Robe Pierre A, Nguyen-Khac Minh, Jolois Olivier, Rogister Bernard, Merville Marie-Paule, Bours Vincent
Department of Neurosurgery, University of Liège, Liège, Belgium.
BMC Cancer. 2005 Apr 2;5:32. doi: 10.1186/1471-2407-5-32.
HSV-tk/ ganciclovir (GCV) gene therapy has been extensively studied in the setting of brain tumors and largely relies on the bystander effect. Large studies have however failed to demonstrate any significant benefit of this strategy in the treatment of human brain tumors. Since dexamethasone is a frequently used symptomatic treatment for malignant gliomas, its interaction with the bystander effect and the overall efficacy of HSV-TK gene therapy ought to be assessed.
Stable clones of TK-expressing U87, C6 and LN18 cells were generated and their bystander effect on wild type cells was assessed. The effects of dexamethasone on cell proliferation and sensitivity to ganciclovir were assessed with a thymidine incorporation assay and a MTT test. Gap junction mediated intercellular communication was assessed with microinjections and FACS analysis of calcein transfer. The effect of dexamethasone treatment on the sensitivity of TK-expressing to FAS-dependent apoptosis in the presence or absence of ganciclovir was assessed with an MTT test. Western blot was used to evidence the effect of dexamethasone on the expression of Cx43, CD95, CIAP2 and BclXL.
Dexamethasone significantly reduced the bystander effect in TK-expressing C6, LN18 and U87 cells. This inhibition results from a reduction of the gap junction mediated intercellular communication of these cells (GJIC), from an inhibition of their growth and thymidine incorporation and from a modulation of the apoptotic cascade.
The overall efficacy of HSV-TK gene therapy is adversely affected by dexamethasone co-treatment in vitro. Future HSV-tk/ GCV gene therapy clinical protocols for gliomas should address this interference of corticosteroid treatment.
单纯疱疹病毒胸苷激酶/更昔洛韦（HSV-tk/GCV）基因疗法已在脑肿瘤治疗中得到广泛研究，且很大程度上依赖旁观者效应。然而，大型研究未能证明该策略在治疗人类脑肿瘤方面有任何显著益处。由于地塞米松是恶性胶质瘤常用的对症治疗药物，因此应评估其与旁观者效应的相互作用以及HSV-TK基因疗法的整体疗效。
构建稳定表达胸苷激酶的U87、C6和LN18细胞克隆，并评估其对野生型细胞的旁观者效应。采用胸腺嘧啶核苷掺入试验和MTT试验评估地塞米松对细胞增殖及对更昔洛韦敏感性的影响。通过微量注射和钙黄绿素转移的流式细胞术分析评估间隙连接介导的细胞间通讯。采用MTT试验评估地塞米松处理对在有或无更昔洛韦情况下表达胸苷激酶细胞对FAS依赖性凋亡敏感性的影响。使用蛋白质免疫印迹法证明地塞米松对Cx43、CD95、CIAP2和BclXL表达的影响。
地塞米松显著降低了表达胸苷激酶的C6、LN18和U87细胞中的旁观者效应。这种抑制作用源于这些细胞间隙连接介导的细胞间通讯（GJIC）减少、生长和胸腺嘧啶核苷掺入受到抑制以及凋亡级联反应受到调节。
体外联合使用地塞米松会对HSV-TK基因疗法的整体疗效产生不利影响。未来针对胶质瘤的HSV-tk/GCV基因疗法临床方案应考虑皮质类固醇治疗的这种干扰作用。
