p53 localization in normal and human tumor cells

描述（由申请人提供）：核细胞质穿梭已成为p53活性的重要决定因素。在多种癌症和正常细胞中，p53通过细胞质中的异常隔离而失活，包括神经母细胞瘤、乳腺癌和应激内皮细胞等。目前的模型表明，这种细胞质定位是由MDM2介导的过度核输出引起的，随后p53与一种或多种细胞质“锚定”蛋白之间存在关联。抑制核输出或阻断锚蛋白结合的策略可能促进p53核积累并增强对当前细胞毒性治疗的敏感性。我们已经建立了一个检测系统，MDM2在瞬时转染的细胞中促进p53核输出。DNA损伤剂阻断p53核输出。我们将描述DNA损伤应激对p53核输出的影响，p53磷酸化在这种影响中的作用，以及是否需要ATM或ATR激酶来抑制应激后p53的输出。此外，我们将检测两种模型细胞类型（人脐静脉内皮细胞（HUVECs）和乳腺癌细胞）中p53的活性，其中野生型p53由于过度的核输出和细胞质隔离而失活。在应激的HUVECs中，某些化合物可能会阻断p53与其细胞质锚蛋白的结合，也可能阻断p53：锚蛋白在乳腺癌细胞中的结合。我们正在测试这些化合物对p53定位和细胞对放疗和其他化疗药物的敏感性的影响。

DESCRIPTION (provided by applicant): Nuclear-cytoplasmic shuttling has emerged as an important determinant of p53 activity. Various cancers and normal cells have been described in which p53 is inactivated through abnormal sequestration in the cytoplasm, including neuroblastoma, breast cancer, and stressed endothelial cells, among others. Current models suggest this cytoplasmic localization results from excessive nuclear export that is mediated by MDM2, followed by association between p53 and one or more cytoplasmic "anchor" proteins. Strategies to inhibit nuclear export or block anchor-protein binding may promote p53 nuclear accumulation and enhance sensitivity to current cytotoxic therapies. We have established an assay system in which MDM2 promotes p53 nuclear export in transiently transfected cells. DNA damaging agents block p53 nuclear export in this system. We will characterize the effect of DNA damaging stress on p53 nuclear export, the role of p53 phosphorylation in this effect, and whether the ATM or ATR kinases are required to inhibit p53 export following stress. In addition, we will examine p53 activity in two model cell types (human umbilical vein endothelial cells (HUVECs) and breast cancer cells) where wild-type p53 is inactivated due to excessive nuclear export and cytoplasmic sequestration. Certain compounds are predicted to block binding between p53 and its cytoplasmic anchor in stressed HUVECs, and may also block p53:anchor protein binding in breast cancer cells. We are testing the effect of these compounds on p53 localization and cellular sensitivity to radiation and other chemotherapeutic agents.