ATR and Chk1 Suppress a Caspase-3–Dependent Apoptotic Response Following DNA Replication Stress
Open Access
- 2 January 2009
- journal article
- research article
- Published by Public Library of Science (PLoS) in PLoS Genetics
- Vol. 5 (1), e1000324
- https://doi.org/10.1371/journal.pgen.1000324
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. The integrity of the genetic information in cells is protected by elaborate mechanisms that ensure that an accurate DNA copy is passed from generation to generation. These mechanisms repair errors in DNA sequence or stop growth if DNA structure is compromised. However, if the level of DNA damage is too severe, cells may also respond by inducing death rather than attempt repair. Relatively little is known about how cells decide whether to repair damage or commit to death. The purpose of our work was to identify genes that control this decision-making process while cells are duplicating DNA. We show that two genes play a major role in this process; however, our work also suggests considerable complexity in this death response as different death pathways are triggered in response to different forms of DNA damage. Since DNA replication inhibitors are used widely in the treatment of cancer, our work may enable us to more effectively kill cancer cells in treatment protocols employing these agents.Keywords
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