The development of colon cancer involves the loss of genes development which control the normal and proliferative potential of cells. These tumor suppressor genes (TSG) are almost invariably inactivated by nonsense or frame shift mutations in the coding region. This observation is so consistent that currently the main way of identifying tumor suppressor genes is to demonstrate the presence of mutations in the tumor cells. Unfortunately, positional cloning methods and candidate gene approaches to find TSGs have been very labor intensive and unsuccessful. Clearly, an unbiased, robust and high-throughput method to detect these mutations would accelerate the discovery of these genes and promote a better understanding of colon tumorigenesis as well as provide many new novel targets for therapeutic intervention. Mutations that inactivate TSG are usually nonsense or frameshift mutations that frequently result in the rapid degradation of the mutant mRNA through the mechanism of nonsense mediated decay (NMD). Inhibition of NMD results in an increase of mutant mRNA in the cell which can then be detected using cDNA arrays. Unfortunately, the drugs used to inhibit NMD also result in mRNA increases for a large number of transcripts due to stress response. This fact complicates the identification of the mutated genes. The goal of this proposal is to use various drug combinations, siRNA, dominant-negative regulators of NMD as well as nonsense suppressor tRNA constructs to develop a robust and high-throughput method to identify mutant genes in colon cancer cells. Alternatively, to minimize the interference of stress response genes we will apply simple drug treatment to inhibit NMD in subclones of different degree of malignancy of the same cell lines. Since the stress response in the subclones of the same cell lines will be similar, the differences in response to drug treatment can indicate the presence of the mutations. Such an approach will help to identify metastasis suppressor genes in colon cancer.
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