Regulation of p53 in stem cells

P53肿瘤抑制蛋白是一种抗增殖蛋白，可诱导细胞周期停滞和细胞凋亡。尽管P53的增殖率很高，但在胚胎干细胞中却非常丰富。因此，我们假设P53在胚胎干细胞中处于不活跃状态。此外，P53主要定位于干细胞的细胞质中，这可能是也可能不是其“功能不活跃”的原因。在这项提案中，我们将分析P53在(静止的)干细胞中是否活跃，如果是这样，为什么胚胎干细胞在P53水平很高的情况下仍有如此高的增殖率。此外，我们将确定P53在干细胞中是否真的是细胞质的，确定其细胞质锚，并确定这种细胞质定位在多大程度上有助于控制(静止的)干细胞中的P53活性。为了研究P53在(静止的)干细胞中是否转录活性，我们将用报告构建的方法稳定地转染胚胎干细胞。最终的数据将通过来自P53阳性和敲除小鼠的胚胎干细胞微阵列以及野生型干细胞中P53的功能失活来进一步证实。为了确定细胞质中P53的S活性，我们将研究其与Bcl2家族促凋亡和抗凋亡成员的关系。为了确定P53是否为细胞质，我们将胚胎干细胞分裂成细胞核和胞浆裂解物，并定量测定每个隔室中P53分子的数量。这项实验的结果将通过定量免疫荧光显微镜以及蛋白质降解和核出口抑制剂的使用来证实。如果P53是细胞质的，我们将确定它与细胞质锚的关联。最后，我们将消除干细胞中的这些细胞质锚，以确定其细胞质定位对其在(静止的)干细胞中的“功能失活”的贡献。

The p53 tumour suppressor protein is an anti-proliferative protein that induces cell cycle arrest and apoptosis. Despite a high proliferation rate, p53 is highly abundant in embryonic stem cells. We there-fore hypothesize that p53 is kept in an inactive state in embryonic stem cells. Furthermore, p53 is mostly localised in the cytoplasm of stem cells which may or may not contribute to its “functional inactivity”. In this proposal, we will analyse whether p53 is active in (resting) stem cells and if so, why embryonic stem cells have such a high proliferation rate despite having high levels of p53. We will, furthermore, determine whether p53 is truly cytoplasmic in stem cells, identify its cytoplasmic anchors and determine to which extent this cytoplasmic localisation contributes to the control of p53 activity in (resting) stem cells. To investigate whether p53 is transcriptionally active in (resting) stem cells, we will stably transfect embryonic stem cells with a reporter construct. The resulting data will be further con-firmed by microarrays of embryonic stem cells from p53-positive and knock-out mice as well as by functional inactivation of p53 in wild type stem cells. For determining p53’s activity in the cytoplasm, we will investigate its association with pro- and anti-apoptotic members of the Bcl-2 family. To deter-mine whether p53 is cytoplasmic or not, we will fractionate embryonic stem cells into nucleus and cytoplasmic lysate and quantitatively determine the number of p53 molecules in each compartment. The results of this experiment will be confirmed by quantitative immunofluorescence microscopy as well as by the use of inhibitors of protein degradation and nuclear export. If p53 is cytoplasmic, we will determine its association with cytoplasmic anchors. Finally, we will eliminate these cytoplasmic anchors in stem cells to determine the contribution of its cytoplasmic localisation to its “functional inactivation” in (resting) stem cells.