title: Cytolethal distending toxin (CDT) is a radiomimetic agent and induces persistent levels of DNA double-strand breaks in human fibroblasts.
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.
authors.affiliation: Department of Toxicology, University Medical Center Mainz, Germany.
authors.forename: Jörg
authors.initials: J
authors.lastname: Fahrer
authors.affiliation: Department of Toxicology, University Medical Center Mainz, Germany.
authors.forename: Johannes
authors.initials: J
authors.lastname: Huelsenbeck
authors.affiliation: Department of Toxicology, University Medical Center Mainz, Germany.
authors.forename: Henriette
authors.initials: H
authors.lastname: Jaurich
authors.affiliation: Department of Toxicology, University Medical Center Mainz, Germany.
authors.forename: Bastian
authors.initials: B
authors.lastname: Dörsam
authors.affiliation: Department of Cell and Molecular Biology, Karolinska Institute, Stockholm, Sweden.
authors.forename: Teresa
authors.initials: T
authors.lastname: Frisan
authors.affiliation: Department of Toxicology, University Medical Center Mainz, Germany.
authors.forename: Marcus
authors.initials: M
authors.lastname: Eich
authors.affiliation: Department of Toxicology, University Medical Center Mainz, Germany.
authors.forename: Wynand P
authors.initials: WP
authors.lastname: Roos
authors.affiliation: Department of Toxicology, University Medical Center Mainz, Germany.
authors.forename: Bernd
authors.initials: B
authors.lastname: Kaina
authors.affiliation: Department of Toxicology, University Medical Center Mainz, Germany; Institute of Toxicology, Heinrich Heine University Düsseldorf, Germany. Electronic address: fritz@uni-duesseldorf.de.
authors.forename: Gerhard
authors.initials: G
authors.lastname: Fritz
mesh_terms: D064007:Ataxia Telangiectasia Mutated Proteins / Q000235:genetics / Q000378:metabolism; D001427:Bacterial Toxins / Q000494:pharmacology*; D002460:Cell Line; D000071877:Checkpoint Kinase 1; D064447:Checkpoint Kinase 2 / Q000378:metabolism; D053903:DNA Breaks, Double-Stranded; D059766:DNA End-Joining Repair / Q000187:drug effects* / Q000528:radiation effects*; D005347:Fibroblasts / Q000378:metabolism; D006657:Histones / Q000378:metabolism; D006801:Humans; D018613:Microscopy, Confocal; D011494:Protein Kinases / Q000378:metabolism; D011839:Radiation, Ionizing; D011994:Recombinant Proteins / Q000494:pharmacology; D011995:Recombination, Genetic; D013995:Time
chemical_list: D001427:Bacterial Toxins; C468783:H2AX protein, human; D006657:Histones; D011994:Recombinant Proteins; C067044:cytolethal distending toxin; D011494:Protein Kinases; D064447:Checkpoint Kinase 2; C576324:ATM protein, human; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C000605867:CHEK1 protein, human; C578634:CHEK2 protein, human; D000071877:Checkpoint Kinase 1
keywords: Cytolethal distending toxin; DNA damage response; DNA double-strand breaks; DNA repair; Gamma irradiation