Jones Kara R, Gewirtz David A, Yannone Steven M, Zhou Shaoming, Schatz David G, Valerie Kristoffer, Povirk Lawrence F
Department of Pharmacology and Toxicology, Virginia Commonwealth University, P.O. Box 980230, Richmond, VA 23298, USA.
Mol Cancer Ther. 2005 Oct;4(10):1541-7. doi: 10.1158/1535-7163.MCT-05-0193.
Incomplete DNA repair or misrepair can contribute to the cytotoxicity of DNA double-strand breaks. Consequently, interference with double-strand break repair, by pharmacologic or genetic means, is likely to sensitize tumor cells to ionizing radiation. The current studies were designed to inhibit the nonhomologous end joining repair pathway by interfering with the function of the XRCC4/ligase IV complex. A PCR-generated fragment of the XRCC4 gene, encompassing the homodimerization and ligase IV-binding domains, was inserted into a plasmid vector (pFLAG-CMV-2) expressing the FLAG peptide and the cassette encoding FLAG-tagged XRCC4 fragment was cloned into an adenoviral vector. Both the plasmid and the corresponding adenovirus elicited robust expression of a truncated XRCC4 protein designed to compete in a dominant-negative fashion with full-length XRCC4 for binding to ligase IV. Binding of the XRCC4 fragment to ligase IV in vivo was confirmed by immunoprecipitation. Clonogenic survival assays showed that the adenovirus expressing the truncated XRCC4 protein sensitizes MDA-MB-231 breast tumor cells to ionizing radiation, presumably through interference with the functional activity of ligase IV, leading to inhibition of the final ligation step in end joining. These studies support the potential clinical utility of combining radiation therapy with agents that inhibit DNA double-strand break repair.
DNA修复不完全或错误修复可导致DNA双链断裂的细胞毒性。因此，通过药理学或遗传学手段干扰双链断裂修复，可能会使肿瘤细胞对电离辐射敏感。当前的研究旨在通过干扰XRCC4/连接酶IV复合物的功能来抑制非同源末端连接修复途径。将包含同源二聚化和连接酶IV结合结构域的XRCC4基因的PCR扩增片段插入表达FLAG肽的质粒载体（pFLAG-CMV-2）中，并将编码FLAG标签的XRCC4片段的盒式结构克隆到腺病毒载体中。质粒和相应的腺病毒均能强烈表达截短的XRCC4蛋白，该蛋白设计用于以显性负性方式与全长XRCC4竞争结合连接酶IV。体内XRCC4片段与连接酶IV的结合通过免疫沉淀得以证实。克隆形成存活试验表明，表达截短的XRCC4蛋白的腺病毒使MDA-MB-231乳腺肿瘤细胞对电离辐射敏感，推测是通过干扰连接酶IV的功能活性，导致末端连接中最终连接步骤的抑制。这些研究支持了将放射治疗与抑制DNA双链断裂修复的药物联合使用的潜在临床应用价值。
