The predicted discovery of tumor suppressor genes (TSGs) more than 25 years ago opened an important new avenue of cancer research. The study of TSGs should not only accelerate basic cancer research, but also assist in the early diagnosis, prognostication, and treatment of human cancers. A rapidly increasing number of putative TSGs have now been identified, and at least one has been mapped to each autosomal chromosome. Since this appears to be an immensely important class of genes, l will focus on the isolation and characterization of novel TSGs in human esophageal cancer. Previous methods of cloning TSGs usually involved positional cloning, i.e., from the chromosomal locus to the gene. This approach is not only time-consuming, but it also necessitates confirmation of the function of the gene after it is obtained positionally. My method will work from the gene outward, utilizing a functional screen to identify potential TSGs. The approach l take will be to construct a cDNA eukaryotic expression library from normal human esophageal epithelium and utilize a large number of random clones from it to transfect esophageal cancer cells. Gene(s) found to inhibit growth of esophageal cancer cells will be partially sequenced and compared to the GenEMBL database. Once isolated, tumor- specific alterations such as point mutations or microdeletions in cloned TSGs will be investigated in primary human esophageal carcinomas. Functions of gene(s) isolated in this fashion will be further tested by checking protein-protein interactions and analyses of promoter regions. Cellular interacting targets of the genes, as well as factors regulating them, will also be explored.
