Pharmacological activation of a novel p53-dependent S-phase checkpoint involving CHK-1

M Ashcroft,A Maya-Mendoza,J Yang,D A Jackson,A Ahmed

doi:10.1038/cddis.2011.42

Abstract

We have recently shown that induction of the p53 tumour suppressor protein by the small-molecule RITA (reactivation of p53 and induction of tumour cell apoptosis; 2,5-bis(5-hydroxymethyl-2-thienyl)furan) inhibits hypoxia-inducible factor-1α and vascular endothelial growth factor expression in vivo and induces p53-dependent tumour cell apoptosis in normoxia and hypoxia. Here, we demonstrate that RITA activates the canonical ataxia telangiectasia mutated/ataxia telangiectasia and Rad3-related DNA damage response pathway. Interestingly, phosphorylation of checkpoint kinase (CHK)-1 induced in response to RITA was influenced by p53 status. We found that induction of p53, phosphorylated CHK-1 and γH2AX proteins was significantly increased in S-phase. Furthermore, we found that RITA stalled replication fork elongation, prolonged S-phase progression and induced DNA damage in p53 positive cells. Although CHK-1 knockdown did not significantly affect p53-dependent DNA damage or apoptosis induced by RITA, it did block the ability for DNA integrity to be maintained during the immediate response to RITA. These data reveal the existence of a novel p53-dependent S-phase DNA maintenance checkpoint involving CHK-1.

Highlights

The p53 tumour suppressor protein is a potent negative regulator of hypoxia-inducible factor-1a (HIF-1a), mediating both apoptotic[3,4] and antiangiogenic effects when overexpressed.[5,6] HIF-1a accumulation in hypoxia is blocked by overexpression[5] or activation[6] of p53, and HIF-1-dependent transcription negatively correlates with p53 status.[7] p53 is mutated in about 50% of human cancers, and several agents have been described that can reactivate mutant[8,9] or activate wild-type p5310–12 in tumour cells
We demonstrate that RITA activates the canonical ataxia telangiectasia mutated/ataxia telangiectasia and Rad3-related (ATM/ataxia telangiectasia and Rad3 related (ATR)) DNA damage cascade and induces DNA damage in p53 positive cells
We have found that RITA can mediate significant tumour cell apoptosis in normoxia and hypoxia in a p53-dependent manner and activate a DNA damage response in vitro and in vivo.[15]

Summary

Introduction

The p53 tumour suppressor protein is a potent negative regulator of HIF-1a, mediating both apoptotic[3,4] and antiangiogenic effects when overexpressed.[5,6] HIF-1a accumulation in hypoxia is blocked by overexpression[5] or activation[6] of p53, and HIF-1-dependent transcription negatively correlates with p53 status.[7] p53 is mutated in about 50% of human cancers, and several agents have been described that can reactivate mutant[8,9] or activate wild-type p5310–12 in tumour cells. Our study identifies a novel p53-dependent S-phase checkpoint involving CHK-1

Methods

Results

Conclusion