EXAMINATION OF UNDEFINED MOLECULAR ALTERATIONS IN GLIOMAS

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The acquisition and progression of the tumorigenic capabilities of neoplastic cells involves genetic alterations leading to the activation of oncogenes and the loss of function of tumor suppressor genes. The cumulative objective of this project is to identify and characterize a tumor suppressor gene localized to the long arm of chromosome 4 that we have recently identified to be involved in the malignant progression of human gliomas. A number of molecular alterations have previously been identified to occur in gliomas, although additional sites of genetic damage would be anticipated to account for the multiple alterations required for oncogenesis. Recently, we have demonstrated the unexpected involvement of a chromosome 4 tumor suppressive locus. This was accomplished by the microcell-mediated transfer of a chromosome 4 into a glioma cell and demonstration of a suppression of the hybrids cells tumorigenic phenotype. Control chromosomal transfers had no phenotypic effects. The inserted chromosome 4 was fragmented, thus only a small segment of the chromosome was retained in the suppressed hybrid cells. This fragment has been partially characterized and shown to contain three regions of chromosome 4; two regions of approximately 1-2 CM and another approximately 7-8 CM. These regions have been molecularly identified and YAC contigs have been generated spanning the involved regions. Initial allelotyping analyses of gliomas and head and neck squamous cell carcinomas specimens, the latter of which have previously been reported to involve deletions on chromosome 4, have implicated one of the retained regions to display consistent loss of heterozygosity (LOH). The localization of two human cancers exhibiting consistent LOH and our chromosomal fragment to independently identify the same small region of chromosome 4, provides strong evidence for the presence of a tumor suppressor gene. To identify the responsible gene, a modified positional cloning strategy will be pursued. Initially, the region will be extensively analyzed for losses or other possible landmarks that may identify the critical region containing the tumor suppressor gene. Also, the possible deletion or rearrangement of the suppressive region in hybrid cells will be functionally and molecularly assessed. Isolated cosmids localized to the various regions will be utilized to further identify the critical region and for generation of cosmid contigs. Once the critical region is defined, expressed gene sequences within the region will be identified and analyzed for alterations in involved neoplasms. This combination of functional and molecular approaches has identified a novel tumor suppressive locus and provided us with an unique means to identify the responsible gene.