REGULATION OF P53 SUBCELLULAR DISTRIBUTION BY MDM2/ARF

P53抑癌基因的失活是肿瘤恶性转化的重要步骤；超过50%的人类肿瘤因突变或缺失而丧失P53的功能。其余肿瘤中的野生型p53也可能受到其他机制的损害，如MDM2癌蛋白的过度表达，或肿瘤抑制因子ARF的丢失。野生型P53在某些肿瘤(95%以上的未分化神经母细胞瘤和37%以上的炎性乳腺肿瘤)中仅定位于细胞质，这可能是P53失活的一种独特机制。在研究胞质野生型p53的肿瘤细胞系中，我们发现MDM2表达的反义抑制、软霉素B抑制核输出途径、MDM2抑制因子ARF的表达或自发分化都可以诱导核P53积聚。这些结果表明，在没有基因扩增或过度表达的情况下，MDM2负责抑制这些肿瘤中的核P53积聚。我们推测，在神经母细胞瘤中，MDM2通过促进其核输出来调节P53的亚细胞定位，而核输出又受ARF和细胞分化的调节。我们建议在以下实验中检验这一假设：[1]。确定ARF表达缺失是否导致神经母细胞瘤细胞系中的P53核排斥。[2]。确定分化后的P53核转位是否由MDM2或ARF的表达和功能改变所致。[3]。确定分化后P53和MDM2的磷酸化状态是否发生改变，从而抑制MDM2介导的核输出和降解。[4]。确定分化后P53的核聚集是否增加了P53的活性和对DNA损伤的敏感性。这些实验将有助于更好地理解核排斥失活P53的机制，ARF对MDM2的调节，以及分化对P53的调节。

Inactivation of the p53 tumor suppressor is an important step in malignant transformation; over 50 percent of human tumors lose p53 function by mutations or deletion. Wild type p53 in the remaining tumors may also be impaired by other mechanisms such as overexpression of the MDM2 oncoprotein, or loss of the tumor suppressor ARF. Wild type p53 in a subset of tumors (greater than 95 percent of undifferentiated neuroblastomas and 37 percent of inflammatory breast tumors) is localized exclusively to the cytoplasm, and this may represent a unique mechanism of p53 inactivation. In studying tumor cell lines with cytoplasmic wild type p53, we have found that nuclear p53 accumulation can be induced by antisense inhibition of MDM2 expression, inhibition of the nuclear export pathway by leptomycin B, expression of the MDM2 inhibitor ARF, or spontaneous differentiation. These results suggest that MDM2 is responsible for inhibiting nuclear p53 accumulation in these tumors, in the absence of gene amplification or overexpression. We hypothesize that in neuroblastomas, MDM2 regulates p53 subcellular localization by promoting its nuclear export, which is in turn regulated by ARF and cell differentiation. We propose to test this hypothesis in the following experiments: [1]. Determine whether loss of ARF expression causes p53 nuclear exclusion in neuroblastoma cell lines. [2]. Determine whether nuclear translocation of p53 after differentiation is caused by changes of MDM2 or ARF expression and function. [3]. Determine whether the phosphorylation status of p53 and MDM2 is altered after differentiation, which inhibits MDM2-mediated nuclear export and degradation. [4]. Determine whether nuclear accumulation of p53 after differentiation increases p53 activity and sensitivity to DNA damage. These experiments will lead to a better understanding of the mechanism of p53 inactivation by nuclear exclusion, the regulation of MDM2 by ARF, and the regulation of p53 by differentiation.