Activity of the CYP17 promoter in bovine adrenocortical cells before and after phenotypic switching

Abstract

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 17α-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. However, the −2544 construct had a lower response to cholera toxin than the −488 construct in cells from both PDLs, indicating the potential existence of a negative regulatory element. The lower expression of the −2544 construct was confirmed in assays on individual clones of cells as well as pooled clones. These data indicate that the loss of cyclic AMP-inducible CYP17 expression during bovine adrenocortical cell senescence likely does not result from a loss of positive regulatory factors that act within 2.5 kb upstream of the CYP17 promoter.