Differential regulation of cyclin A, cyclin B and p21 concentrations in a growth-restricted human fibroblast cell line.

When the culture temperature was shifted from 35 degrees C to 39 degrees C, human fibroblasts immortalized by the temperature-sensitive simian virus 40 T antigen became larger and acquired the morphological characteristics of senescent fibroblasts. After culture at 39 degrees C for 48 h, most cells had ceased to proliferate. A rapid depletion of cells with S-phase DNA content was observed after the temperature shift. To elucidate the mechanism governing this rapid arrest of proliferation, we studied the expression of genes involved in the regulation of cell cycle progression. Cyclin A, cyclin B and p34cdc2 concentrations were not changed during growth restriction, whereas p21 was rapidly induced in these growth-restricted cells. Transient expression of exogenous p21 in cells cultured at 35 degrees C led to growth restriction and morphological changes characteristic of senescence. Furthermore, we studied the reversibility of growth restriction induced by the temperature increase. The results showed that senescent morphology and growth arrest were not reversible. In these cells the p21 concentration remained high and p34cdc2 remained undetectable. This indicates that p21 accumulation might be responsible for the maintenance of senescence. Our findings provide information on the use of growth restriction of immortalized fibroblasts induced by a temperature shift as a model system to study senescence.