Functional analysis of cell cycle regulators by in vitro gene targeting using DT40 cells

DNA损伤后基因组完整性的维持依赖于DNA修复和检查点系统的协调作用。检查点的失效会导致基因组的不稳定，从而导致多种DNA序列的改变和染色体的异常，并易于发生癌症。通过对酵母和果蝇的遗传学分析，可以鉴定出各种调节检查点的分子。我们的实验室已经确定了这些分子在哺乳动物中的同源物。为了研究这些分子在脊椎动物细胞中的功能，我们使用超重组鸡B细胞系DT40进行了体外基因敲除。本课题的主要研究结果如下：1)后期促进复合体(APC)被两种调控蛋白--CDC20和CDH1激活。在酵母和果蝇中，CDH1-APC活性针对从有丝分裂结束到G1期的有丝分裂周期蛋白。为了研究cdh1在脊椎动物细胞中的功能，我们获得了破坏I…的鸡dt40细胞的克隆。更多的人在他们的CDH1基因座上。CDH1对细胞活力和细胞周期进程是必不可少的。然而，CDH1的缺失导致有丝分裂周期蛋白在G1期的非程序性积聚，导致雷帕霉素诱导的G1期停滞被取消。此外，我们还发现，CDH1^&lt；-/-&gt；细胞不能维持DNA损伤诱导的G2期停滞，而CDH1-APC被X射线辐射诱导的DNA损伤激活。因此，CDH1-APC的激活在依赖CDK抑制剂的G1期和DNA损伤诱导的G2期阻滞中都起着至关重要的作用。2)在果蝇中，瘤基因是基于其作为肿瘤抑制因子的能力而被鉴定的。我们以前发现了一种人类的疣的同源物，称为疣，并表明在有丝分裂过程中，人的疣激酶定位于有丝分裂器官。我们产生了疣(-/-)DT40克隆，并发现这些克隆的后期有丝分裂显著延长。此外，许多细胞不能进行胞质分裂。这些发现表明，疣与脊椎动物细胞的晚期有丝分裂事件有关，其功能的失活可能会导致正常的有丝分裂进程失败，导致染色体不稳定。较少

Maintenance of genomic integrity after DNA damage depends on the coordinated action of the DNA repair and checkpoint systems. The failure of checkpoint leads to genomic instability, which causes multiple DNA sequence alterations and chromosomal aberrations, and disposition to cancer.Genetic analyses of yeast and Drosophila allowed identification of various molecules regulating checkpoints. Our laboratory has identified mammalian homologues of those molecules. To investigate the function of those molecules in vertebrate cells, we performed in vitro gene knock-out using the hyper-recombinogenic chicken B-cell line DT40. Followings are major findings we obtained in this project :1) Anaphase-promoting complex (APC) is activated by two regulatory proteins, Cdc20 and Cdh1. In yeast and Drosophila, Cdh1-APC activity targets mitotic cyclins from the end of mitosis to the G1 phase. To investigate the function of Cdh1 in vertebrate cells, we generated clones of the chicken DT40 cells disrupted i … More n their Cdh1 loci. Cdh1 was dispensable for viability and cell cycle progression. However, loss of Cdh1 induced unscheduled accumulation of mitotic cyclins in G1 resulting in abrogation of G1l arrest caused by treatment with rapamycin, an inducer of p27^<kip1>. Furthermore, we found that Cdh1^<-/-> cells fail to maintain DNA-damage induced G2 arrest and that Cdh1-APC is activated by X-radiation-induced DNA damage. Thus, activation of Cdh1-APC plays a crucial role in both Cdk-inhibitor-dependent G1 arrest and DNA-damage-induced G2 arrest.2) The warts gene was identified based on its ability to act as a tumor suppressor in Drosophila. We previously identified a human homologue of the warts, termed WARTS and showed that the human WARTS kinase is localized to mitotic apparatus during mitosis. We generated WARTS (-/-) DT40 clones and found that late mitosis is significantly prolonged in those clones. Furthermore, many of the cells fail to undergo cytokinesis. These findings suggest that WARTS is involved in late mitotic events in vertebrate cells and that inactivation of its function may result in failure in normal mitotic progression, leading to chromosomal instability. Less