REGULATION OF CELL GROWTH BY THE WILMS TUMOR GENE

描述：(改编自研究人员摘要)肾母细胞瘤 抑制基因WT1编码锌指转录因子WT1。一个 已经在Wilms的亚群中发现了WT1基因的一些突变 肿瘤、间皮瘤、卵巢肿瘤和急性髓系白血病 WT1的功能障碍可能在许多肿瘤中起重要作用。然而， 实际上，人们对WT1调控的内源基因一无所知， 因此，WT1触发的信号通路以及WT1是如何突变的 影响这些途径导致Wilms‘s肿瘤的机制还有待发现。 调查者研究的长期目标是 WT1在发育过程中的正常功能与其功能障碍 导致肾母细胞瘤发生的WT1突变。调查人员 最近克隆了一种在细胞中表达超过十倍的基因 在WT1水平检测不到的细胞中表达WT1。这个基因是 鉴定为视网膜母细胞瘤(Rb)抑制相关蛋白(AP) (RbAp46)，一种与Rb物理相互作用的核蛋白。这个 最近，研究人员克隆并表达了人乳头瘤病毒的全长c DNA RbAp46，并分析其对肿瘤细胞的作用。值得注意的是， 外源性RbAp46抑制了四分之四的肿瘤细胞系的生长， 提示RbAp46本身具有生长抑制活性。这个 调查人员现在计划建立两者之间的正相关关系 WT1和RbAp46在不同肿瘤细胞和小鼠体内的表达水平 用Northern和原位杂交技术研究不同发育阶段的组织 杂交分析。他们计划分析RbAp46的启动子区域 确定WT1是否直接调节RbAp46的表达。他们 计划探索RbAp46作为WT1功能调节因子的可能作用 通过表达外源RbAp46或下调RbAp46在细胞内的表达。这个 研究人员计划探索RbAp46作为一种 分析生长抑制因子对细胞周期的影响及其影响 在ras信号通路上。调查人员计划执行结构 和功能分析确定RbAp46的结构域(S) 功能。研究人员计划确定相互作用的蛋白质 与RbAp46通过免疫共沉淀和体内GST捕获试验， 如有必要，可采用酵母双杂交策略。希望这些都是 研究将为Wilms‘s病的治疗干预奠定基础 肿瘤和其他肿瘤也是如此。

DESCRIPTION: (Adapted from investigator's abstract) The Wilms' tumor suppressor gene, wt1, encodes a zinc finger transcription factor, WT1. A number of mutations to the wt1 gene have been identified in subsets of Wilms tumor, mesothelioma, ovarian tumor and acute myeloid leukemia, suggesting that dysfunction of WT1 may be important in many tumors. However, effectively nothing is known about the endogenous genes regulated by WT1, and thus the signaling pathways triggered by WT1 and how mutated WT1 influences these pathways to result in Wilms' tumor remain to be discovered. The long-term objective of the investigator's research is to characterize the normal functions of WT1 during development and the dysfunctions of mutated WT1 that lead to the genesis of Wilms' tumor. The investigators recently cloned a gene whose expression is over tenfold greater in cells expressing WT1 than in cells with undetectable levels of WT1. The gene was identified as retinoblastoma (Rb) suppressor associated protein (AP) (RbAp46), a nuclear protein that physically interacts with Rb. The investigators have recently cloned and expressed the full-length cDNA of RbAp46, and analyzed its effect on tumor cells. Remarkably, expression of exogenous RbAp46 suppressed growth in four out of four tumor cell lines, suggesting that RbAp46 itself has growth inhibitory activity. The investigators now plan to establish the positive correlation between the levels of WT1 and RbAp46 expression in different tumor cells and in mouse tissues from different stages of development using Northern and in situ hybridization analysis. They plan to analyze the promoter region of RbAp46 to determine whether WT1 directly regulates the expression of RbAp46. They plan to explore the possible roles of RbAp46 as a mediator of WT1 function by expressing exogenous RbAp46 or by down-regulating RbAp46 in cells. The investigators plan to probe the mechanisms by which RbAp46 functions as a growth inhibitor by analyzing its impact on the cell cycle and its influence on the ras signaling pathway. The investigators plan to perform structure and function analysis to identify the domain(s) of RbAp46 required for its function. The investigators plan to identify the proteins which interact with RbAp46 by co-immunoprecipitation and an in vivo GST capture assay and, if necessary, by a yeast two-hybrid strategy. It is hoped that these studies will lay the foundation of therapeutic intervention for the Wilms' tumor and other tumors as well.