Regulation of p53 in stem cells

干细胞中 p53 的调节

• 批准号: 202426319
• 负责人: Privatdozentin Dr. Christine Blattner
• 金额: --
• 依托单位: Institut für Biologische und Chemische Systeme (IBCS)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2011
• 资助国家: 德国
• 起止时间: 2010-12-31 至 2013-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/202426319?language=en
• 关键词: Regulation; p53; stem; cells

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.

p53肿瘤抑制蛋白是诱导细胞周期停滞和凋亡的抗增殖蛋白。尽管增殖率高，但p53在胚胎干细胞中非常丰富。因此，我们假设p53在胚胎干细胞中保持在非活性状态。此外，p53主要位于干细胞的细胞质中，这可能有助于或可能不有助于其“功能失活”。在这项提案中，我们将分析p53在（静息）干细胞中是否活跃，如果是这样，为什么胚胎干细胞尽管具有高水平的p53，但仍具有如此高的增殖率。此外，我们将确定p53在干细胞中是否真的是胞质的，确定其胞质锚定，并确定在何种程度上这种胞质定位有助于控制（静息）干细胞中的p53活性。为了研究p53在（静息）干细胞中是否具有转录活性，我们将用报告基因构建体稳定转染胚胎干细胞。所得到的数据将通过p53阳性和敲除小鼠胚胎干细胞的微阵列以及野生型干细胞中p53的功能失活进一步证实。为了确定p53在细胞质中的活性，我们将研究其与Bcl-2家族的促凋亡和抗凋亡成员的关系。为了确定p53是否是胞质的，我们将胚胎干细胞破碎成细胞核和胞质裂解液，并定量测定每个隔室中p53分子的数量。该实验的结果将通过定量免疫荧光显微镜以及通过使用蛋白质降解和核输出的抑制剂来证实。如果p53是细胞质，我们将确定其与细胞质锚的关联。最后，我们将消除干细胞中的这些细胞质锚点，以确定其细胞质定位对其（静息）干细胞中“功能失活”的贡献。