Changes in p21Cip1 and p27Kip1 expression are not required for cell cycle entry and progression to S phase in Swiss 3T3 cells

Abstract The cyclin-dependent kinase inhibitors, p21 Cip1 and p27 Kip1 , play an important role in the regulation of progression through G 1 to S phase in mammalian cells. Here we report that confluent 3T3 cells expressed p21 Cip1 and p27 Kip1 predominantly in the nucleus, and the level of both proteins declined as the cells entered the cell cycle and progressed through G 1 in response to serum growth factors. However, when confluent cells were serum starved prior to treatment, no downregulation of p21 Cip1 or p27 Kip1 expression was observed. Notably, serum starvation did not significantly influence the capacity of the cells to progress to the S phase. It was observed that serum starvation reduced cell density. Further, when cells were plated at a range of different densities, starved of serum to render them quiescent and then subsequently treated with serum, a reduction in p21 Cip1 and p27 Kip1 expression was observed in cells plated at high density but not in those at low density. Again, the extent and timing of progression to S phase was not influenced by cell density. To establish the potential role of cell:cell contact in the observed density-dependent regulation of p21 Cip1 and p27 Kip1 expression, cells were plated onto micorarrays of adhesive islands that prevented individual cells from making any contact with other cells. Under these conditions serum growth factors induced p21 Cip1 and p27 Kip1 downregulation, and hence, there is no requirement for cell:cell contact. Together, these data indicate that there are conditions under which 3T3 cells can progress to the S phase without downregulation of p21 Cip1 and p27 Kip1 . The significance of these observations and mechanisms by which density-dependent regulation of p21 Cip1 and p27 Kip1 expression may occur are discussed.