Mdm2 Is Required for Inhibition of Cdk2 Activity by p21, Thereby Contributing to p53-Dependent Cell Cycle Arrest

Abstract

p53 is extensively posttranslationally modified in response to various types of cellular stress. Such modifications have been implicated in the regulation of p53 protein levels as well as its DNA binding and transcriptional activities. Treatment of cells with doxorubicin causes phosphorylation and acetylation of p53, transcriptional upregulation of p21 and other target genes, and growth arrest. In contrast, downregulation of Mdm2 by a small interfering RNA (siRNA) approach led to increased levels of p53 lacking phosphorylation at serine 15 and acetylation at lysine 382. Levels of binding of p53 to the p21 promoter were comparable following treatment with doxorubicin or Mdm2 siRNA. Moreover, p53 was transcriptionally active and capable of inducing or repressing a variety of its target genes. Surprisingly, p53 upregulated by Mdm2 siRNA had no effect on cell cycle progression. Although comparable in level to that achieved by treatment with the p53 activators actinomycin D and nutlin-3, the increases in p53 and p21 after downregulation of Mdm2 were not sufficient to trigger cell cycle arrest. This version of p21 was capable of interacting with cyclin-dependent kinase 2 (Cdk2) but failed to inhibit its activity. Taken together, these results argue that Mdm2 is needed for full inhibition of Cdk2 activity by p21, thereby positively contributing to p53-dependent cell cycle arrest.

We thank A. W. Lin (Roswell Park Cancer Institute) for the p21 shRNA stable cell line and G. Lozano (M. D. Anderson Cancer Center) for the p53-null mouse embryo fibroblasts. We are grateful to Matthew O'Connell and Zhen-Qiang Pan for their technical advice and helpful discussions and for sharing reagents. Present and past members of the Manfredi laboratory Lois Resnick-Silverman, Dana Lukin, Anthony Mastropietro, Selvon St. Clair, Wendy Liu, Sejal Patel, Shohreh Varmeh-Ziaie, Pierre-Jacques Hamard, Luis Carvajal, and Kester Haye are thanked for their help and support. Quantitative PCR was performed at the Mount Sinai shared research facility.

These studies were supported by a grant from the National Cancer Institute (R01 CA86001).