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.
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