Renewal inhibition of human mammary cell growth in vitro: cortisol and the recruitment of cells to terminal differentiation.

Cortisol and insulin stimulated exponential growth of normal human mammary epithelium in short-term monolayer culture. The response of cells depended on their organization into "growth units" on the surface of the culture dish; single cells did not respond. Growth of cells in the units ceased after only 3-4 doublings, ending in terminal differentiation. The 3-4 divisions that occurred in response to insulin and cortisol and that resulted in terminal differentiation, were not inhibited by short-range signals normally transmitted at population confluence. When growing above confluence density in response to hormones, cells reduced volume to accommodate the "terminal differentiation" divisions while still largely preserving a monolayer. The longevity of populations of normal cells (9-14 divisions), which occurred in 3 or 4 passages, exceeded the average longevity of individual cells in one passage (3-4 divisions). This disparity between real and apparent longevity was due to the inhibition of growth of divisional cells within growth units, which occurred in concert with terminal differentiation of other cells in these units. Inhibited cells could be recruited to undergo terminal differentiation divisions in response to cortisol and insulin, but only when the growth units were disrupted and terminally differentiated cells were eliminated, which occurred at subculture. We refer to the inhibition of growth that occurs in growth units as "renewal inhibition" to distinguish it from population-wide "confluence inhibition" and to emphasize three other aspects: (1) it occurred in terminally differentiating growth units; (2) it occurred in the continued presence of an inductive hormonal stimulus for differentiative growth; and (3) it conserved less differentiated cells for recruitment to terminal differentiation. There are parallels between renewal inhibition in vitro and the signals that restrain growth of mammary cells in 'growth buds" in vivo to preserve their capacity for multiple cycles of secretory differentiation. Differences in the behavior of normal and malignant breast cells in vitro suggest that renewal inhibition, rather than confluence inhibition, may be an important locus of growth control alteration in malignant transformation.