Previous efforts to determine whether or not the transcription factor and tumor suppressor protein p53 is required for DNA damage‐induced apoptosis in pluripotent embryonic stem cells (ESCs) produced contradictory conclusions. To resolve this issue, p53+/+ and p53−/− ESCs derived by two different methods were used to quantify time‐dependent changes in nuclear DNA content; annexin‐V binding; cell permeabilization; and protein expression, modification, and localization. The results revealed that doxorubicin (Adriamycin [ADR]) concentrations 10 to 40 times less than commonly used in previous studies induced the DNA damage‐dependent G2‐checkpoint and completed apoptosis within the same time frame, regardless of the presence or absence of p53, p21, and PUMA. Increased ADR concentrations delayed initiation of apoptosis in p53−/− ESCs, but the rates of apoptosis remained equivalent. Similar results were obtained by inducing apoptosis with either staurosporine inhibition of kinase activities or WX8 disruption of lysosome homeostasis. Differentiation of ESCs by LIF deprivation revealed p53‐dependent formation of haploid cells, increased genomic stability, and suppression of the G2‐checkpoint. Minimal induction of DNA damage now resulted in p53‐facilitated apoptosis, but regulation of pluripotent gene expression remained p53‐independent. Primary embryonic fibroblasts underwent p53‐dependent total cell cycle arrest (a prelude to cell senescence), and p53‐independent apoptosis occurred in the presence of 10‐fold higher levels of ADR, consistent with previous studies. Taken together, these results reveal that the multiple roles of p53 in cell cycle regulation and apoptosis are first acquired during pluripotent stem cell differentiation.

中文翻译：

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在依赖 p53 的胚胎干细胞分化之前，细胞周期停滞和凋亡不依赖于 p53

之前确定转录因子和肿瘤抑制蛋白 p53 是否是 DNA 损伤诱导的多能胚胎干细胞 (ESC) 细胞凋亡所必需的努力产生了相互矛盾的结论。为了解决这个问题，使用两种不同方法衍生的 p53+/+ 和 p53-/- ESC 来量化核 DNA 含量的时间依赖性变化；膜联蛋白-V 结合；细胞透化；和蛋白质表达、修饰和定位。结果表明，无论 p53 存在与否，阿霉素（阿霉素 [ADR]）浓度比以往研究中常用的浓度低 10 到 40 倍，可诱导 DNA 损伤依赖性 G2 检查点并在同一时间范围内完成细胞凋亡， p21 和彪马。增加的 ADR 浓度延迟了 p53-/- ESC 细胞凋亡的开始，但细胞凋亡率保持不变。通过用星形孢菌素抑制激酶活性或 WX8 破坏溶酶体稳态来诱导细胞凋亡，获得了类似的结果。通过 LIF 剥夺对 ESC 的分化揭示了 p53 依赖性单倍体细胞的形成、基因组稳定性的增加和 G2 检查点的抑制。DNA 损伤的最小诱导现在导致 p53 促进细胞凋亡，但多能基因表达的调节仍然不依赖 p53。原代胚胎成纤维细胞经历了 p53 依赖性总细胞周期停滞（细胞衰老的前奏），并且在 ADR 水平高 10 倍的情况下发生了 p53 非依赖性细胞凋亡，这与之前的研究一致。综合起来，