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To facilitate studies on di erentiation of adrenocortical cells and regulation of steroidogenic genes, we established cell lines from adrenals of adult transgenic mice harboring a temperature-sensitive large T-antigen gene of simian virus 40. Adrenal glands of the mice exhibited normal cortical zonation including a functionally undi erentiated cell-layer between the aldosterone-synthesizing zona glomerulosa cells and the corticosterone-synthesizing zona fasciculata cells. | Eur. J. Biochem. 269 69-81 2002 FEBS 2002 Conditionally immortalized adrenocortical cell lines at undifferentiated states exhibit inducible expression of glucocorticoid-synthesizing genes Kuniaki Mukai1 Hideko Nagasawa Reiko Agake-Suzuki1 Fumiko Mitani1 Keiko Totani1 Nobuaki Yanai2 Masuo Obinata2 Makoto Suematsu1 and Yuzuru Ishimura1 1Department of Biochemistry and Integrative Medical Biology School of Medicine Keio University Shinjuku-ku Tokyo Japan 2Institute of Development Aging and Cancer Tohoku University Sendai Japan To facilitate studies on differentiation of adrenocortical cells and regulation of steroidogenic genes we established cell lines from adrenals of adult transgenic mice harboring a temperature-sensitive large T-antigen gene of simian virus 40. Adrenal glands of the mice exhibited normal cortical zonation including a functionally undifferentiated cell-layer between the aldosterone-synthesizing zona glomerulosa cells and the corticosterone-synthesizing zona fasciculata cells. At a permissive temperature 33 C established cell lines AcA201 AcE60 and AcA101 expressed steroidogenic genes encoding steroidogenic factor-1 cholesterol side-chain cleavage P450scc and steroidogenic acute regulatory protein which are expressed throughout adrenal cortices and gonads. Genes encoding 3b-hydroxysteroid dehydrogenase and steroid 21-hydroxylase P450c21 which catalyze the intermediate steps for syntheses of both aldosterone and corticosterone were inducible in the three cell lines in temperature- and or dibutyryl cAMP-dependent manners. Notably these cell lines displayed distinct expression patterns of the steroid 11 b-hydroxylase P45011b gene responsible for the zone-specific synthesis of corticosterone. AcA201 cells expressed the P45011b gene at 33 C showing the property of the zona fasciculata cells while AcE60 cells expressed it upon a shift to a nonpermissive temperature 39 C . On the other hand AcA101 expressed the P45011fi gene at 39 C synergistically with .