HMGB1 and HMGB2 proteins are abundantly expressed in human embryonic stem cells (hESCs) and hESC-derived progenitor cells (neuroectodermal cells, hNECs), though their functional roles in pluripotency and the mechanisms underlying their differentiation in response to the anticancer drug etoposide remain to be elucidated. Here, we show that HMGB1 and/or HMGB2 knockdown (KD) by shRNA in hESCs did not affect the cell stemness/pluripotency regardless of etoposide treatments, while in hESC-derived neuroectodermal cells, treatment resulted in differential effects on cell survival and the generation of rosette structures. The objective of this work was to determine whether HMGB1/2 proteins could modulate the sensitivity of hESCs and hESC-derived progenitor cells (hNECs) to etoposide. We observed that HMGB1 KD knockdown (KD) and, to a lesser extent, HMGB2 KD enhanced the sensitivity of hESCs to etoposide. Enhanced accumulation of 53BP1 on telomeres was detected by confocal microscopy in both untreated and etoposide-treated HMGB1 KD hESCs and hNECs, indicating that the loss of HMGB1 could destabilize telomeres. On the other hand, decreased accumulation of 53BP1 on telomeres in etoposide-treated HMGB2 KD hESCs (but not in HMGB2 KD hNECs) suggested that the loss of HMGB2 promoted the stability of telomeres. Etoposide treatment of hESCs resulted in a significant enhancement of telomerase activity, with the highest increase observed in the HMGB2 KD cells. Interestingly, no changes in telomerase activity were found in etoposide-treated control hNECs, but HMGB2 KD (unlike HMGB1 KD) markedly decreased telomerase activity in these cells. Changes in telomerase activity in the etoposide-treated HMGB2 KD hESCs or hNECs coincided with the appearance of DNA damage markers and could already be observed before the onset of apoptosis. Collectively, we have demonstrated that HMGB1 or HMGB2 differentially modulate the impact of etoposide treatment on human embryonic stem cells and their progenitor cells, suggesting possible strategies for the enhancement of the efficacy of this anticancer drug.

中文翻译：

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Nonhistone蛋白HMGB1和HMGB2差异地调节人类胚胎干细胞和祖细胞对抗癌药物依托泊苷的反应

HMGB1和HMGB2蛋白在人胚胎干细胞（hESCs）和hESC衍生的祖细胞（神经外胚层细胞，hNECs）中大量表达，尽管它们在多能性中的功能性作用以及它们对抗癌药依托泊苷的反应所起的分化机制仍然存在。阐明。在这里，我们显示HMGB1和/或HMGB2不论依托泊苷如何处理，hESC中shRNA的敲低（KD）均不影响细胞干性/多能性，而在hESC衍生的神经外胚层细胞中，处理导致对细胞存活率和玫瑰花结结构产生不同的影响。这项工作的目的是确定HMGB1 / 2蛋白是否可以调节hESCs和hESC衍生的祖细胞（hNECs）对依托泊苷的敏感性。我们观察到HMGB1 KD敲除（KD），并在较小程度上，HMGB2 KD增强了hESC对依托泊苷的敏感性。通过共聚焦显微镜在未处理和依托泊苷处理的HMGB1 KD hESCs和hNECs中检测到53BP1在端粒上的积累增强，表明HMGB1的丢失可能会使端粒不稳定。另一方面，依托泊苷处理的HMGB2 KD hESCs（但不是在HMGB2 KD hNECs）中端粒上53BP1的积累减少表明HMGB2的丧失促进了端粒的稳定性。依托泊苷对hESC的处理导致端粒酶活性显着增强，在HMGB2 KD细胞中观察到最高的升高。有趣的是，在依托泊苷处理的对照hNECs中未发现端粒酶活性的变化，但HMGB2 KD（与HMGB1 KD不同）显着降低了这些细胞中的端粒酶活性。依托泊苷处理的HMGB2端粒酶活性的变化KD hESCs或hNECs与DNA损伤标志物的出现相吻合，并且可以在细胞凋亡开始之前被观察到。集体地，我们已经证明HMGB1或HMGB2差异调节依托泊苷治疗对人类胚胎干细胞及其祖细胞的影响，提示了增强这种抗癌药功效的可能策略。