REGULATION OF P53 SUBCELLULAR DISTRIBUTION BY MDM2/ARF

MDM2/ARF 对 P53 亚细胞分布的调节

• 批准号: 6350461
• 负责人: JIANDONG CHEN
• 金额: $ 19.78万
• 依托单位: UNIVERSITY OF SOUTH FLORIDA
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-02-15 至 2004-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6350461
• 关键词: DNA damage; antisense nucleic acid; cell differentiation; cell nucleus; gene expression; genetic transcription; neuroblastoma; oncoproteins; p53 gene /protein; phosphoproteins; phosphorylation; tissue /cell culture; tumor suppressor proteins; ubiquitin

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.

p53肿瘤抑制因子的失活是恶性转化的重要步骤;超过50%的人类肿瘤因突变或缺失而失去p53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MDM 2癌蛋白的过度表达或肿瘤抑制因子ARF的缺失。 野生型p53在肿瘤的一个子集（超过95%的未分化的神经母细胞瘤和37%的炎性乳腺肿瘤）是专门定位于细胞质，这可能是一个独特的机制p53失活。 在研究具有胞质野生型p53的肿瘤细胞系中，我们发现，可以通过MDM 2表达的反义抑制、通过来普霉素B抑制核输出途径、MDM 2抑制剂ARF的表达或自发分化来诱导核p53积累。 这些结果表明，MDM 2是负责抑制核p53积累在这些肿瘤中，在基因扩增或过度表达的情况下。 我们假设在神经母细胞瘤中，MDM 2通过促进p53的核输出调节p53的亚细胞定位，而核输出又受到ARF和细胞分化的调节。 我们将在以下实验中验证这一假设：[1]。确定ARF表达缺失是否导致神经母细胞瘤细胞系中p53核排斥。[2]的文件。确定分化后p53的核转位是否由MDM 2或ARF表达和功能的变化引起。[3]的文件。确定分化后p53和MDM 2的磷酸化状态是否发生改变，从而抑制MDM 2介导的核输出和降解。[4]的文件。确定分化后p53的核积累是否增加p53活性和对DNA损伤的敏感性。 这些实验将导致更好地了解核排斥的p53失活机制，ARF对MDM 2的调节，以及分化对p53的调节。