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DNA-PKcs deficiency leads to persistence of oxidatively induced clustered DNA lesions in human tumor cells
Authors:Prakash Peddi  Charles W Loftin  Jennifer S Dickey  Jessica M Hair  Kara J Burns  Khaled Aziz  Dave C Francisco  Mihalis I Panayiotidis  Olga A Sedelnikova  William M Bonner  Thomas A Winters  Alexandros G Georgakilas
Institution:1. Department of Biology, Thomas Harriot College of Arts and Sciences, East Carolina University, Greenville, NC 27858, USA;2. Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20895, USA;3. Department of Pathology, Medical School, University of Ioannina, University Campus, 45110 Ioannina, Greece;4. Nuclear Medicine Department, Warren Grant Magnuson Clinical Center, National Institutes of Health, Bethesda, MD 20892, USA;1. Department of Animal Physiology and Ecotoxicology, University of Silesia, Bankowa 9, PL 40-007 Katowice, Poland;2. Department of Physiology in Zabrze, Medical University of Silesia, Jordana 19, PL 41-808 Zabrze, Poland;3. Department of Internal Medicine, Sealy Center for Molecular Medicine, University of Texas Medical Branch, Galveston, Texas 77555;4. Departments of Neurology and Neuroscience and Cell Biology, University of Texas Medical Branch, Galveston, Texas 77555;8. Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, Texas 77555;5. Department of Transgenic Mouse Core Facility, University of Texas Medical Branch, Galveston, Texas 77555;6. Department of Radiation Oncology, Houston Methodist Research Institute, Houston, Texas 77030;12. Department of Cancer and DNA Damage Responses, Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720;1. Division of Rheumatology, Department of Medicine, Toronto Western Hospital, University of Toronto, Ground Floor, East Wing, 399 Bathurst Street, Toronto, Ontario, Canada M5T 2S8;2. Division of Rheumatology, Department of Medicine, Mount Sinai Hospital, University of Toronto, Toronto, Ontario, Canada;3. Institute of Health Policy, Management and Evaluation, University of Toronto, Toronto, Ontario, Canada;4. Auckland City Hospital, Auckland, New Zealand;5. Department of Rheumatic Diseases, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands;6. Rheumatology Centre, Sint Maartenskliniek, Nijmegen, The Netherlands;7. Division of Rheumatology, Department of Medicine, St Joseph Health Care, University of Western Ontario, London, Ontario, Canada;8. Division of Rheumatology, Department of Medicine, Jewish General Hospital, McGill University, Montreal, Quebec, Canada;9. Rheumatology Department, University of Basel, Basel, Switzerland;10. Department of Rheumatology AVC, University of Florence, Firenze, Italy;11. Department of BioMedicine, University of Florence, Firenze, Italy;12. Division of Rheumatology AOUC, Department of Medicine & Denothecentre, University of Florence, Firenze, Italy;13. Centre for Rheumatology and Connective Tissue Diseases, Royal Free Hospital, London, UK;14. Department of Rheumatology, University Hospital Zurich, Zurich, Switzerland;15. Dipartimento di Scienze Cliniche e Molecolari, Clinica Medica, Università Politecnica delle Marche, Ancona, Italy;p. Musculoskeletal Research Group, Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, UK;q. The University of Texas Health Science Center at Houston, Houston, TX, USA;r. Division of Rheumatology, Clinical Immunology and Allergy, Department of Medicine, Georgetown University School of Medicine, USA;s. Scleroderma Research Consultants, Avon, CT, USA;t. Department of Medicine, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA;u. Division of Rheumatology and Clinical Immunology, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA;v. Servicio de Reumatología, Hospital Universitario 12 de Octubre, Madrid, Spain;w. Department of Rheumatology, German Rheumatology Research Center, Leibniz Institute, Berlin, Germany;x. Division of Immunology and Rheumatology, Department of Medicine, Stanford University, Stanford, CA, USA;y. Department of Dermatology, Stanford University, Stanford, CA, USA;z. Division of Clinical Immunology and Rheumatology, The University of Alabama at Birmingham, Birmingham, AL, USA;11. Division of Rheumatology, The University of Pennsylvania, Philadelphia, PA, USA;22. Division of Rheumatology & Immunology, Department of Medicine, Medical University of South Carolina, SC, USA;33. Division of Rheumatology, Northwestern University Feinberg School of Medicine, Chicago, IL, USA;44. Rheumatology A Department, Paris Descartes University, Cochin Hospital, France;55. Department of Rheumatology and Clinical Immunology, Justus-Liebig University Giessen, Kerckhoff Clinic, Bad Nauheim, Germany;66. Scleroderma Program, Division of Rheumatology, University of Michigan, Ann Arbor, MI, USA;1. Software Engineering Department, Jordan University of Science and Technology, Irbid, Jordan;2. Faculty of Information Technology, Benghazi University, Libya
Abstract:DNA-dependent protein kinase (DNA-PK) is a key non-homologous-end-joining (NHEJ) nuclear serine/threonine protein kinase involved in various DNA metabolic and damage signaling pathways contributing to the maintenance of genomic stability and prevention of cancer. To examine the role of DNA-PK in processing of non-DSB clustered DNA damage, we have used three models of DNA-PK deficiency, i.e., chemical inactivation of its kinase activity by the novel inhibitors IC86621 and NU7026, knockdown and complete absence of the protein in human breast cancer (MCF-7) and glioblastoma cell lines (MO59-J/K). A compromised DNA-PK repair pathway led to the accumulation of clustered DNA lesions induced by γ-rays. Tumor cells lacking protein expression or with inhibited kinase activity showed a marked decrease in their ability to process oxidatively induced non-DSB clustered DNA lesions measured using a modified version of pulsed-field gel electrophoresis or single-cell gel electrophoresis (comet assay). In all cases, DNA-PK inactivation led to a higher level of lesion persistence even after 24–72 h of repair. We suggest a model in which DNA-PK deficiency affects the processing of these clusters first by compromising base excision repair and second by the presence of catalytically inactive DNA-PK inhibiting the efficient processing of these lesions owing to the failure of DNA-PK to disassociate from the DNA ends. The information rendered will be important for understanding not only cancer etiology in the presence of an NHEJ deficiency but also cancer treatments based on the induction of oxidative stress and inhibition of cluster repair.
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