Dissociation of senescence-associated changes in differentiated gene expression and replicative senescence in...

In bovine adrenocortical cells senescing in culture, the decline in expression of cyclic AMP-inducible 17 alpha-hydroxylase in mass cultures and clones of bovine adrenocortical cells results from a decrease in the number of cells in the population capable of expressing this gene. As the population of cells decreases in proliferative potential, the fraction of the population that expresses 17 alpha-hydroxylase after induction with cyclic AMP decreases, until the population comprises entirely nonexpressing cells. We used in situ hybridization to study the relationship between the loss of the ability to replicate and the loss of expression of this differentiated function gene. Colonies of cells from early passage cultures comprised complex mosaics of expressing and nonexpressing cells, indicating a high-frequency switching event. At an individual cell level, there was a complete independence of replicative senescence and loss of 17 alpha-hydroxylase expression. Groups of cells with a morphology characteristic of replicative senescence, occurring frequently in early-passage cultures, comprised both expressing and nonexpressing cells, as did cells that appeared to be in rapid growth. This was confirmed by prior incubation of cells with bromodeoxyuridine to label cycling cells; both cycling and noncycling populations comprised mixtures of expressing and nonexpressing cells. In contrast, replication was required for the loss of 17 alpha-hydroxylase expression, since cells rendered incapable of division by treatment with mitomycin C maintained 17 alpha-hydroxylase expression over long time periods. Thus, the process that results stochastically in replicative senescence and the process that results in loss of 17 alpha-hydroxylase expression are independent, but they have similar time-courses and both require replication for phenotypic switching to take place.