Tumor suppressor genes are the targets of inactivating mutations and chromosomal deletions in human cancers, but only a subset have been molecularly cloned, and the mechanisms by which they promote aberrant growth and differentiation are still largely undefined. Neuroblastoma, the most common extracranial solid tumor of childhood, arises in sympathetic neural crest cells and is characterized by two major genetic abnormalities, deletions of the short arm of chromosome 1 and amplification of the NMYC oncogene. This proposal focuses on the molecular cloning and functional analysis of two putative tumor suppressors that have been localized to chromosome bands 1p35-36: NB1 in neuroblastomas with single-copy NMYC genes and NB2 in those with amplified NMYC genes. A novel """"""""hybridization-string"""""""" fluorescence in situ hybridization (FISH) assay will be employed to pinpoint genomic regions containing the NB1 and NB2 genes, through analysis of large numbers of tumors for translocation breakpoints or informative interstitial deletions that bisect and inactivate these loci. Once the regions have been localized, expressed sequences will be characterized and assembled into cDNAs corresponding to genes located in the target regions. Detection of intragenic inactivating point mutations or microdeletions by sequence analysis of the coding regions of NB1 and NB2 will implicate these genes as bona fide tumor suppressors. The mechanisms responsible for the inactivation of NB1 and NB2 in childhood neuroblastoma will be studied by a combination of FISH and DNA sequencing, and germline DNAs will be examined to learn if mutations in one allele of these loci can be inherited. It will also be important to determine the intracellular locations, developmental patterns of expression in the mouse, and biochemical properties of the NB1 and NB2 products. Finally, disruption of these genes by homologous recombination in embryonic stem cells, to generate chimeric mouse models, will help to clarify in vivo the roles of NB1 and NB2 in development and as tumor suppressors in tissues of the sympathetic nervous system.
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