A SYNTHETIC LETHAL STRATEGY TARGETING ANEUPLOID CELLS

癌细胞含有多种突变，这些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，有一组基因发生突变，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快速鉴定，并可导致恶性肿瘤特征性的非整倍体增加。 该提案的目标是（i）在哺乳动物细胞中建立一种新的阴性选择方案（合成致死性），以及（ii）应用该策略来鉴定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。 确定的次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。将用编码部分人cDNA文库的标记的附加型质粒转染同基因有丝分裂检查点熟练和缺陷细胞。 标签构成短的、独特的寡核苷酸，其有助于通过互补寡核苷酸微阵列定量检测质粒编码的基因。 部分cDNA构建体用于通过显性负或反义机制抑制相应野生型蛋白的功能/表达。 通过比较转染后立即回收的质粒与几代细胞后存在的质粒，鉴定赋予致死表型或抑制细胞生长的单个质粒。 将通过阵列杂交检测对有丝分裂检查点缺陷细胞具有选择性毒性的构建体，并通过序列分析鉴定cDNA。 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，所提出的研究的目的是建立一个新的系统，在哺乳动物细胞中的负选择，应用该系统来识别显性负突变体，优先致命的有丝分裂检查点缺陷的细胞，并表征这些选定的蛋白质结构的功能参与纺锤体组装。 被分离的部分cDNA克隆的表达抑制的蛋白质代表在有丝分裂检查点缺陷细胞中必需的蛋白质，并且构成潜在的化疗药物靶标。

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.