Cancer cells contain multiple mutations that contribute to their phenotypic differences from normal cells. Among the genetic alterations observed are mutations in a set of genes that guarantees the correct partitioning of chromosomes during cell division. Mutations in the mitotic checkpoint are being rapidly identified in a variety of tumors and can result in the increased aneuploidy that characterizes malignancies. The goals of this proposal are to (i) establish a novel negative selection scheme (synthetic lethality) in mammalian cells and (ii) apply this strategy to identify secondary molecular targets that are preferentially lethal when inactivated in mitotic checkpoint defective cells. The identified secondary targets will enable the development of new chemotherapeutic drugs that selectively target tumors. Isogeneic mitotic checkpoint proficient and deficient cells will be transfected with tagged episomal plasmids that encode a partial human cDNA library. The tags constitute short, unique oligonucleotide, that facilitate the quantitative detection of plasmid encoded genes, by complementary oligonucleotide micro- arrays. The partial cDNA constructs serve to inhibit the function/expression of the corresponding wild-type protein through a dominant negative or anti-sense mechanism. Individual plasmids that confer a lethal phenotype or suppress cell growth will be identified by comparing the plasmids recovered immediately following transfection to that present after several cell generations. Constructs that are selectively toxic to mitotic checkpoint defective cells will be detected array hybridization and the cDNA will be identified by sequence analysis. The relationship of the expressed constructs to the mitotic checkpoint will be characterized by transient transfection studies utilizing both the full length and partial cDNA clones. In summary, the aims of the proposed studies are to establish a novel system for negative selection in mammalian cells, apply this system to identify dominant negative mutants that are preferentially lethal in mitotic checkpoint defective cells, and to characterize the functional involvement of these selected protein constructs in spindle assembly. The proteins inhibited by the expression of isolated partial cDNA clones represent proteins that are essential in mitotic checkpoint deficient cells and constitute potential chemotherapeutic drug targets.
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