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In cancer cells, the function of the tumor suppressor protein p53 is usually blocked. Impairment of the p53 pathway results in tumor cells with endogenous overexpression of Mdm2 via a naturally occurring single nucleotide polymorphism (SNP) in the mdm2 gene at position 309. Here we report that in mdm2 SNP309 cells, inactivation of p53 results in a chromatin-associated Mdm2-p53 complex without clearance of p53 by protein degradation. Nuclear accumulation of p53 protein in mdm2 SNP309 cells results after 6 h of camptothecin, etoposide, or mitomycin C treatment, with the p53 protein phosphorylated at Ser15. Chromatin immunoprecipitation demonstrated p53 and Mdm2 bound to p53 responsive elements. Interestingly, although the p53 protein was able to bind to DNA, quantitative PCR showed compromised transcription of endogenous target genes. Additionally, exogenously introduced p53 was incapable of activating transcription from p53 responsive elements in SNP309 cells, confirming the trans-acting nature of the inhibitor. Inhibition of Mdm2 by siRNA resulted in transcriptional activation of these p53 targets. Our data suggest that overproduction of Mdm2, resulting from a naturally occurring SNP, inhibits chromatin-bound p53 from activating the transcription of its target genes.

Original publication

DOI

10.1074/jbc.M505203200

Type

Journal article

Journal

J Biol Chem

Publication Date

22/07/2005

Volume

280

Pages

26776 - 26787

Keywords

Antineoplastic Agents, Cell Line, Tumor, Chromatin, Homozygote, Humans, Nuclear Proteins, Phosphorylation, Polymorphism, Single Nucleotide, Protein Binding, Proto-Oncogene Proteins, Proto-Oncogene Proteins c-mdm2, Transcriptional Activation, Tumor Suppressor Protein p53