Mutations in the human Adematous polyposis coli gene (APC) cause polyps that eventually give rise to intestinal tumors. Little is known about the function of APC protein, other than that it binds to and down regulates b-catenin, a major component of the Wnt-1 signaling pathway. The goal of this project is to define the cellular role of APC and the nature of its interaction with Wnt-1 signaling, in both vertebrate and invertebrate cells. We will develop antibodies and other reagents to follow mammalian components of the Wnt-1/APC signaling pathway. These include Wnt-1, a, b, and g catenins, p120, GSK-3b and APC. Using these reagents, we will develop assays for the consequences of Wnt signaling on the phosphorylation of APC, its intracellular levels and its physical association with other components of the pathway. By screening Drosophila expression libraries with human APC antibodies, we have identified a partial cDNA encoding Drosophila APC-like molecule. We will complete sequence analysis of the N and C terminal fragments of the gene and characterize its genomic organization and pattern of expression during embryonic and larval development. Antibodies to Drosophila APC will be used to determine the intracellular localization of the protein and its physical association with the fly homologue of Beta-catenin before and after Wnt-1 signaling. Point mutations in the D- APC will be generated in large scale mutagenesis experiments, and the phenotype of homozygotes lacking all APC function will be determined at the cellular and organismal level. To examine the parallels between APC activity in flies and humans in greater detail, in vitro mutated forms of D-APC corresponding to truncations associated with human tumors will be expressed in specific Drosophila cell types using transgenes with tissue specific promoters. We will develop assays to test the role of APC (and any other components identified in the Drosophila pathway) in vertebrate tissue culture cells. Wildtype and mutant forms of Drosophila APC will be expressed in vertebrate cell lines that will allow assaying their effect of response of vertebrate cells to Wnt- 1. Any dominant effects of misexpression of APC in Drosophila will be used in suppressor mutagenesis screens to identify second site mutations in genes that play a role in function of APC or its effect on the Wnt1/wg pathway. Lastly, the reagents developed to characterize the Wnt-1/APC pathway will be used to characterize a range of samples from human cancers. These studies will focus on the role of a-catenin, since its levels appear to be decreased in a wide variety of human cancers.
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