p53 Regulation of G₂ Checkpoint Is Retinoblastoma Protein Dependent

Abstract

In the present study, we investigated the role of p53 in G₂ checkpoint function by determining the mechanism by which p53 prevents premature exit from G₂ arrest after genotoxic stress. Using three cell model systems, each isogenic, we showed that either ectopic or endogenous p53 sustained a G₂arrest activated by ionizing radiation or adriamycin. The mechanism was p21 and retinoblastoma protein (pRB) dependent and involved an initial inhibition of cyclin B1-Cdc2 activity and a secondary decrease in cyclin B1 and Cdc2 levels. Abrogation of p21 or pRB function in cells containing wild-type p53 blocked the down-regulation of cyclin B1 and Cdc2 expression and led to an accelerated exit from G₂after genotoxic stress. Thus, similar to what occurs in p21 and p53 deficiency, pRB loss can uncouple S phase and mitosis after genotoxic stress in tumor cells. These results indicate that similar molecular mechanisms are required for p53 regulation of G₁ and G₂ checkpoints.

ACKNOWLEDGMENTS

Caroline D. Scatena and Luo Jia Tang contributed equally to this work.

We thank K. Cho for the RKO-NEO and RKO-E7 cells lines, B. Vogelstein for the isogenic set of HCT116 cells, J. Lee for the cyclin B1 promoter-CAT construct, the staff of the S. Hiebert laboratory for assistance with the EMSA, and E. Nishida and H. Piwnica-Worms for cyclin B1 and Cdc2 cDNAs, respectively. We thank the members of the Pietenpol laboratory for critical reading of the manuscript.

This work was supported by a Burroughs Wellcome New Investigator in Toxicology award (J.A.P.), NIH grant CA70856 (J.A.P.), NIEHS institutional training grant ES07028 (C.D.S.), NIH grant CA68485 (core services), and NIEHS grants ES07028 and ES00267 (core services).