Cytolethal distending toxin (CDT) is a radiomimetic agent and induces, persistent levels of DNA.double-strand breaks in human fibroblasts
Cytolethal distending toxin (CDT) is a radiomimetic agent and induces, persistent levels of DNA.double-strand breaks in human fibroblasts
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DOI：
10.1016/j.dnarep.2014.03.002
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发表时间：
2014-06-01
期刊：
影响因子：
3.8
通讯作者：
Fritz, Gerhard
中科院分区：
文献类型：
--
作者：
Fahrer, Joerg;Huelsenbeck, Johannes;Fritz, Gerhard
细胞致死性膨胀毒素（CDT）是由多种病原菌产生的一种独特的基因毒素。三联蛋白毒素通过内吞作用内化到哺乳动物细胞中，然后逆行转运到ER。在易位进入细胞核后，CDT由于其固有的核酸内切酶活性而催化DNA双链断裂（DSB）的形成。在本研究中，我们比较了DNA损伤反应（DDR）在人成纤维细胞触发的重组CDT的电离辐射（IR），一个众所周知的DSB诱导剂。此外，我们剖析了参与CDT诱导的DNA损伤的检测和修复的途径。基于qRT-PCR阵列的mRNA和蛋白质印迹分析显示由CDT和IR引起的DDR模式的部分重叠，ATM-Chk 2和ATR-Chkl轴两者均强烈活化。在其体外DNase I样活性的质粒DNA，中性和碱性彗星试验显示，主要诱导的DSB在CDT处理的成纤维细胞，而照射细胞产生的SSB和碱不稳定的网站更高的金额。使用共聚焦显微镜，动态的DSB替代标记物. γ-H2 AX的CDT或IR的脉冲处理后进行监测。在照射细胞中观察到的快速诱导和消失的γ-H2 AX焦点相反，CDT诱导的γ-H2 AX焦点的数量形成延迟和持续。CDT处理后也产生53 BP 1病灶，并与γ-H2 AX病灶共定位。我们进一步证明ATM缺陷细胞对CDT诱导的DNA损伤非常敏感，这反映在细胞死亡率增加以及伴随的caspase-3和PARP-1裂解。最后，我们提供了新的证据，同源重组（HR）和非同源末端连接（NHEJ）保护对CDT引起的DSB。总之，研究结果表明，CDT的功能作为拟辐射剂，因此，是一个有吸引力的工具，选择性地诱导持久水平的DSB和揭示相关的细胞反应。(C)2014爱思唯尔有限公司版权所有。
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-Chkl 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.y-H2AX was monitored after pulse treatment with CDT or IR. In contrast to the fast induction and disappearance of -y-H2AX-foci observed in irradiated cells, the number of -y-H2AXfoci induced by CDT were formed with a delay and persisted. 53BP1 foci were also generated following CDT treatment and co-localized with -y-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. (C) 2014 Elsevier B.V. All rights reserved.