A Chromatin-associated and Transcriptionally Inactive p53-Mdm2 Complex Occurs in mdm2 SNP309 Homozygous Cells

Nicoleta C Arva,Tamara R Gopen,Kathryn E Talbott,Latoya E Campbell,Agustin Chicas,David E White,Gareth L Bond,Arnold J Levine,Jill Bargonetti

doi:10.1074/jbc.m505203200

Abstract

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.

Highlights

In cancer cells, the function of the tumor suppressor protein p53 is usually blocked
Endogenous Overexpression of Mdm2 via a Naturally Occurring single nucleotide polymorphism (SNP) Inhibits Apoptosis following Chemotherapeutic Drug Treatment—We previously documented induced p53 stabilization and subsequent apoptosis in ML-1 cells treated for 6 h with CPT, ETOP, or mitomycin C (MC), and no apoptosis induction in the p53deficient human cell line K562 treated with the same drugs [21]
The drugs CPT, ETOP, and MC increased the level of p53 in ML-1 cells as well as in MANCA and A875 cells (Fig. 1B, lanes 1–12), whereas no p53 was expressed in the p53-negative control cell line K562 (Fig. 1B, lanes 13–16)

Summary

Introduction

The function of the tumor suppressor protein p53 is usually blocked. Impairment of the p53 pathway results in tumor cells with endogenous overexpression of Mdm via a naturally occurring single nucleotide polymorphism (SNP) in the mdm gene at position 309. Our data suggest that overproduction of Mdm, resulting from a naturally occurring SNP, inhibits chromatin-bound p53 from activating the transcription of its target genes. In doing so we found that p53 in cell lines homozygous for SNP309 could be significantly stabilized after 6 h of DNA damage treatment with different drugs This p53 remained associated with the cellular Mdm protein in the nucleus and was not sequestered to the nucleolus. This p53 bound to chromatin in conjunction with Mdm and was unable to activate transcription of downstream target genes. We addressed the inhibitory actions of Mdm by showing that a DNA-bound form of p53 is blocked for trans-activation activity in the presence of high levels of Mdm

Methods

Results

Conclusion