REGULATION OF CELL GROWTH BY THE WILMS TUMOR GENE

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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.