Arimondo Paola B, Thomas Craig J, Oussedik Kahina, Baldeyrou Brigitte, Mahieu Christine, Halby Ludovic, Guianvarc'h Dominique, Lansiaux Amélie, Hecht Sidney M, Bailly Christian, Giovannangeli Carine
UMR 5153 CNRS-Muséum National d'Histoire Naturelle USM0503, INSERM UR565, 43 rue Cuvier, 75231 Paris Cédex 05, France.
Mol Cell Biol. 2006 Jan;26(1):324-33. doi: 10.1128/MCB.26.1.324-333.2006.
Topoisomerase I is a ubiquitous DNA-cleaving enzyme and an important therapeutic target in cancer chemotherapy for camptothecins (CPTs). These drugs stimulate DNA cleavage by topoisomerase I but exhibit little sequence preference, inducing toxicity and side effects. A convenient strategy to confer sequence specificity consists of the linkage of topoisomerase poisons to DNA sequence recognition elements. In this context, triple-helix-forming oligonucleotides (TFOs) covalently linked to CPTs were investigated for the capacity to direct topoisomerase I-mediated DNA cleavage in cells. In the first part of our study, we showed that these optimized conjugates were able to regulate gene expression in cells upon the use of a Photinus pyralis luciferase reporter gene system. Furthermore, the formation of covalent topoisomerase I/DNA complexes by the TFO-CPT conjugates was detected in cell nuclei. In the second part, we elucidated the molecular specificity of topoisomerase I cleavage by the conjugates by using modified DNA targets and in vitro cleavage assays. Mutations either in the triplex site or in the DNA duplex receptor are not tolerated; such DNA modifications completely abolished conjugate-induced cleavage all along the DNA. These results indicate that these conjugates may be further developed to improve chemotherapeutic cancer treatments by targeting topoisomerase I-induced DNA cleavage to appropriately chosen genes.
拓扑异构酶I是一种普遍存在的DNA切割酶，是喜树碱（CPT）在癌症化疗中的重要治疗靶点。这些药物刺激拓扑异构酶I进行DNA切割，但对序列的偏好性很小，会导致毒性和副作用。赋予序列特异性的一种便捷策略是将拓扑异构酶毒素与DNA序列识别元件相连。在这种情况下，研究了与CPT共价连接的三链螺旋形成寡核苷酸（TFO）在细胞中指导拓扑异构酶I介导的DNA切割的能力。在我们研究的第一部分，我们表明，在使用萤火虫荧光素酶报告基因系统时，这些优化的偶联物能够调节细胞中的基因表达。此外，在细胞核中检测到了TFO-CPT偶联物形成的共价拓扑异构酶I/DNA复合物。在第二部分中，我们通过使用修饰的DNA靶标和体外切割试验，阐明了偶联物对拓扑异构酶I切割作用的分子特异性。三链体部位或DNA双链受体中的突变是不被容忍的；这种DNA修饰完全消除了偶联物沿DNA诱导的切割。这些结果表明，这些偶联物可能会进一步开发，通过将拓扑异构酶I诱导的DNA切割靶向适当选择的基因来改善癌症化疗。
