Cottin S, Ghani K, Caruso M
Centre de Recherche en Cancérologie de l'Université Laval, L'Hôtel-Dieu de Québec, Centre Hospitalier Universitaire de Québec, Québec, Canada.
Cancer Gene Ther. 2008 Dec;15(12):823-31. doi: 10.1038/cgt.2008.49. Epub 2008 Jul 4.
Herpes simplex virus thymidine kinase (TK) gene transfer followed by ganciclovir (GCV) administration is an approach investigated for glioblastoma treatment. The bystander effect (BE) enhances the cytotoxic effect of this strategy by allowing the diffusion of phosphorylated GCV from TK-expressing cells toward neighboring TK negative cells. This transfer of toxic metabolites is mainly mediated via gap junctions that are composed of connexins. Downregulation and/or cytoplasmic localization of connexins are common in tumors, and should be detrimental to the success of the TK/GCV strategy. In this study, we investigated the level of expression, the localization and the functionality of connexin43 (Cx43) in three glioblastoma cell lines. We showed that Cx43 was predominantly located in lysosomes and late endosomes, with only few gap junctions present at the cell surface. Surprisingly, the gap-junctional intercellular communication (GJIC) and the BE capacity were preserved, and in two of the cell lines analyzed, it was at least twice as high as compared to a control HeLa transfectant that expresses high levels of Cx43 at the cell membrane. Experiments performed in the presence of alpha-glycyrrhetinic acid or small interfering RNA confirmed that Cx43 was responsible for the GJIC and the BE. Our results indicate for the first time that the very limited numbers of gap junctions present in glioblastoma cells are highly functional. We thus conclude that the TK/GCV strategy is still a valuable therapeutic option to be developed for the treatment of glioblastoma patients.
单纯疱疹病毒胸苷激酶（TK）基因转移后给予更昔洛韦（GCV）是一种针对胶质母细胞瘤治疗的研究方法。旁观者效应（BE）通过使磷酸化的GCV从表达TK的细胞向邻近的TK阴性细胞扩散，增强了该策略的细胞毒性作用。这种有毒代谢物的转移主要通过由连接蛋白组成的间隙连接介导。连接蛋白的下调和/或细胞质定位在肿瘤中很常见，这应该不利于TK/GCV策略的成功。在本研究中，我们研究了三种胶质母细胞瘤细胞系中连接蛋白43（Cx43）的表达水平、定位和功能。我们发现Cx43主要位于溶酶体和晚期内体中，细胞表面仅存在少量间隙连接。令人惊讶的是，间隙连接细胞间通讯（GJIC）和BE能力得以保留，并且在分析的两个细胞系中，其至少是在细胞膜上高水平表达Cx43的对照HeLa转染细胞的两倍。在存在α-甘草次酸或小干扰RNA的情况下进行的实验证实，Cx43负责GJIC和BE。我们的结果首次表明，胶质母细胞瘤细胞中存在的非常有限数量的间隙连接具有高度功能。因此，我们得出结论，TK/GCV策略仍然是一种有价值的治疗选择，有待开发用于治疗胶质母细胞瘤患者。
