The identification of human tumor suppressor genes has led to new insights into the mechanisms of human cancer development. Some of the first tumor suppressor genes were identified through studies of pediatric malignancies including the RB and WTI genes. In the case of Wilms' tumors, further investigations have led to the discovery of other potential tumor suppressor genes on chromosomes 11, 16 as well as an unmapped familial form. In a complementary fashion, he has taken a functional approach by using a biological assay, tumor suppression, to map the locations of functional tumor suppressor genes via monochromosome transfer. He has now narrowed the location of a second Wilms' tumor suppressor gene, WT2, to an approximately 350 kb region on 11p15.5. Other studies have placed translocations in Beckwith-Weidemann Syndrome patients and loss of heterozygosity for Wilms tumor samples in this same region. In addition, this region of the human genome contains genes subject to inactivation by genomic imprinting. Intriguingly, many Wilms' tumors show a loss of imprinting for genes in 11p15.5 leading to either their inactivation or increased expression. The role of these epigenetic events such as imprinting in Wilms' tumor and other human cancer development remains unknown. Therefore, the isolation and characterization of the WT2 tumor suppressor gene would constitute a major advance in understanding these influences. During the last funding period, he developed a PAC/BAC/PI contig across the WT2 tumor suppressor gene region and began the identification of candidate genes in the area. In this competitive renewal application, he proposes to isolate the WT2 gene and characterize its status in the development of Wilms' tumor.
In Specific Aim A, he will identify as many genes as possible for the WT2 tumor suppressor region by solution hybrid capture and analysis of genomic DNA sequence.
In Specific Aim B, he will screen each candidate gene for correlative expression with tumor suppression in a microcell hybrid model system. He also will search for genomic alterations in primary tumor samples and examine the expression pattern in normal tissues. He hopes to limit the number of strong candidate genes by these criteria to five or less. In the last specific aim, he will identify the WT2 gene by screening for mutations and loss of expression in primary tumor samples. He will also transfer the gene into the G401 cell line to demonstrate functional tumor suppressor activity. The availability of the WT2 gene will broaden the understanding of tumor suppressor gene functions, provide important clues about the process of normal mammalian tissue development including genomic imprinting and impact upon treatment and detection of human cancer.
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