizmann Institute of Science, Rehovot, Israel.
Amsterdam
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A
D000818:Animals; D002453:Cell Cycle; D002461:Cell Line, Transformed; D000242:Cyclic AMP / Q000096:biosynthesis / Q000494:pharmacology; D004279:DNA, Viral; D005260:Female; D015967:Gene Expression Regulation, Viral; D011905:Genes, ras; D012739:Gonadal Steroid Hormones / Q000037:antagonists & inhibitors / Q000096:biosynthesis*; D006107:Granulosa Cells / Q000166:cytology / Q000378:metabolism* / Q000648:ultrastructure; D011374:Progesterone / Q000096:biosynthesis; D013539:Simian virus 40 / Q000235:genetics*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection
D004279:DNA, Viral; D012739:Gonadal Steroid Hormones; D011374:Progesterone; D000242:Cyclic AMP; D013755:Tetradecanoylphorbol Acetate