Regulation of p53 in stem cells

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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.