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

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.     

DNA damage checkpoints in stem cells, ageing and cancer

DNA damage induces cell-intrinsic checkpoints, including p53 and retinoblastoma (RB), as well as upstream regulators (exonuclease 1 (EXO1), ataxia telangiectasia mutated (ATM), ATR, p16(INK4a) and p19(ARF)) and downstream targets (p21, PUMA (p53 upregulated modulator of apoptosis) and sestrins). Clearance of damaged cells by cell-intrinsic checkpoints suppresses carcinogenesis but as a downside may impair stem cell and tissue maintenance during ageing. Modulating the activity of DNA damage checkpoints can either accelerate or decelerate tissue ageing and age-related carcinogenesis. The outcome depends on cell-intrinsic and cell-extrinsic mechanisms that regulate the clearance of damaged cells and on the molecular context in ageing tissues, including the level of DNA damage accumulation itself.     

A dynamic model for the p53 stress response networks under ion radiation

Summary. P53 controls the cell cycle arrest and cell apoptosis through interaction with the downstream genes and their signal pathways. To stimulate the investigation into the complicated responses of p53 under the circumstance of ion radiation (IR) in the cellular level, a dynamic model for the p53 stress response networks is proposed. The model can be successfully used to simulate the dynamic processes of generating the double-strand breaks (DSBs) and their repairing, ataxia telangiectasia mutated (ATM) activation, as well as the oscillations occurring in the p53-MDM2 feedback loop.     

DNA replication stress in CHK1-depleted tumour cells triggers premature (S-phase) mitosis through inappropriate activation of Aurora kinase B

The disruption of DNA replication in cells triggers checkpoint responses that slow-down S-phase progression and protect replication fork integrity. These checkpoints are also determinants of cell fate and can help maintain cell viability or trigger cell death pathways. CHK1 has a pivotal role in such S-phase responses. It helps maintain fork integrity during replication stress and protects cells from several catastrophic fates including premature mitosis, premature chromosome condensation and apoptosis. Here we investigated the role of CHK1 in protecting cancer cells from premature mitosis and apoptosis. We show that premature mitosis (characterized by the induction of histone H3 phosphorylation, aberrant chromatin condensation, and persistent RPA foci in arrested S-phase cells) is induced in p53-deficient tumour cells depleted of CHK1 when DNA synthesis is disrupted. These events are accompanied by an activation of Aurora kinase B in S-phase cells that is essential for histone H3 Ser10 phosphorylation. Histone H3 phosphorylation precedes the induction of apoptosis in p53−/− tumour cell lines but does not appear to be required for this fate as an Aurora kinase inhibitor suppresses phosphorylation of both Aurora B and histone H3 but has little effect on cell death. In contrast, only a small fraction of p53+/+ tumour cells shows this premature mitotic response, although they undergo a more rapid and robust apoptotic response. Taken together, our results suggest a novel role for CHK1 in the control of Aurora B activation during DNA replication stress and support the idea that premature mitosis is a distinct cell fate triggered by the disruption of DNA replication when CHK1 function is suppressed.     

CHK2‐independent induction of telomere dysfunction checkpoints in stem and progenitor cells

Telomere shortening limits the proliferation of primary human fibroblasts by the induction of senescence, which is mediated by ataxia telangiectasia mutated‐dependent activation of p53. Here, we show that CHK2 deletion impairs the induction of senescence in mouse and human fibroblasts. By contrast, CHK2 deletion did not improve the stem‐cell function, organ maintenance and lifespan of telomere dysfunctional mice and did not prevent the induction of p53/p21, apoptosis and cell‐cycle arrest in telomere dysfunctional progenitor cells. Together, these results indicate that CHK2 mediates the induction of senescence in fibroblasts, but is dispensable for the induction of telomere dysfunction checkpoints at the stem and progenitor cell level in vivo.     

HERC2/USP20 coordinates CHK1 activation by modulating CLASPIN stability

CLASPIN is an essential mediator in the DNA replication checkpoint, responsible for ATR (ataxia telangiectasia and Rad3-related protein)-dependent activation of CHK1 (checkpoint kinase 1). Here we found a dynamic signaling pathway that regulates CLASPIN turn over. Under unperturbed conditions, the E3 ubiquitin ligase HERC2 regulates the stability of the deubiquitinating enzyme USP20 by promoting ubiquitination-mediated proteasomal degradation. Under replication stress, ATR-mediated phosphorylation of USP20 results in the disassociation of HERC2 from USP20. USP20 in turn deubiquitinates K48-linked-polyubiquitinated CLASPIN, stabilizing CLASPIN and ultimately promoting CHK1 phosphorylation and CHK1-directed checkpoint activation. Inhibition of USP20 expression promotes chromosome instability and xenograft tumor growth. Taken together, our findings demonstrated a novel function of HERC2/USP20 in coordinating CHK1 activation by modulating CLASPIN stability, which ultimately promotes genome stability and suppresses tumor growth.     

An essential function for NBS1 in the prevention of ataxia and cerebellar defects

Nijmegen breakage syndrome (NBS), ataxia telangiectasia and ataxia telangiectasia-like disorder (ATLD) show overlapping phenotypes such as growth retardation, microcephaly, cerebellar developmental defects and ataxia. However, the molecular pathogenesis of these neurological defects remains elusive. Here we show that inactivation of the Nbn gene (also known as Nbs1) in mouse neural tissues results in a combination of the neurological anomalies characteristic of NBS, ataxia telangiectasia and ATLD, including microcephaly, growth retardation, cerebellar defects and ataxia. Loss of Nbn causes proliferation arrest of granule cell progenitors and apoptosis of postmitotic neurons in the cerebellum. Furthermore, Nbn-deficient neuroprogenitors show proliferation defects (but not increased apoptosis) and contain more chromosomal breaks, which are accompanied by ataxia telangiectasia mutated protein (ATM)-mediated p53 activation. Notably, depletion of p53 substantially rescues the neurological defects of Nbn mutant mice. This study gives insight into the physiological function of NBS1 (the Nbn gene product) and the function of the DNA damage response in the neurological anomalies of NBS, ataxia telangiectasia and ATLD.     

FBH1 promotes DNA double-strand breakage and apoptosis in response to DNA replication stress

Proper resolution of stalled replication forks is essential for genome stability. Purification of FBH1, a UvrD DNA helicase, identified a physical interaction with replication protein A (RPA), the major cellular single-stranded DNA (ssDNA)–binding protein complex. Compared with control cells, FBH1-depleted cells responded to replication stress with considerably fewer double-strand breaks (DSBs), a dramatic reduction in the activation of ATM and DNA-PK and phosphorylation of RPA2 and p53, and a significantly increased rate of survival. A minor decrease in ssDNA levels was also observed. All these phenotypes were rescued by wild-type FBH1, but not a FBH1 mutant lacking helicase activity. FBH1 depletion had no effect on other forms of genotoxic stress in which DSBs form by means that do not require ssDNA intermediates. In response to catastrophic genotoxic stress, apoptosis prevents the persistence and propagation of DNA lesions. Our findings show that FBH1 helicase activity is required for the efficient induction of DSBs and apoptosis specifically in response to DNA replication stress.     

Cytosolic DNA triggers mitochondrial apoptosis via DNA damage signaling proteins independently of AIM2 and RNA polymerase III

A key host response to limit microbial spread is the induction of cell death when foreign nucleic acids are sensed within infected cells. In mouse macrophages, transfected DNA or infection with modified vaccinia virus Ankara (MVA) can trigger cell death via the absent in melanoma 2 (AIM2) inflammasome. In this article, we show that nonmyeloid human cell types lacking a functional AIM2 inflammasome still die in response to cytosolic delivery of different DNAs or infection with MVA. This cell death induced by foreign DNA is independent of caspase-8 and carries features of mitochondrial apoptosis: dependence on BAX, APAF-1, and caspase-9. Although it does not require the IFN pathway known to be triggered by infection with MVA or transfected DNA via polymerase III and retinoid acid-induced gene I-like helicases, it shows a strong dependence on components of the DNA damage signaling pathway: cytosolic delivery of DNA or infection with MVA leads to phosphorylation of p53 (serines 15 and 46) and autophosphorylation of ataxia telangiectasia mutated (ATM); depleting p53 or ATM with small interfering RNA or inhibiting the ATM/ATM-related kinase family by caffeine strongly reduces apoptosis. Taken together, our findings suggest that a pathway activating DNA damage signaling plays an important independent role in detecting intracellular foreign DNA, thereby complementing the induction of IFN and activation of the AIM2 inflammasome.     

Human cytomegalovirus disrupts both ataxia telangiectasia mutated protein (ATM)- and ATM-Rad3-related kinase-mediated DNA damage responses during lytic infection

Many viruses (herpes simplex virus type 1, polyomavirus, and human immunodeficiency virus type 1) require the activation of ataxia telangiectasia mutated protein (ATM) and/or Mre11 for a fully permissive infection. However, the longer life cycle of human cytomegalovirus (HCMV) may require more specific interactions with the DNA repair machinery to maximize viral replication. A prototypical damage response to the double-stranded ends of the incoming linear viral DNA was not observed in fibroblasts at early times postinfection (p.i.). Apparently, a constant low level of phosphorylated ATM was enough to phosphorylate its downstream targets, p53 and Nbs1. p53 was the only cellular protein observed to relocate at early times, forming foci in infected cell nuclei between 3.5 and 5.5 h p.i. Approximately half of these foci localized with input viral DNA, and all localized with viral UL112/113 prereplication site foci. No other DNA repair proteins localized with the virus or prereplication foci in the first 24 h p.i. When viral replication began in earnest, between 24 and 48 h p.i., there were large increases in steady-state levels and phosphorylation of many proteins involved in the damage response, presumably triggered by ATM-Rad3-related kinase activation. However, a sieving process occurred in which only certain proteins were specifically sequestered into viral replication centers and others were particularly excluded. In contrast to other viruses, activation of a damage response is neither necessary nor detrimental to infection, as neither ATM nor Mre11 was required for full virus replication and production. Thus, by preventing simultaneous relocalization of all the necessary repair components to the replication centers, HCMV subverts full activation and completion of both double-stranded break and S-phase checkpoints that should arrest all replication within the cell and likely lead to apoptosis.     

BID binds to replication protein A and stimulates ATR function following replicative stress

Proapoptotic BH3-interacting death domain agonist (BID) regulates apoptosis and the DNA damage response. Following replicative stress, BID associates with proteins of the DNA damage sensor complex, including replication protein A (RPA), ataxia telangiectasia and Rad3 related (ATR), and ATR-interacting protein (ATRIP), and facilitates an efficient DNA damage response. We have found that BID stimulates the association of RPA with components of the DNA damage sensor complex through interaction with the basic cleft of the N-terminal domain of the RPA70 subunit. Disruption of the BID-RPA interaction impairs the association of ATR-ATRIP with chromatin as well as ATR function, as measured by CHK1 activation and recovery of DNA replication following hydroxyurea (HU). We further demonstrate that the association of BID with RPA stimulates the association of ATR-ATRIP to the DNA damage sensor complex. We propose a model in which BID associates with RPA and stimulates the recruitment and/or stabilization of ATR-ATRIP to the DNA damage sensor complex.     

A DR4:tBID axis drives the p53 apoptotic response by promoting oligomerization of poised BAX

The cellular response to p53 activation varies greatly in a stimulus- and cell type-specific manner. Dissecting the molecular mechanisms defining these cell fate choices will assist the development of effective p53-based cancer therapies and also illuminate fundamental processes by which gene networks control cellular behaviour. Using an experimental system wherein stimulus-specific p53 responses are elicited by non-genotoxic versus genotoxic agents, we discovered a novel mechanism that determines whether cells undergo proliferation arrest or cell death. Strikingly, we observe that key mediators of cell-cycle arrest (p21, 14-3-3σ) and apoptosis (PUMA, BAX) are equally activated regardless of outcome. In fact, arresting cells display strong translocation of PUMA and BAX to the mitochondria, yet fail to release cytochrome C or activate caspases. Surprisingly, the key differential events in apoptotic cells are p53-dependent activation of the DR4 death receptor pathway, caspase 8-mediated cleavage of BID, and BID-dependent activation of poised BAX at the mitochondria. These results reveal a previously unappreciated role for DR4 and the extrinsic apoptotic pathway in cell fate choice following p53 activation.     

53BP1 deficiency combined with telomere dysfunction activates ATR-dependent DNA damage response

TRF1 protects mammalian telomeres from fusion and fragility. Depletion of TRF1 leads to telomere fusions as well as accumulation of γ-H2AX foci and activation of both the ataxia telangiectasia mutated (ATM)- and the ataxia telangiectasia and Rad3 related (ATR)-mediated deoxyribonucleic acid (DNA) damage response (DDR) pathways. 53BP1, which is also present at dysfunctional telomeres, is a target of ATM that accumulates at DNA double-strand breaks and favors nonhomologous end-joining (NHEJ) repair over ATM-dependent resection and homology-directed repair (homologous recombination [HR]). To address the role of 53BP1 at dysfunctional telomeres, we generated mice lacking TRF1 and 53BP1. 53BP1 deficiency significantly rescued telomere fusions in mouse embryonic fibroblasts (MEFs) lacking TRF1, but they showed evidence of a switch from the NHEJ- to HR-mediated repair of uncapped telomeres. Concomitantly, double-mutant MEFs showed evidence of hyperactivation of the ATR-dependent DDR. In intact mice, combined 53BP1/TRF1 deficiency in stratified epithelia resulted in earlier onset of DNA damage and increased CHK1 phosphorylation during embryonic development, leading to aggravation of skin phenotypes.