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阻止致癌转化： 激活p53依赖性生长停滞或凋亡，主要通过螯合 Mdm 2癌基因在核仁中的表达，并阻断Mdm 2介导的p53降解。 最近的研究结果表明：（1）p53依赖性 生长停滞，2）ARF可以通过其他途径抑制生长， 涉及p53或Mdm 2。本建议旨在确定和描述其他 ARF诱导的生长停滞的介质和抑制剂。最终目标 目的是阐明ARF抑制生长的机制。目标1将决定 鉴定的ARF结合蛋白Mdm 2和两种新蛋白的作用 在ARF介导的生长中， 镇压Mdm 2拮抗p53非依赖性活性的机制 通过检测p53/Mdm 2-null细胞中的ARF功能来定义ARF的 表达Mdm 2突变体。Parfs和ARF中的交互域将 通过诱变鉴定，而Parfs是生长的假设， 将在细胞中测试ARF诱导的生长停滞所需的抑制剂， 过度表达或缺乏功能性Parfs。目标2将定义两个角色 ARE相关磷蛋白，p58和p65，在ARF信号通路。我们将 检测p58和p65是否在ARF反应中磷酸化， 以p53依赖性方式选择性结合ARF的生长抑制形式。 p58和p65的身份将通过检测p58是否为58 kDa形式的Mdm 2（因为它是磷蛋白）和生物化学纯化 通过亲和色谱法测定50-70 kDa的ARF相关蛋白。在目标3中， 将使用逆转录病毒cDNA文库进行无偏遗传筛选， 鉴定和表征超越p53依赖性新基因， p53非依赖性ARF诱导的细胞阻滞。这些研究将促进我们的基本 了解ARF在不同信号通路中的功能，因此， 提供与开发适当的抗癌药物相关的知识 战略布局

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.