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Cytolethal distending toxin (CDT) is a radiomimetic agent and induces persistent levels of DNA double-strand breaks in human fibroblasts
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2014 (English)In: DNA Repair, ISSN 1568-7864, E-ISSN 1568-7856, Vol. 18, p. 31-43Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
Elsevier, 2014. Vol. 18, p. 31-43
Keywords [en]
Cytolethal distending toxin, DNA damage response, DNA double-strand breaks, DNA repair, Gamma irradiation
National Category
Cell and Molecular Biology Cell Biology Microbiology Genetics
Identifiers
URN: urn:nbn:se:umu:diva-165139DOI: 10.1016/j.dnarep.2014.03.002ISI: 000337658600004PubMedID: 24680221OAI: oai:DiVA.org:umu-165139DiVA, id: diva2:1369406
Available from: 2019-11-12 Created: 2019-11-12 Last updated: 2019-11-12Bibliographically approved

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Frisan, Teresa

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