p53 localization in normal and human tumor cells

描述（由申请人提供）：核质穿梭已成为p53活性的重要决定因素。已经描述了各种癌症和正常细胞，其中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，这种细胞质定位的结果从过度的核输出，是由MDM 2介导的，其次是p53和一个或多个细胞质“锚”蛋白之间的关联。抑制核输出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性疗法的敏感性。我们已经建立了一个检测系统，其中MDM2促进p53核输出在瞬时转染细胞。DNA损伤剂在该系统中阻断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激酶来抑制应激后的p53输出。此外，我们将研究p53活性在两种模型细胞类型（人脐静脉内皮细胞（HUVEC）和乳腺癌细胞），其中野生型p53是由于过度的核输出和胞质隔离失活。预测某些化合物阻断应激HUVEC中p53与其细胞质锚之间的结合，并且还可阻断乳腺癌细胞中p53：锚蛋白结合。我们正在测试这些化合物对p53定位和细胞对放射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.