Loss of expression of a differentiated function gene, steroid 17α-hydroxylase, as adrenocortical cells senesce in culture

Senescence in cultured adrenocortical cells involves changes in expression of differentiated functions as well as changes in responses to mitogenic stimulation. Steroid 17α-hydroxylase (steroid 17α-monooxygenase, EC 1.14.99.9) is an adrenal-specific enzyme, the expression of which is dependent on the presence of stimulators of cyclic AMP production, such as cholera toxin. Dot-blot hybridization of RNA from bovine adrenocortical cells that had been incubated with cholera toxin showed a marked decline in 17α-hydroxylase mRNA levels as a function of population doubling level, closely paralleling the decline in induction of 17α-hydroxylase enzyme activity. The lower levels of 17α-hydroxylase induction did not result from a requirement for a longer time period for induction or from a specific defect in response to cholera toxin and were not caused by a general failure of enzyme induction in response to cyclic AMP. The decreased growth rate in older cells result from a general decline in response to several growth factors. However, the decline in 17α-hydroxylase induction did not result from a loss of response of the cells to mitogens, since quiescent cells at a low population doubling level showed stimulation of 17α-hydroxylase mRNA by cholera toxin to levels similar to those in nonquiescent cultures and added mitogens either had no effect on 17α-hydroxylase mRNA levels or decreased them. There was, however, a specific posttranscriptional effect of insulin on 17α-hydroxylase. The loss of 17α-hydroxylase induction is unlikely to result from overgrowth of a minority cell type lacking the ability to induce 17α-hydroxylase, because adrenocortical cell clones that had high levels of 17α-hydroxylase induction gave rise to cells with lower levels of induction on subcloning. Thus, loss of 17α-hydroxylase activity in adrenocortical cellular senescence results from a primary failure of accumulation of 17α-hydroxylase mRNA after incubation with the inducing agent.