Department of Toxicology, University Medical Center, Mainz, Germany.
Institute of Medical Biostatistics, Epidemiology and Informatics, University Medical Center, Mainz, Germany.
Cell Mol Life Sci. 2021 Jul;78(14):5587-5604. doi: 10.1007/s00018-021-03864-0. Epub 2021 Jun 8.
To clarify whether differential compartmentalization of Survivin impacts temozolomide (TMZ)-triggered end points, we established a well-defined glioblastoma cell model in vitro (LN229 and A172) and in vivo, distinguishing between its nuclear and cytoplasmic localization. Expression of nuclear export sequence (NES)-mutated Survivin (SurvNESmut-GFP) led to impaired colony formation upon TMZ. This was not due to enhanced cell death but rather due to increased senescence. Nuclear-trapped Survivin reduced homologous recombination (HR)-mediated double-strand break (DSB) repair, as evaluated by γH2AX foci formation and qPCR-based HR assay leading to pronounced induction of chromosome aberrations. Opposite, clones, expressing free-shuttling cytoplasmic but not nuclear-trapped Survivin, could repair TMZ-induced DSBs and evaded senescence. Mass spectrometry-based interactomics revealed, however, no direct interaction of Survivin with any of the repair factors. The improved TMZ-triggered HR activity in Surv-GFP was associated with enhanced mRNA and stabilized RAD51 protein expression, opposite to diminished RAD51 expression in SurvNESmut cells. Notably, cytoplasmic Survivin could significantly compensate for the viability under RAD51 knockdown. Differential Survivin localization also resulted in distinctive TMZ-triggered transcriptional pathways, associated with senescence and chromosome instability as shown by global transcriptome analysis. Orthotopic LN229 xenografts, expressing SurvNESmut exhibited diminished growth and increased DNA damage upon TMZ, as manifested by PCNA and γH2AX foci expression, respectively, in brain tissue sections. Consequently, those mice lived longer. Although tumors of high-grade glioma patients expressed majorly nuclear Survivin, they exhibited rarely NES mutations which did not correlate with survival. Based on our in vitro and xenograft data, Survivin nuclear trapping would facilitate glioma response to TMZ.
为了阐明 Survivin 的差异区室化是否影响替莫唑胺（TMZ）触发的终点，我们在体外（LN229 和 A172）和体内建立了明确的神经胶质瘤细胞模型，区分其核定位和细胞质定位。表达核输出序列（NES）突变的 Survivin（SurvNESmut-GFP）导致 TMZ 后集落形成受损。这不是由于增强的细胞死亡，而是由于增加的衰老。核固缩的 Survivin 减少了同源重组（HR）介导的双链断裂（DSB）修复，如通过 γH2AX 焦点形成和基于 qPCR 的 HR 测定评估的那样，导致染色体畸变的明显诱导。相反，表达自由穿梭细胞质但不是核固缩 Survivin 的克隆能够修复 TMZ 诱导的 DSB 并逃避衰老。然而，基于质谱的相互作用组学表明， Survivin 与任何修复因子都没有直接相互作用。与 survNESmut 细胞中 RAD51 表达减少相反，提高的 TMZ 触发的 HR 活性与增强的 mRNA 和稳定的 RAD51 蛋白表达相关。值得注意的是，细胞质 Survivin 可以显著补偿 RAD51 敲低下的生存能力。差异 Survivin 定位也导致了独特的 TMZ 触发的转录途径，与衰老和染色体不稳定性相关，如通过全转录组分析所示。表达 survNESmut 的 LN229 原位异种移植瘤在 TMZ 后表现出生长减少和 DNA 损伤增加，这分别表现为脑组织切片中 PCNA 和 γH2AX 焦点的表达。因此，这些老鼠活得更久。尽管高级别神经胶质瘤患者的肿瘤主要表达核 Survivin，但它们很少发生 NES 突变，并且与生存无关。基于我们的体外和异种移植数据，Survivin 的核捕获将促进胶质细胞瘤对 TMZ 的反应。
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