Activity of the CYP17 promoter in bovine adrenocortical cells before and after phenotypic switching.
Cultured bovine adrenocortical cells reach replicative senescence after 100-120 population doublings in culture. Before reaching senescence, cells undergo high frequency phenotypic switching from CYP17+ to CYP17-, where '+' and '-' refer to the ability of intracellular cyclic AMP to induce expression of CYP17 (steroid 17 alpha-hydroxylase). We used luciferase reporter constructs to assess the activity of the CYP17 promoter in bovine adrenocortical cells before and after phenotypic switching. We constructed two plasmids containing -2544 to +29 and -488 to +29 of the 5' region of CYP17 linked to a promoterless luciferase gene. Because of technical difficulties with transient transfection of late-passage bovine adrenocortical cells, these experiments were performed using stable transfection. Cells at early passage (PDL 10) and late passage (PDL 55) were cotransfected with either of these two plasmids ligated to pSV3neo, and G418-resistant pools of clones were derived. The activity of the CYP17 promoter in these transfectants was tested by growing cells in complete medium until semiconfluent and then transferring them into defined medium with cholera toxin and insulin-like growth factor I for 6 h. Luciferase activity was consistently induced by cholera toxin/IGF-I over five passages in pooled clones derived by transfection of early passage cells with the -488 construct. Despite the lack of expression of the endogenous CYP17 gene in transfectants from late-passage cells, induced luciferase activity was higher in late-passage transfectants than early-passage transfectants for both the -2544 and -488 constructs.(ABSTRACT TRUNCATED AT 250 WORDS)
Lala
D S
DS
Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta 30912.
Hornsby
P J
PJ
D000446:Aldehyde-Lyases / Q000096:biosynthesis* / Q000235:genetics; D000818:Animals; D002417:Cattle; D002455:Cell Division; D002478:Cells, Cultured; D016922:Cellular Senescence; D000242:Cyclic AMP / Q000494:pharmacology*; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis* / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics; D008297:Male; D010641:Phenotype; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D013254:Steroid 17-alpha-Hydroxylase / Q000096:biosynthesis* / Q000235:genetics; D015383:Zona Fasciculata / Q000166:cytology / Q000378:metabolism*; D015385:Zona Reticularis / Q000166:cytology / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D003577:Cytochrome P-450 Enzyme System; D000242:Cyclic AMP; D008156:Luciferases; D013254:Steroid 17-alpha-Hydroxylase; D000446:Aldehyde-Lyases