Effects of p21^Cip1/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

ABSTRACT

It has been proposed that the functions of the cyclin-dependent kinase inhibitors p21^Cip1/Waf1 and p27^Kip1 are limited to cell cycle control at the G₁/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 G₁/S transition, contributes to regulation of the G₂/M transition. Both G₁- and G₂-arrested cells were observed in all cell types, with different preponderances. Preponderant G₁ arrest in response to p21 expression correlated with the presence of functional pRb. G₂ 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 G₂-arrested cells. These results suggest that the primary target of the Cip/Kip family of inhibitors leading to efficient G₁ arrest as well as to blockade of DNA replication from either G₁ or G₂ 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 with genotoxic agents that activate the p53/p21 pathway.

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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

ACKNOWLEDGMENTS

We thank Joe Nevins and James DeGregori (Duke University) and John Cogswell (Glaxo Wellcome) for the gift of control p21 and p27 adenoviruses; Glen Nemerow and Dan von Segrenn (Scripps) for advice on adenovirus production, purification, and titer determination; Jean Wang and Laura Whittaker (UCSD) and Tyler Jacks (MIT) for MEFs; Nick Rhind for advice on irradiation and digital microscopy; and Peter Vogt, Paul Russell, Curt Wittenberg, Ben Cravatt, Kevin Sullivan, and Fred Jones for critical reading of the manuscript.

This work was supported by NIH grant GM46006 to S.I.R.