Grignet-Debrus C, Cool V, Baudson N, Velu T, Calberg-Bacq C M
Laboratory of Fundamental Virology and Immunology, Institute of Pathology, University of Liege, Belgium.
Cancer Gene Ther. 2000 Nov;7(11):1456-68. doi: 10.1038/sj.cgt.7700250.
To investigate the factors influencing the bystander effect--a key element in the efficacy of suicide gene therapy against cancer--we compared the effect triggered by four extremely efficient gene/prodrug combinations, i.e., VZVtk/BVDU, the thymidine kinase of Varicella zoster virus associated with (E)-5-(2-bromovinyl)-2'-deoxyuridine; VZVtk/BVaraU, the same enzyme associated with (E)-5-(2-bromovinyl)-1-beta-D-arabinofuranosyluracil; HSVtk/BVDU, the association of the Herpes simplex virus thymidine kinase with BVDU; and the classical HSVtk/GCV (ganciclovir) paradigm. The cells used, the human MDA-MB-435 breast cancer, and the rat 9L glioblastoma lines were equally sensitive in vitro to these four associations. In both cell types, the combinations involving pyrimidine analogues (BVDU, BVaraU) displayed a smaller bystander killing than the combination involving the purine analogue (GCV). In addition, the bystander effect induced by all the tk/prodrug systems was reduced in MDA-MB-435 cells in comparison to 9L cells; albeit, the viral kinases were produced at a higher level in the breast cancer cells. All systems induced apoptotic death in the two cell types, but the MDA-MB-435 cells, deprived of connexin 43, were noncommunicating in striking contrast with the 9L cells. That functional gap junctions have to be increased in order to improve the breast cancer cell response to suicide gene therapy was demonstrated by transducing the Cx43 gene: this modification enhanced the bystander effect associated in vitro with GCV treatment and, by itself, decreased the tumorigenicity of the untreated cells. However, the noncommunicating MDA-MB-435 cells triggered a significant bystander effect both in vitro and in vivo with the HSVtk/GCV system, showing that communication through gap junctions is not the only mechanism involved.
为了研究影响旁观者效应（自杀基因疗法抗癌疗效的关键因素）的因素，我们比较了四种极其有效的基因/前药组合所引发的效应，即水痘带状疱疹病毒胸苷激酶（VZVtk）与（E）-5-（2-溴乙烯基）-2'-脱氧尿苷（BVDU）组合；VZVtk与（E）-5-（2-溴乙烯基）-1-β-D-阿拉伯呋喃糖基尿嘧啶（BVaraU）组合；单纯疱疹病毒胸苷激酶（HSVtk）与BVDU组合；以及经典的HSVtk/更昔洛韦（GCV）模式。所使用的细胞，即人MDA-MB-435乳腺癌细胞和大鼠9L胶质母细胞瘤细胞系，在体外对这四种组合同样敏感。在这两种细胞类型中，涉及嘧啶类似物（BVDU、BVaraU）的组合所表现出的旁观者杀伤作用比涉及嘌呤类似物（GCV）的组合要小。此外，与9L细胞相比，MDA-MB-435细胞中所有tk/前药系统诱导的旁观者效应均有所降低；尽管乳腺癌细胞中病毒激酶的产生水平更高。所有系统均在这两种细胞类型中诱导凋亡性死亡，但缺乏连接蛋白43的MDA-MB-435细胞与9L细胞形成鲜明对比，它们不进行细胞间通讯。通过转导Cx43基因证明，为了改善乳腺癌细胞对自杀基因疗法的反应，必须增加功能性缝隙连接：这种修饰增强了体外与GCV治疗相关的旁观者效应，并且其本身降低了未处理细胞的致瘤性。然而，不进行细胞间通讯的MDA-MB-435细胞在体外和体内通过HSVtk/GCV系统均引发了显著的旁观者效应，这表明通过缝隙连接进行通讯不是唯一涉及的机制。
