Wilms' tumor is an embryonal malignancy of the kidney which has been linked to the inactivation of one or more tumor suppressor genes. Initial clinical observations noted the relatively frequent occurrence of bilateral tumors, implying an underlying genetic susceptibility in some individuals, as well as the existence of rare congenital syndromes that were associated with Wilms' tumor and displayed specific germline chromosome abnormalities. Molecular studies of sporadic Wilms' tumors demonstrated allelic losses involving two distinct loci on chromosome 11, at bands p13 and p15. Using positional cloning techniques, we were involved in the initial isolation of the 11p13 Wilms' tumor gene, which we have called WT1. WT1 encodes a zinc finger protein with significant homology to the Early Growth Response genes (EGR). It is normally expressed in specific cells within the kidney, during a narrow window in development. Four alternatively spliced mRNA transcripts are produced, and one of these has been shown to recognize a DNA sequence similar to the EGR1 consensus and to mediate transcriptional repression in vitro. We have shown that most Wilms' tumors express high levels of WT1, consistent with their tissue of origin, but that a fraction (10%) contain inactivating mutations within the coding sequence. While most of these mutations result in homozygous inactivation of the gene, some Wilms' tumors contain only a single mutated WT1 allele, retaining the second wild type copy. We have recently shown that such a mutated WT1 allele is capable of exerting a """"""""dominant negative"""""""" effect, cooperating with the viral gene E1A in transforming primary kidney cells. We propose to build on our initial observations, focusing on the use of mutational and functional analyses to define the role of WT1 and the types of tumors in which its inactivation contributes to tumorigenesis. We have recently completed the genomic map of WT1, which enables us to PCR amplify exons of the gene from a variety of clinical specimens, and search for the presence of inactivating mutations. We have already shown that WT1 mutations are not limited to Wilms' tumors, but are also found in adult mesotheliomas, asbestos-induced tumors of the pleural lining, a tissue that normally expresses WT1 early in development. We have recently identified Wilms' tumor cell lines bearing WT1 mutations, making it possible to re-introduce the different splice forms of wild type WT1 and test their ability to reverse the transformed phenotype. Finally, we will extend our study of """"""""dominant negative"""""""" WT1 mutations, by using these to suppress normal WT1 function in model systems including in vitro models of cellular differentiation and transgenic mice. Insofar as dominant negative WT1 mutations result from disruption of the DNA binding domain, their effect may be mediated by protein interactions, which we will be able to study using the stable transfectant cell lines that we have developed.
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