Cytolethal distending toxin (CDT) is a radiomimetic agent and induces persistent levels of DNA double-strand breaks in human fibroblasts |
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| Institution: | 1. Department of Toxicology, University Medical Center Mainz, Germany;2. Department of Cell and Molecular Biology, Karolinska Institute, Stockholm, Sweden;3. Institute of Toxicology, Heinrich Heine University Düsseldorf, Germany;1. Median Unit, University of Reims Champagne-Ardenne, Centre National de la Recherche Scientifique, Matrice Extracellulaire et Dynamique Cellulaire, Reims, France;2. ArchimMed SARL, Jouy en Josas, France;3. Department of Pathology, Lariboisiere Hospital, Assistance Publique-Hôpitaux de Paris, Paris, France;4. Research Center for Interventional Imagery, Assistance Publique-Hôpitaux de Paris and National Institute for Agricultural Research, Jouy-En-Josas, France;5. Department of Neuroradiology, Hôpital Lariboisère, Assistance Publique-Hôpitaux de Paris, Paris, France;6. Materials and Complex Systems Laboratory, Centre National de la Recherche Scientifique, Paris, France;1. Department of Biochemistry and Molecular Biology, Harbin Medical University, Harbin, China;2. Department of General Surgery, The Second Affiliated Hospital of Harbin Medical University, Harbin, China;3. Department of Respiratory Medical Oncology, The Affiliated Tumor Hospital of Harbin Medical University, Harbin, China;4. Translational Medicine Research and Cooperation Center of Northern China, Heilongjiang Academy of Medicine Sciences, Harbin, China;1. Institute of Virology, Hunan Agricultural University, Changsha, Hunan 410128, China;2. Hunan Provincial Key Laboratory for Biology and Control of Plant Diseases and Insect Pests, Hunan Agricultural University, Changsha, Hunan 410128, China;3. Department of Microbiology, Miami University, 32 Pearson Hall, Oxford, OH 45056, USA;1. Harvard Center for Population and Development Studies, Harvard University, Cambridge, MA 02138, USA;3. Institute for Molecular Bioscience, Division of Chemistry and Structural Biology, University of Queensland, St. Lucia, Queensland 4067, Australia;4. School of Chemistry and Molecular Biosciences, University of Queensland, St. Lucia, Queensland 4067, Australia |
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| Abstract: | Cytolethal distending toxin (CDT) is a unique genotoxin produced by several pathogenic bacteria. The tripartite protein toxin is internalized into mammalian cells via endocytosis followed by retrograde transport to the ER. Upon translocation into the nucleus, CDT catalyzes the formation of DNA double-strand breaks (DSBs) due to its intrinsic endonuclease activity. In the present study, we compared the DNA damage response (DDR) in human fibroblasts triggered by recombinant CDT to that of ionizing radiation (IR), a well-known DSB inducer. Furthermore, we dissected the pathways involved in the detection and repair of CDT-induced DNA lesions. qRT-PCR array-based mRNA and western blot analyses showed a partial overlap in the DDR pattern elicited by CDT and IR, with strong activation of both the ATM-Chk2 and the ATR-Chk1 axis. In line with its in vitro DNase I-like activity on plasmid DNA, neutral and alkaline Comet assay revealed predominant induction of DSBs in CDT-treated fibroblasts, whereas irradiation of cells generated higher amounts of SSBs and alkali-labile sites. Using confocal microscopy, the dynamics of the DSB surrogate marker γ-H2AX was monitored after pulse treatment with CDT or IR. In contrast to the fast induction and disappearance of γ-H2AX-foci observed in irradiated cells, the number of γ-H2AX-foci induced by CDT were formed with a delay and persisted. 53BP1 foci were also generated following CDT treatment and co-localized with γ-H2AX foci. We further demonstrated that ATM-deficient cells are very sensitive to CDT-induced DNA damage as reflected by increased cell death rates with concomitant cleavage of caspase-3 and PARP-1. Finally, we provided novel evidence that both homologous recombination (HR) and non-homologous end joining (NHEJ) protect against CDT-elicited DSBs. In conclusion, the findings suggest that CDT functions as a radiomimetic agent and, therefore, is an attractive tool for selectively inducing persistent levels of DSBs and unveiling the associated cellular responses. |
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| Keywords: | Cytolethal distending toxin DNA double-strand breaks DNA damage response DNA repair Gamma irradiation |
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