Effects of p21Cip1/Waf1 at both the G1/S and the G2/M cell cycle transitions:: pRb is a critical determinant in blocking DNA replication and in preventing endoreduplication

It has been proposed that the functions of the cyclin-dependent kinase inhibitors p21(Cip/Waf1) and p27(Kip1) are limited to cell cycle control at the G(1)/S-phase transition and in the maintenance of cellular quiescence. To test the validity of this hypothesis, p21 was expressed in a diverse panel of cell lines, thus isolating the effects of p21 activity from the pleiotropic effects of upstream signaling pathways that normally induce p21 expression. The data show that at physiological levels of accumulation, p21, in addition to its role in negatively regulating the G(1)/S transition, contributes to regulation of the G(2)/M transition. Both G(1)- and G(2)-arrested cells were observed in all cell types, with different preponderances. Preponderant G(1) arrest in response to p21 expression correlated with the presence of functional pRb. G(2) arrest was more prominent in pRb-negative cells, The arrest distribution did not correlate with the p53 status, and proliferating-cell nuclear antigen (PCNA) binding activity of p21 did not appear to be involved, since p27, which lacks a PCNA binding domain, produced similar arrest Bs. In addition, DNA endoreduplication occurred in pRb-negative but not in pRb-positive cells, suggesting that functional pRb is necessary to prevent DNA replication in p21 G(2)-arrested cells. These results suggest that the primary target of the Cip/Kip family of inhibitors leading to efficient G(1) arrest as well as to blockade of DNA replication from either G(1) or G(2) phase is the pRb regulatory system, Finally, the tendency of Rb-negative cells to undergo endoreduplication cycles when p21 is expressed may have negative implications in the therapy of Rb-negative cancers,vith genotoxic agents that activate the p53/p21 pathway.