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

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.

Chk2 mediates RITA-induced apoptosis

“…Supporting the observed effects on apoptosis, RITA induced PARP cleavage in p53 þ / þ cells, but not in p53 À/À and Chk2 À/À cells. As reported before, [9][10][11][12] RITA also induced a DNA damage response, evidenced by phosphorylation of ATM-Ser1981, KRAB-interacting protein 1 (KAP1)-Ser824, Chk2-Thr68, p53-Ser15 and p53-Ser46, as well as by downregulation of Hdmx. Interestingly, these effects were strongly attenuated in p53 À/À and Chk2 À/À cells.…”

“…Interestingly, these effects were strongly attenuated in p53 À/À and Chk2 À/À cells. In addition, Chk1-Ser345 phosphorylation, recently implicated in RITA response, 11 and Hdm2 downregulation were all p53 and Chk2 dependent (Figure 4b). It is important to note that not only stress-induced p53 phosphorylation is strongly reduced in Chk2 À/À cells but also p53 stabilization (Figure 4b).…”

“…11 To test whether this effect depends on Chk2 expression, we analyzed 5-ethynyl-2 0 -deoxyuridine (EdU) incorporation in HCT116 p53 þ / þ , p53 À/À and Chk2 À/À cells in a RITA time course (Figure 5a). In p53 þ / þ cells, 4-h RITA treatment already effectively blocked replication.…”

“…10 Importantly, several groups recently reported the induction of a DNA damage response by RITA. [9][10][11][12] The canonical DNA damage response network is traditionally divided into two major pathways, involving the sensor kinases ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3-related (ATR) that activate their respective effector kinases, Chk2 and Chk1. ATR phosphorylates Chk1 on Ser317 and Ser345.…”

“…Recently, RITA was proposed to induce p53-dependent replication stalling, with a role for Chk1 in maintaining DNA integrity, but not in the induction of apoptosis. 11 In this study, we focused on the role of Chk2 in the RITA response and found that Chk2 is essential for the induction of replication arrest and apoptosis by RITA.…”