Chk1 signaling in the G2 DNA damage checkpoint

描述(由申请人提供)：这项建议集中在检查点，该检查点监测G2的DNA损伤，并防止有丝分裂发生，直到DNA修复完成。在G1/S和G2/M转换期有明显的检查点通路发挥作用，延迟进入S期或在DNA损伤的情况下进行有丝分裂。G1/S检查点基因突变，如P53，在肿瘤中普遍存在，促进了基因组的不稳定，促进了肿瘤的演变，并取消了导致化疗耐药的凋亡反应。G2检查点基因的突变非常罕见，这表明它们对肿瘤细胞的生存能力很重要。这项研究的长期目标是全面剖析G2检查点反应的生物学，并在此基础上设计和评估靶向抗癌治疗，特别是对携带P53突变的肿瘤的治疗。这一提议的核心是一系列实验，旨在研究这一检查点的关键组成部分Chk1蛋白激酶的调控。G2检查点和它们调控的细胞周期调节器在进化过程中高度保守。因此，这里描述的实验将在人类细胞、小鼠和裂殖酵母中进行，裂殖酵母长期以来一直被用作G2细胞周期控制的范例。为了了解Chk1的功能和调节机制，酵母系统的遗传学将被用于一系列基于功能丧失和功能获得的Chk1等位基因集合的筛选。这些实验还将涉及已经确定的改变Chk1功能的基因的克隆，以及建立在这种遗传学基础上的检查点信号的生化解剖。检查点受阻细胞重新进入细胞周期的机制(S)也将被调查。同时，将在培养的人类细胞中总结和扩展这些发现，采用生化方法研究野生型和突变型Chk1，以及在S.pombe中被确认为调节Chk1功能的蛋白质的同源物。最后，我们将基于已经在小鼠肿瘤细胞中抑制Chk1信号的细胞系中获得的数据，来研究G2检查点抑制作为一种靶向抗癌治疗的用途。这项研究将产生对细胞周期的基本生物学和基因组的稳定性以及癌症治疗的新方法和新靶点的测试都很重要的数据。

DESCRIPTION (provided by applicant): This proposal focuses on the checkpoint that monitors DNA damage in G2 and prevents mitosis from occurring until DNA repair is completed. There are distinct checkpoint pathways functioning at the G1/S and G2/M transitions that delay entry into S-phase or mitosis in the presence of DNA damage. Mutations in G1/S check point genes, such as p53, are commonplace in tumors, promoting genomic instability, tumor evolution and abolish apoptotic responses leading to chemo resistance. Mutations in G2 check point genes are extremely infrequent, suggesting they are important for tumor cell viability. The long-term goal of this study is to fully dissect the biology of G2 checkpoint responses, and on the basis of this, to devise and assess targeted anti-cancer therapies, especially for the treatment of tumors bearing mutations in p53. Central to this proposal is a series of experiments that aim to investigate regulation of a key component of this checkpoint, the Chk1 protein kinase. The G2 checkpoints and the cell cycle regulators that they regulate are highly conserved in evolution. Therefore, the experiments described here will be carried out in both human cells, mice, and in the fission yeast Schizosaccharomyces pombe, the organism that has long been used as a paradigm of G2 cell cycle control. To understand mechanisms of Chk1 function and regulation, the genetics of the yeast system will be utilized in a series of screens based on a large collection of both loss- and gain-of- function Chk1 alleles. These experiments will also involve the cloning of genes already identified to alter Chk1 function and the biochemical dissection of checkpoint signaling that builds on this genetics. The mechanism(s) by which checkpoint arrested cells re-enter the cell cycle will also be investigated. In parallel, findings will be recapitulated and expanded upon in human cells in culture, taking biochemical approaches to study wildtype and mutant Chk1, and homologs of proteins identified as modulating Chk1 function in S. pombe. Finally, we will build on data already obtained in cell lines to inhibit Chk1 signaling in tumor cells in the mouse to investigate the utility of G2 checkpoint inhibition as a targeted anti-cancer therapy. The study will yield data that will be important in terms of both the basic biology of the cell cycle and stability of the genome and also in the testing of new approaches and new targets for cancer therapy.