Growth Inhibition by the ARF Tumor Suppressor

描述(由申请人提供)：ARF是第二常见的 人类癌症中的失活基因。它通过以下方式防止致癌转化 激活P53依赖的生长停滞或凋亡，主要通过隔离 核仁中mdm2癌基因及阻断mdm2介导的p53降解。 最近的研究结果表明，1)依赖于p53的基因还需要其他因素 生长停滞，2)ARF可以通过其他途径抑制生长，而不是 涉及P53或MDM2。该提案旨在确定和描述其他 ARF诱导生长停滞的中介物和抑制物。终极目标 旨在阐明ARF抑制生长的机制。目标一号将决定 识别的ARF结合蛋白MDM2和两个新蛋白的作用 在ARF介导的生长中，核仁中与ARF相关的(PARF) 压制。MDM2拮抗P53非依赖性活性的机制 ARF的定义将通过检测p53/MDM2缺失细胞中的ARF功能来确定 表达MDM2突变体。PARF和ARF内的相互作用域将是 通过突变鉴定，而PAF是生长的假说 ARF诱导生长停止所需的抑制剂将在以下细胞中进行测试 过度表达或缺乏功能性的PARF。目标2将定义两个角色 ARF信号通路中与ARE相关的磷蛋白p58和p65。我们会 测试p58和p65是否在ARF反应中被磷酸化，以及它们是否 以P53依赖的方式选择性地与ARF的生长抑制形式结合。 P58和p65的身份将通过测试p58是否是58来确定 MDM2的KDA形式(因为它是一种磷蛋白)和生化提纯 亲和层析得到50-70 kDa的ARF相关蛋白。在Aim 3中，一个 将使用逆转录病毒cDNA文库进行无偏基因筛查 识别和表征新的基因，覆盖P53依赖和 P53非依赖性ARF诱导的停搏。这些研究将推进我们的根本 了解ARF在不同信号通路中的功能，因此， 提供与开发合适的抗癌药物相关的知识 战略。

DESCRIPTION (provided by applicant): ARF is the second most commonly inactivated gene in human cancer. It prevents oncogenic transformation by activating p53-dependent growth arrest or apoptosis, primarily by sequestering the Mdm2 oncogene in nucleoli and blocking Mdm2-mediated degradation of p53. Recent findings indicate that 1) other factors are required for p53-dependent growth arrest, and 2) ARF can inhibit growth through other pathways that do not involve p53 or Mdm2. This proposal aims to identify and characterize additional mediators and inhibitors of ARF-induced growth arrest. The ultimate objective is to delineate mechanisms of growth suppression by ARF. Aim 1 will determine the role of identified ARF binding proteins, Mdm2 and two novel proteins (Parfs) that associate with ARF in nucleoli, in ARF-mediated growth suppression. The mechanism by which Mdm2 antagonizes p53-independent activities of ARF will be defined by assaying ARF function in p53/Mdm2-null cells expressing Mdm2 mutants. Interaction domains within Parfs and ARF will be identified through mutagenesis, while the hypothesis that Parfs are growth inhibitors required for ARF-induced growth arrest will be tested in cells which overexpress or lack functional Parfs. Aim 2 will define the role of two ARE-associated phosphoproteins, p58 and p65, in ARF signaling pathways. We will test if p58 and p65 are phosphorylated in response to ARF and if they selectively bind to growth inhibitory forms of ARF in a p53-dependent manner. The identities of p58 and p65 will be determined by testing if p58 is the 58 kDa form of Mdm2 (since it is a phosphoprotein) and biochemically purifying ARF-associated proteins of 50-70 kDa by affinity chromatography. In Aim 3, an unbiased genetic screen using retroviral cDNA libraries will be performed to identify and characterize novel genes that override p53-dependent and p53-independent ARF-induced arrest. These studies will advance our fundamental understanding of ARF function in the different signaling pathways, and as such, provide knowledge that is relevant to developing appropriate anticancer strategies.