p53 localization in normal and human tumor cells

描述(申请人提供)：核质穿梭已成为P53活性的重要决定因素。在各种癌症和正常细胞中，P53通过胞浆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介导的过度核输出导致的，随后是P53与一个或多个细胞质“锚”蛋白之间的关联。抑制核出口或阻断锚定蛋白结合的策略可能会促进P53核积聚，并增强对当前细胞毒治疗的敏感性。我们已经建立了一种检测系统，在该系统中，MDM2促进瞬时转染细胞中的p53核输出。DNA损伤剂在这个系统中阻止P53核输出。我们将描述DNA损伤应激对P53核输出的影响，P53磷酸化在这一效应中的作用，以及应激后是否需要ATM或ATR激酶来抑制P53核输出。此外，我们将检测两种模型细胞(人脐静脉内皮细胞和乳腺癌细胞)中的P53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。据预测，某些化合物可以阻断应激状态下HUVECs中P53与其胞质锚之间的结合，也可能阻断乳腺癌细胞中P53：锚蛋白的结合。我们正在测试这些化合物对p53定位和细胞对辐射和其他化疗药物敏感性的影响。

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.