García Carolina Paola, Videla Richardson Guillermo Agustín, Dimopoulos Nicolás Alexis, Fernandez Espinosa Damián Darío, Miriuka Santiago Gabriel, Sevlever Gustavo Emilio, Romorini Leonardo, Scassa María Elida
Laboratorios de Investigación Aplicada a Neurociencias, LIAN-CONICET, Fundación FLENI, Ruta 9, Km 53, (B1625XAF) Escobar, Buenos Aires, Argentina.
PLoS One. 2016 Mar 31;11(3):e0152607. doi: 10.1371/journal.pone.0152607. eCollection 2016.
Human embryonic stem cells (hESCs) are hypersensitive to genotoxic stress and display lower survival ability relative to their differentiated progeny. Herein, we attempted to investigate the source of this difference by comparing the DNA damage responses triggered by the topoisomerase I inhibitor camptothecin, in hESCs, human induced pluripotent stem cells (hiPSCs) and hESCs-derived neuroprogenitors (NP). We observed that upon camptothecin exposure pluripotent stem cells underwent apoptosis more swiftly and at a higher rate than differentiated cells. However, the cellular response encompassing ataxia-telangiectasia mutated kinase activation and p53 phosphorylation both on serine 15 as well as on serine 46 resulted very similar among the aforementioned cell types. Importantly, we observed that hESCs and hiPSCs express lower levels of the anti-apoptotic protein Bcl-2 than NP. To assess whether Bcl-2 abundance could account for this differential response we treated cells with ABT-263, WEHI-539 and ABT-199, small molecules that preferentially target the BH3-binding pocket of Bcl-xL and/or Bcl-2 and reduce their ability to sequester pro-apoptotic factors. We found that in the absence of stress stimuli, NP exhibited a higher sensitivity to ABT- 263 and WEHI-539 than hESCs and hiPSCs. Conversely, all tested cell types appeared to be highly resistant to the Bcl-2 specific inhibitor, ABT-199. However, in all cases we determined that ABT-263 or WEHI-539 treatment exacerbated camptothecin-induced apoptosis. Importantly, similar responses were observed after siRNA-mediated down-regulation of Bcl-xL or Bcl-2. Taken together, our results suggest that Bcl-xL contrary to Bcl-2 contributes to ensure cell survival and also functions as a primary suppressor of DNA double-strand brake induced apoptosis both in pluripotent and derived NP cells. The emerging knowledge of the relative dependence of pluripotent and progenitor cells on Bcl-2 and Bcl-xL activities may help to predict cellular responses and potentially manipulate these cells for therapeutic purposes in the near future.
人类胚胎干细胞（hESCs）对基因毒性应激高度敏感，相对于其分化后代，其生存能力较低。在此，我们试图通过比较拓扑异构酶I抑制剂喜树碱在hESCs、人诱导多能干细胞（hiPSCs）和hESCs衍生的神经祖细胞（NP）中引发的DNA损伤反应，来研究这种差异的来源。我们观察到，在喜树碱暴露后，多能干细胞比分化细胞更快且以更高的速率发生凋亡。然而，在上述细胞类型中，包括共济失调毛细血管扩张突变激酶激活以及丝氨酸15和丝氨酸46上的p53磷酸化在内的细胞反应非常相似。重要的是，我们观察到hESCs和hiPSCs中抗凋亡蛋白Bcl-2的表达水平低于NP。为了评估Bcl-2丰度是否可以解释这种差异反应，我们用ABT-263、WEHI-539和ABT-199处理细胞，这些小分子优先靶向Bcl-xL和/或Bcl-2的BH3结合口袋，并降低它们隔离促凋亡因子的能力。我们发现,在没有应激刺激的情况下，NP对ABT-263和WEHI-539的敏感性高于hESCs和hiPSCs。相反，所有测试的细胞类型似乎对Bcl-2特异性抑制剂ABT-199具有高度抗性。然而，在所有情况下，我们都确定ABT-263或WEHI-539处理会加剧喜树碱诱导的凋亡。重要的是，在通过siRNA介导下调Bcl-xL或Bcl-2后观察到了类似的反应。综上所述，我们的结果表明，与Bcl-2相反，Bcl-xL有助于确保细胞存活，并且在多能细胞和衍生的NP细胞中均作为DNA双链断裂诱导的凋亡的主要抑制因子发挥作用。多能细胞和祖细胞对Bcl-2和Bcl-xL活性的相对依赖性这一新知识，可能有助于预测细胞反应，并有可能在不久的将来为治疗目的而操纵这些细胞。
