A SYNTHETIC LETHAL STRATEGY TARGETING ANEUPLOID CELLS

癌细胞包含多种突变，导致其与正常细胞的表型差异。在观察到的遗传改变中，有一组基因发生了突变，这些基因保证了细胞分裂过程中染色体的正确分配。有丝分裂检查点的突变在各种肿瘤中被迅速发现，并可能导致恶性肿瘤特征的非整倍体增加。本提案的目标是：(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），（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性的次级分子靶点。确定的次级靶点将使开发选择性靶向肿瘤的新化疗药物成为可能。同基因有丝分裂检查点熟练和缺陷的细胞将被标记的编码部分人类cDNA文库的外胚质粒转染。这些标签构成短而独特的寡核苷酸，通过互补寡核苷酸微阵列，便于质粒编码基因的定量检测。部分cDNA结构通过显性负或反义机制抑制相应野生型蛋白的功能/表达。通过将转染后立即恢复的质粒与几代细胞后立即恢复的质粒进行比较，可以确定赋予致死表型或抑制细胞生长的单个质粒。对有丝分裂检查点缺陷细胞有选择性毒性的构建体将通过阵列杂交检测，cDNA将通过序列分析鉴定。表达的构建体与有丝分裂检查点的关系将通过利用全长和部分cDNA克隆的瞬时转染研究来表征。总之，本研究的目的是在哺乳动物细胞中建立一个新的负选择系统，应用该系统识别在有丝分裂检查点缺陷细胞中优先致死的显性负突变体，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中必需的蛋白质，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.