When grown for long periods in culture, bovine adrenocortical cells lose the expression of a differentiated function gene, steroid 17α-hydroxylase. Previously, we documented a decline in 17α-hydroxylase mRNA with increasing culture passage level after induction with cyclic AMP (P. J. Hornsby et al., 1987, Proc. Natl. Acad. Sci. USA84, 1580). We used in situ hybridization to investigate the loss of expression of this gene during cellular senescence at an individual cell level. In primary cultures, cells were uniformly positive for hybridization with cDNA for 17α-hydroxylase after cyclic AMP induction. After two passages, cultures comprised a mixture of hybridizing and nonhybridizing cells. Cells appeared either to hybridize at a level comparable to that in primary cultures or to be nonhybridizing. When in situ hybridization was combined with immunofluorescence, cells positive for immunofluorescence were also positive for hybridization. Senescing mass cultures showed decreasing numbers of positive cells, and after 30 passages cultures comprised entirely nonhybridizing cells. Thus, the previously observed decline in overall 17α-hydroxylase mRNA levels results from a decline in the fraction of expressing cells in the culture, and the rate of loss of expressing cells is in agreement with the rate of loss of total 17α-hydroxylase mRNA. Primary clones, even when isolated at an early stage of clonal expansion, had mixtures of subclones of hybridizing and nonhybridizing cells. On recloning, hybridizing subclones usually produced uniformly nonhybridizing sub-subclones. Some subclones within primary clones had a morphology associated with replicative senescence (flattened cells with sparse intercellular contacts), yet had high numbers of hybridizing cells. We conclude that, in both mass and clonal populations, cells initially expressing 17α-hydroxylase rapidly give rise to clones of nonexpressing cells. Such cells are continually derived by a stochastic process from cells originally expressing the gene.
ASJC Scopus subject areas
- Cell Biology