A SYNTHETIC LETHAL STRATEGY TARGETING ANEUPLOID CELLS

癌细胞含有多种突变，这些突变导致了它们与正常细胞的表型差异。在观察到的基因变化中，有一组基因突变，这些基因保证了细胞分裂过程中染色体的正确划分。在各种肿瘤中，有丝分裂检查点的突变正在迅速被发现，并可能导致恶性肿瘤特征的非整倍体增加。这项建议的目标是(I)在哺乳动物细胞中建立一种新的负选择方案(合成致死性)，以及(Ii)应用该策略来识别在有丝分裂检查点缺陷细胞中失活时优先致死的二级分子靶标。已确定的二级靶点将使选择性靶向肿瘤的新化疗药物的开发成为可能。同基因有丝分裂检查点熟练的和缺陷的细胞将被编码部分人类cdna文库的标记的附体质粒转染。标签构成短的、唯一的寡核苷酸，便于通过互补的寡核苷酸微阵列定量检测编码的基因。部分构建的cDNA通过显性的负或反义机制来抑制相应野生型蛋白的功能/表达。通过比较转基因后立即恢复的质粒与几代细胞后出现的质粒，可以鉴定出具有致命性表型或抑制细胞生长的个别质粒。对有丝分裂检查点缺陷细胞具有选择性毒性的构建体将被检测到阵列杂交，并将通过序列分析来鉴定该基因。表达的构建体与有丝分裂检查点的关系将通过使用全长和部分cDNA克隆的瞬时转基因研究来表征。综上所述，本研究的目的是在哺乳动物细胞中建立一种新的负选择系统，应用该系统来鉴定在有丝分裂检查点缺陷细胞中优先致死的显性负性突变体，并表征这些被选择的蛋白质结构在纺锤体组装中的功能参与。被分离的部分cDNA克隆表达抑制的蛋白质代表有丝分裂检查点缺陷细胞所必需的蛋白质，并构成潜在的化疗药物靶点。

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.