Abstract
The related PIK-like kinases Ataxia-Telangiectasia Mutated (ATM) and ATM- and Rad3-related (ATR) play major roles in the regulation of cellular responses to DNA damage or replication stress. The pro-apoptotic role of ATM and p53 in response to ionizing radiation (IR) has been widely investigated. Much less is known about the control of apoptosis following DNA replication stress. Recent work indicates that Chk1, the downstream phosphorylation target of ATR, protects cells from apoptosis induced by DNA replication inhibitors as well as IR. The aim of the work reported here was to determine the roles of ATM- and ATR-protein kinase cascades in the control of apoptosis following replication stress and the relationship between Chk1-suppressed apoptotic pathways responding to replication stress or IR. ATM and ATR/Chk1 signalling pathways were manipulated using siRNA-mediated depletions or specific inhibitors in two tumour cell lines or fibroblasts derived from patients with inherited mutations. We show that depletion of ATM or its downstream phosphorylation targets, NBS1 and BID, has relatively little effect on apoptosis induced by DNA replication inhibitors, while ATR or Chk1 depletion strongly enhances cell death induced by such agents in all cells tested. Furthermore, early events occurring after the disruption of DNA replication (accumulation of RPA foci and RPA34 hyperphosphorylation) in ATR- or Chk1-depleted cells committed to apoptosis are not detected in ATM-depleted cells. Unlike the Chk1-suppressed pathway responding to IR, the replication stress-triggered apoptotic pathway did not require ATM and is characterized by activation of caspase 3 in both p53-proficient and -deficient cells. Taken together, our results show that the ATR-Chk1 signalling pathway plays a major role in the regulation of death in response to DNA replication stress and that the Chk1-suppressed pathway protecting cells from replication stress is clearly distinguishable from that protecting cells from IR.
Highlights
Cells respond to DNA damage by triggering cell cycle arrest, DNA repair, or death
We show that two genes play a major role in this process; our work suggests considerable complexity in this death response as different death pathways are triggered in response to different forms of DNA damage
We show that depletion or loss of Ataxia-Telangiectasia Mutated (ATM), NBS1 or BID has little or no significant effect on the induction of apoptosis in two human tumour cell lines or immortalized human fibroblasts treated with DNA replication inhibitors, even though depletion of ATR or Chk1 in these cells led to high levels of cell death
Summary
Cells respond to DNA damage by triggering cell cycle arrest, DNA repair, or death. The related PIK-like kinases ATM (AtaxiaTelangiectasia Mutated) and ATR (ATM- and Rad3-related) are major coordinators of this damage response [1]. ATM is central to the DNA double-strand break (DSB) response It delays DNA synthesis and the onset of mitosis following DSB induction by agents such as ionizing radiation (IR) through a complex signalling cascade that includes p53, Chk and NBS1 as phosphorylation targets [2,3,4]. This signalling cascade plays a major role in the onset of apoptosis following IR through the p53-mediated transcriptional activation of pro-apoptotic proteins such as BAX and PUMA [5,6,7]. Chk may be important for the ATM-independent pathway as mouse cells with knockouts of both Chk and ATM show levels of apoptosis similar to those found in p532/2 cells [9]
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