Nuclear Accumulation of p21^Cip1 at the Onset of Mitosis: a Role at the G₂/M-Phase Transition

ABSTRACT

Cell cycle arrest in G₁ in response to ionizing radiation or senescence is believed to be provoked by inactivation of G₁ cyclin-cyclin-dependent kinases (Cdks) by the Cdk inhibitor p21^{Cip1/Waf1/Sdi1}. We provide evidence that in addition to exerting negative control of the G₁/S phase transition, p21 may play a role at the onset of mitosis. In nontransformed fibroblasts, p21 transiently reaccumulates in the nucleus near the G₂/M-phase boundary, concomitant with cyclin B1 nuclear translocation, and associates with a fraction of cyclin A-Cdk and cyclin B1-Cdk complexes. Premitotic nuclear accumulation of cyclin B1 is not detectable in cells with low p21 levels, such as fibroblasts expressing the viral human papillomavirus type 16 E6 oncoprotein, which functionally inactivates p53, or in tumor-derived cells. Moreover, synchronized E6-expressing fibroblasts show accelerated entry into mitosis compared to wild-type cells and exhibit higher cyclin A- and cyclin B1-associated kinase activities. Finally, primary embryonic fibroblasts derived from p21^−/− mice have significantly reduced numbers of premitotic cells with nuclear cyclin B1. These data suggest that p21 promotes a transient pause late in G₂ that may contribute to the implementation of late cell cycle checkpoint controls.

ACKNOWLEDGMENTS

We thank James Brugarolas and Tyler Jacks (Cambridge, Mass.) for p21^−/− MEFs, Jacques Piette (IGM, Montepellier, France) for p53⁻ primary fibroblasts, Clare McGowan and Paul Russell (Scripps Research Institute, La Jolla, Calif.) for anti-cyclin B1 antibodies, Martha Henze and Ludger Hengst (Scripps Research Institute) for anti-p21 antibody, Emma Lees and Ed Harlow (MGH, Boston, Mass.) for monoclonal cyclin D1, cyclin E, and cyclin A antibodies, Jerry Shay (University of Texas SW Medical Center, Dallas) for HPV16 E6-transduced IMR-90 cells, and Patrick Turowski (CRBM, Montpellier, France) for supplying some synchronized Hs68 cells. V.D. extends thanks to all members of Jacques Pouysségur’s laboratory (Centre de Biochimie, Nice, France) for their hospitality and for many discussions in the course of this work and to Marcel Dorée, Daniel Fisher, and Annick Péléraux (CRBM) for their helpful comments on the manuscript. Finally, special thanks go to Anne Brunet (Centre de Biochimie, Nice, France) for constant encouragement and for critically reading the original versions of the manuscript.

This work was partially supported by Association pour la Recherche sur le Cancer grant ARC-6852 to V.D. and Public Health Service grant GM46004 to S.I.R.