Quantitative, noninvasive imaging of radiation-induced DNA double-strand breaks in vivo.
Quantitative, noninvasive imaging of radiation-induced DNA double-strand breaks in vivo.
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DOI：
10.1158/0008-5472.can-10-2540
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发表时间：
2011-06-15
期刊：
影响因子：
11.2
通讯作者：
Li CY
中科院分区：
文献类型：
--
作者：
Li W;Li F;Huang Q;Shen J;Wolf F;He Y;Liu X;Hu YA;Bedford JS;Li CY
DNA双链断裂是DNA损伤的主要形式，也是放射治疗和一些化疗药物杀死癌细胞的关键机制。尽管其重要性，测量DNA双链断裂仍然是一项繁琐的任务，通常通过凝胶电泳或免疫荧光染色进行。在这里，我们报告了一种新的方法，图像和定量DNA双链断裂活哺乳动物细胞通过双片段荧光素酶重建。将萤火虫荧光素酶基因的N端和C端片段分别与H2 AX和MDC 1基因融合。我们的策略是基于这样一个确定的事实，即在DNA双链断裂的位点，H2 AX蛋白被磷酸化并与MDC 1蛋白物理缔合，从而将N-和C-荧光素酶片段聚集在一起并重建荧光素酶活性。我们的策略允许连续的，非侵入性定量的DNA双链断裂的细胞照射的X射线和56 Fe离子。此外，它允许在两周内在受照射的肿瘤中非侵入性地评价DNA双链断裂（DSB）。令人惊讶的是，我们检测到第二波的DSB诱导在照射肿瘤细胞的辐射暴露后几天，除了最初的快速诱导DSB。我们的结论是，我们的新的分裂荧光素酶为基础的γ-H2 AX-MDC 1相互作用的成像方法是一个强大的新工具，在体内研究DNA双链断裂修复动力学，具有相当大的优势，需要在一个较长的时间内观察的实验。
DNA double strand breaks is a major form of DNA damage and a key mechanism through which radiotherapy and some chemotherapeutic agents kill cancer cells. Despite its importance, measuring DNA double strand breaks is still a tedious task that is normally carried out by gel electrophoresis or immunofluorescence staining. Here we report a novel approach to image and quantify DNA double strand breaks in live mammalian cells through bi-fragment luciferase reconstitution. N- and C- terminal fragments of firefly luciferase gene were fused with H2AX and MDC1 genes, respectively. Our strategy was based on the established fact that at the sites of DNA double strand breaks, H2AX protein is phosphoryated and physically associates with the MDC1 protein, thus bringing together N- and C- luciferase fragments and reconstituting luciferase activity. Our strategy allowed serial, non-invasive quantification of DNA double strand breaks in cells irradiated with x-rays and 56Fe ions. Furthermore, it allowed for the evaluation of DNA double strand breaks (DSBs) non-invasively in vivo in irradiated tumors over two weeks. Surprisingly, we detected a second wave of DSB induction in irradiated tumor cells days after radiation exposure in addition to the initial rapid induction of DSBs. We conclude that our new split-luciferase based method for imaging γ-H2AX-MDC1 interaction is a powerful new tool to study DNA double strand break repair kinetics in vivo with considerable advantage for experiments requiring observations over an extended period of time.