ATM kinase activity modulates cFLIP protein levels: potential interplay between DNA damage signalling and TRAIL-induced apoptosis

Ataxia telangiectasia (A-T) is a rare cancer-predisposing genetic disease, caused by the lack of functional ATM kinase, a major actor of the double strand brakes (DSB) DNA-damage response. A-T patients show a broad and diverse phenotype, which includes an increased rate of lymphoma and leukemia development. Fas-induced apoptosis plays a fundamental role in the homeostasis of the immune system and its defects have been associated with autoimmunity and lymphoma development. We therefore investigated the role of ATM kinase in Fas-induced apoptosis. Using A-T lymphoid cells, we could show that ATM deficiency causes resistance to Fas-induced apoptosis. A-T cells up-regulate FLIP protein levels, a well-known inhibitor of Fas-induced apoptosis. Reconstitution of ATM kinase activity was sufficient to decrease FLIP levels and to restore Fas sensitivity. Conversely, genetic and pharmacologic ATM kinase inactivation resulted in FLIP protein up-regulation and Fas resistance. Both ATM and FLIP are aberrantly regulated in Hodgkin lymphoma. Importantly, we found that reconstitution of ATM kinase activity decreases FLIP protein levels and restores Fas sensitivity in Hodgkin lymphoma–derived cells. Overall, these data identify a novel molecular mechanism through which ATM kinase may regulate the immune system homeostasis and impair lymphoma development.

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Correction of ATM mutations in iPS cells from two ataxia-telangiectasia patients restores DNA damage and oxidative stress responses

Human Molecular Genetics ◽

10.1093/hmg/ddaa023 ◽

2020 ◽

Vol 29 (6) ◽

pp. 990-1001 ◽

Cited By ~ 5

Author(s):

Dmitry A Ovchinnikov ◽

Sarah L Withey ◽

Hannah C Leeson ◽

U Wang Lei ◽

Ashmitha Sundarrajan ◽

...

Keyword(s):

Oxidative Stress ◽

Dna Damage ◽

Stress Responses ◽

Ataxia Telangiectasia ◽

Compound Heterozygous ◽

Gene Correction ◽

Atm Kinase ◽

Atm Protein ◽

Induced Apoptosis ◽

And Oxidative Stress

Abstract Patients with ataxia-telangiectasia (A-T) lack a functional ATM kinase protein and exhibit defective repair of DNA double-stranded breaks and response to oxidative stress. We show that CRISPR/Cas9-assisted gene correction combined with piggyBac (PB) transposon-mediated excision of the selection cassette enables seamless restoration of functional ATM alleles in induced pluripotent stem cells from an A-T patient carrying compound heterozygous exonic missense/frameshift mutations, and from a patient with a homozygous splicing acceptor mutation of an internal coding exon. We show that the correction of one allele restores expression of ~ 50% of full-length ATM protein and ameliorates DNA damage-induced activation (auto-phosphorylation) of ATM and phosphorylation of its downstream targets, KAP-1 and H2AX. Restoration of ATM function also normalizes radiosensitivity, mitochondrial ROS production and oxidative-stress-induced apoptosis levels in A-T iPSC lines, demonstrating that restoration of a single ATM allele is sufficient to rescue key ATM functions. Our data further show that despite the absence of a functional ATM kinase, homology-directed repair and seamless correction of a pathogenic ATM mutation is possible. The isogenic pairs of A-T and gene-corrected iPSCs described here constitute valuable tools for elucidating the role of ATM in ageing and A-T pathogenesis.

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Deregulation of DNA Damage Signal Transduction by Herpesvirus Latency-Associated M2

Journal of Virology ◽

10.1128/jvi.02732-05 ◽

2006 ◽

Vol 80 (12) ◽

pp. 5862-5874 ◽

Cited By ~ 41

Author(s):

Xiaozhen Liang ◽

Mary T. Pickering ◽

Nam-Hyuk Cho ◽

Heesoon Chang ◽

Michael R. Volkert ◽

...

Keyword(s):

Dna Damage ◽

Immune Surveillance ◽

Atm Kinase ◽

Kinase Activation ◽

Cycle Arrest ◽

Damage Response ◽

Gamma Herpesvirus ◽

Infected Cells ◽

Induced Apoptosis ◽

Host Immune Surveillance

ABSTRACT Infected cells recognize viral replication as a DNA damage stress and elicit a DNA damage response that ultimately induces apoptosis as part of host immune surveillance. Here, we demonstrate a novel mechanism where the murine gamma herpesvirus 68 (γHV68) latency-associated, anti-interferon M2 protein inhibits DNA damage-induced apoptosis by interacting with the DDB1/COP9/cullin repair complex and the ATM DNA damage signal transducer. M2 expression constitutively induced DDB1 nuclear localization and ATM kinase activation in the absence of DNA damage. Activated ATM subsequently induced Chk activation and p53 phosphorylation and stabilization without eliciting H2AX phosphorylation and MRN recruitment to foci upon DNA damage. Consequently, M2 expression inhibited DNA repair, rendered cells resistant to DNA damage-induced apoptosis, and induced a G1 cell cycle arrest. Our results suggest that γHV68 M2 blocks apoptosis-mediated intracellular innate immunity, which might ultimately contribute to its role in latent infection.

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Poly(ADP-ribose) polymerase-1 inhibits ATM kinase activity in DNA damage response

Biochemical and Biophysical Research Communications ◽

10.1016/j.bbrc.2004.05.031 ◽

2004 ◽

Vol 319 (2) ◽

pp. 596-602 ◽

Cited By ~ 17

Author(s):

Fumiaki Watanabe ◽

Hidesuke Fukazawa ◽

Mitsuko Masutani ◽

Hiroshi Suzuki ◽

Hirobumi Teraoka ◽

...

Keyword(s):

Dna Damage ◽

Dna Damage Response ◽

Kinase Activity ◽

Atm Kinase ◽

Damage Response

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Reduced ATM kinase activity and an attenuated p53 response to DNA damage in carcinogen-induced preneoplastic hepatic lesions in the rat

Carcinogenesis ◽

10.1093/carcin/22.12.2023 ◽

2001 ◽

Vol 22 (12) ◽

pp. 2023-2031 ◽

Cited By ~ 9

Author(s):

Ilona Silins ◽

Niklas Finnberg ◽

Annika Ståhl ◽

Johan Högberg ◽

Ulla Stenius

Keyword(s):

Dna Damage ◽

Kinase Activity ◽

Atm Kinase ◽

P53 Response ◽

Hepatic Lesions

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Role of P53 protein in activation of atm- and parp-mediated dna damage repair (DDR) pathways induced by topoisomerase type II inhibitors

Kazan medical journal ◽

10.17750/kmj2016-245 ◽

2016 ◽

Vol 97 (2) ◽

pp. 245-249

Author(s):

B R Ramazanov ◽

R R Khusnutdinov ◽

A R Galembikova ◽

P D Dunaev ◽

S V Boichuk

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Cell Death ◽

Cell Lines ◽

Kinase Activity ◽

P53 Protein ◽

Human Fibroblasts ◽

Repair System ◽

Type Ii ◽

Atm Kinase

Aim. To study the mechanisms of doxorubicin genotoxic effects in terms of poly-(ADP)-ribose-polymerase (PARP) and the ATM-kinase (Ataxia Telangiectasia Mutated) inhibition in cell lines with different p53 status.Methods. The study was conducted on BJ and BJp53DD human fibroblasts cell lines, cultured in DMEM medium supplemented with fetal bovine serum, L-glutamine and antibiotics. Inhibition of PARP and ATM-kinase activity was attained by adding synthetic inhibitors Nu1025 and Ku55933 respectively. Chemotherapy drug doxorubicin was used to induce deoxyribonucleic acid (DNA) damages. Cell viability analysis was performed using MTS-test. Repair system proteins and apoptotic markers expression was assessed by western blotting. Cells distribution by cell cycle phases was performed by flow cytometry.Results. Adding PARP and ATM-kinase inhibitors to the BJ p53DD cell line culture resulted in a significant reduction in the viable cells number amid DNA damage induction caused by doxorubicin. Cell death in these samples occurs according to the apoptosis mechanism, what was confirmed by the increase in hypodiploid cells number and increased expression of cleaved forms of PARP-1 and caspase-3. The above-described effects of the type II topoisomerase inhibitor doxorubicin were significantly higher in BJ fibroblasts line with non-functional p53 protein (p53DD) compared with conventional BJ human fibroblasts line.Conclusion. In the context of the failure of p53-dependent mechanisms of cell cycle regulation in BJ p53DD human fibroblasts, PARP and ATM-kinase activity inhibition leads to increased cell death by apoptosis mechanism induced by the doxorubicin action.

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Skin-Derived Precursors against UVB-Induced Apoptosis via Bcl-2 and Nrf2 Upregulation

BioMed Research International ◽

10.1155/2016/6894743 ◽

2016 ◽

Vol 2016 ◽

pp. 1-11 ◽

Cited By ~ 6

Author(s):

Jianqiao Zhong ◽

Li Li

Keyword(s):

Stem Cells ◽

Dna Damage ◽

Mrna Levels ◽

Uvb Radiation ◽

Critical Factors ◽

Ki67 Expression ◽

Protein Levels ◽

Qrt Pcr ◽

Ki67 Staining ◽

Induced Apoptosis

Bcl-2 and Nrf2 are critical factors in protecting cells against UVB-induced apoptosis. Hair-follicle-bulge stem cells could resist ionization through Bcl-2 upregulation. Skin-derived precursors (SKPs) dwelling on the bulge may be against UVB irradiation. Initially, SKPs were isolated and identified. Then, SKPs were exposed to UVB and grew in medium for 24 hours. CCK-8 assay, TUNEL, and Ki67 staining evaluated cells apoptosis/proliferation, while SA-βgal staining evaluated cells senescence and pH2AX immunostaining evaluated DNA damage. Meanwhile, Bcl-2, Nrf2, HO-1, Bax, and Bak expressions were assessed by qRT-PCR and western blot. Two weeks later, floating spheres appeared and were identified as SKPs. After UVB radiation, SKPs maintained spherical colonies and outnumbered unirradiated ones, showing high Ki67 expression and low TUNEL, SA-βgal, and pH2AX expression. Fibroblasts (FBs), however, displayed deformation, senescence, and reduction, with increased TUNEL, SA-βgal, and pH2AX expression. Moreover, Bcl-2 and Nrf2 mRNA expression were significantly higher than Bak and Bax in irradiated SKPs. Conversely, Bcl-2 and Nrf2 mRNA levels greatly decreased compared with Bax and Bak in irradiated FBs. Interestingly, SKPs showed higher protein levels of Bcl-2, Nrf2, and HO-1 than FBs. SKPs exert a beneficial effect on resisting UVB-induced apoptosis, which may be associated with Bcl-2 and Nrf2 upregulation.

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Autophosphorylation at serine 1981 stabilizes ATM at DNA damage sites

The Journal of Cell Biology ◽

10.1083/jcb.200906064 ◽

2009 ◽

Vol 187 (7) ◽

pp. 977-990 ◽

Cited By ~ 115

Author(s):

Sairei So ◽

Anthony J. Davis ◽

David J. Chen

Keyword(s):

Dna Damage ◽

Cellular Response ◽

Kinase Activity ◽

Critical Role ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Ataxia Telangiectasia Mutated ◽

Atm Kinase ◽

Strand Breaks ◽

Damage Response

Ataxia telangiectasia mutated (ATM) plays a critical role in the cellular response to DNA damage. In response to DNA double-strand breaks (DSBs), ATM is autophosphorylated at serine 1981. Although this autophosphorylation is widely considered a sign of ATM activation, it is still not clear if autophosphorylation is required for ATM functions including localization to DSBs and activation of ATM kinase activity. In this study, we show that localization of ATM to DSBs is differentially regulated with the initial localization requiring the MRE11–RAD50–NBS1 complex and sustained retention requiring autophosphorylation of ATM at serine 1981. Autophosphorylated ATM interacts with MDC1 and the latter is required for the prolonged association of ATM to DSBs. Ablation of ATM autophosphorylation or knock-down of MDC1 protein affects the ability of ATM to phosphorylate downstream substrates and confer radioresistance. Together, these data suggest that autophosphorylation at serine 1981 stabilizes ATM at the sites of DSBs, and this is required for a proper DNA damage response.

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p53 induces ARTS to promote mitochondrial apoptosis

Cell Death and Disease ◽

10.1038/s41419-021-03463-8 ◽

2021 ◽

Vol 12 (2) ◽

Author(s):

Qian Hao ◽

Jiaxiang Chen ◽

Junming Liao ◽

Yingdan Huang ◽

Yu Gan ◽

...

Keyword(s):

Dna Damage ◽

Target Gene ◽

Luciferase Reporter ◽

Mitochondrial Apoptosis ◽

Luciferase Reporter Gene ◽

Γ Irradiation ◽

Multiple Stress ◽

Protein Levels ◽

The Arts ◽

Induced Apoptosis

AbstractApoptosis related protein in TGF-β signaling pathway (ARTS) was originally discovered in cells undergoing apoptosis in response to TGF-β, but ARTS also acts downstream of many other apoptotic stimuli. ARTS induces apoptosis by antagonizing the anti-apoptotic proteins XIAP and Bcl-2. Here we identified the pro-apoptotic Sept4/ARTS gene as a p53-responsive target gene. Ectopic p53 and a variety of p53-inducing agents increased both mRNA and protein levels of ARTS, whereas ablation of p53 reduced ARTS expression in response to multiple stress conditions. Also, γ-irradiation induced p53-dependent ARTS expression in mice. Consistently, p53 binds to the responsive DNA element on the ARTS promoter and transcriptionally activated the promoter-driven expression of a luciferase reporter gene. Interestingly, ARTS binds to and sequesters p53 at mitochondria, enhancing the interaction of the latter with Bcl-XL. Ectopic ARTS markedly augments DNA damage stress- or Nutlin-3-triggered apoptosis, while ablation of ARTS preferentially impairs p53-induced apoptosis. Altogether, these findings demonstrate that ARTS collaborates with p53 in mitochondria-engaged apoptosis.

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Identification of ATM Mutations in Multiple Myeloma Tumours.

Blood ◽

10.1182/blood.v106.11.1562.1562 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1562-1562

Author(s):

Belinda Austen ◽

Maria Podinovskaia ◽

Jane Starcynski ◽

Giancarlo Barone ◽

Anne Reiman ◽

...

Keyword(s):

Multiple Myeloma ◽

Dna Damage ◽

Kinase Activity ◽

Plasma Cells ◽

Poor Response ◽

Primary Therapy ◽

Atm Kinase ◽

Sequence Change ◽

Atm Gene ◽

Atm Protein

Abstract Ataxia Telangiectasia (AT) patients have biallelic inactivation of the ATM gene and exhibit a 200 fold increased frequency of lymphoid tumours, commonly occurring at a young age. In AT families the relative risk for developing myeloma is 4.49 for heterozygous ATM carriers compared to normal controls, and multiple myeloma has been reported in an AT patient with an unusually mild form of the disease associated with prolonged survival. Normal plasma cells have high ATM expression similar to that seen in lymphocytes from the mantle zone of lymph node follicles. Although somatic ATM mutations have been found in a number of adult lymphoid malignancies including T-PLL, mantle cell lymphoma and CLL, there is no data on the occurrence of ATM mutations in multiple myeloma tumours. We screened multiple myeloma bone marrow aspirates for ATM expression by immunohistochemistry and for ATM mutations using denaturing high performance liquid chromatography analysis (HPLC). By immunohistochemistry, markedly reduced ATM expression was seen in the malignant plasma cells in 3 out of 45 tumours. These three tumours were analysed for mutations by HPLC but only one of the three patients had an identified mutation. In this tumour with the lowest ATM expression, the mutation 7181C/T (S2394L) was identified. We next modelled this mutation in an in vitro system to confirm that the S2394L substitution led to a form of ATM protein that had defective ATM kinase activity. We cloned the ATM gene, carrying the sequence change, into ATM negative cell lines and measured ATM kinase activity by assessing the phosphorylation of ATM targets following DNA damage with ionising irradiation. By this method, we were able to confirm that the S2394L sequence change resulted in a form of ATM protein that has absent kinase activity. A further 42 multiple myeloma tumours were analysed for ATM mutations by HPLC. We identified 4 more tumours that had mutations in the ATM gene, namely 8053T/A (S2685T), 6995T/C (L2332P), 4724G/A (R1575H) and IVS40-1G/C. Notably, the changes 8053T/A and 6995T/C are located within highly conserved ATM protein domains and the change IVS40-1G/C has been found in AT patients. Following DNA damage, deficiency of ATM leads to increased levels of unrepaired DNA breaks leading to genomic instability and also to defects in p53 dependent apoptosis. Loss of ATM activity through mutations may be contributing to multiple myeloma development and this idea is supported by the increased incidence of myeloma in AT carriers. ATM is essential for the activation of p53 dependent apoptosis in response to chemotherapy, and ATM mutations are associated with a poor outcome and chemoresistance in CLL patients. Four of the myeloma patients with ATM mutations in this study had aggressive disease including three with a poor response to primary therapy. In summary, we have shown for the first time that ATM mutations can occur at a low frequency in sporadic multiple myeloma tumours, identifying 5 patients in this study. Preliminary data suggests they may be associated with a poor response to treatment although this needs further confirmation.

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