Spatiotemporal Regulation of Chk1 in Cell Biology, Cancer Etiology and Therapy

描述（由申请人提供）：DNA，位于我们细胞内的生命蓝图，不断受到环境和细胞内DNA损伤剂的攻击。如果不加以纠正，这些错误可能会导致癌症的发展。幸运的是，细胞已经建立了精致的DNA监视网络，称为细胞周期检查点，用于检测和修复受损的DNA。细胞周期检查点的核心是一种蛋白激酶Chk1，它对细胞活力和哺乳动物胚胎发育至关重要。有趣的是，越来越多的证据表明Chk1也可能有利于肿瘤的生长，因为Chk1在恶性肿瘤中表达增加，其水平与肿瘤分级呈正相关。最重要的是，我们最近发现Chk1主要参与癌细胞对化疗药物的耐药性。这些发现强调了Chk1在癌症病因学和治疗中的重要性。因此，更好地了解Chk1调控不仅提供了DNA损伤反应的见解，而且可能导致癌症治疗新策略的发展。本实验室和其他人最近的研究揭示了一个令人兴奋的Chk1时空调节模型，其中DNA损伤诱导磷酸化和Chk1从染色质快速释放到细胞核，随后进入细胞质。Chk1的染色质释放失败会损害细胞周期检查点。另一方面，活跃Chk1蛋白的细胞积累导致癌细胞对临床使用的化疗药物喜树碱（CPT）产生耐药性。这可能是因为chk1富集的细胞能够更好地处理CPT引起的DNA损伤。因此，一个普遍的假设是Chk1从一个细胞室到另一个细胞室的动态动员对于响应DNA损伤的细胞周期检查点的激活以及化疗反应至关重要。为了验证这一假设，我们首先希望了解Chk1在细胞中染色质->核->细胞质动员的详细分子机制。我们将首先确定调节Chk1（一种非dna结合蛋白）的染色质结合和解离的因素（目的1）。其次，我们将定义Chk1如何从细胞核输出到细胞质（目的2）。此外，我们将探讨Chk1时空调节的功能意义。为此，我们将利用该实验室建立的一种新型体外系统，该系统高度概括了细胞周期检查点，以及我们用Chk1突变蛋白特异性定位在特定细胞室中产生的独特细胞系（目的2）。最后，我们将把这项研究从纯粹的生化和分子研究扩展到一些将为癌症病因学和化疗提供新见解的东西，通过测试两个假设：一个是阻断Chk1的时空动员使癌细胞对化疗药物敏感，另一个是Chk1是人类肿瘤对化疗治疗反应的预测标志物（目的3）。

DESCRIPTION (provided by applicant): DNA, the blueprint of life located inside our cells, is under continuous assault by both environmental and intracellular DNA damaging agents. If not corrected, these errors may lead to the development of cancer. Fortunately, cells have built up elegant DNA surveillance networks, termed cell cycle checkpoints, to detect and repair damaged DNA. Central to cell cycle checkpoints is a protein kinase, Chk1, which is essential for cell viability and mammalian embryonic development. Intriguingly, increasing evidence suggests that Chk1 may benefit tumor growth as well, as Chk1 expression is increased in malignant tumors and its level positively correlates with tumor grade. Most importantly, we recently showed that Chk1 is centrally involved in the resistance of cancer cells to chemotherapeutic drugs. These findings highlight the importance of Chk1 in cancer etiology and therapy. Therefore, a better understanding of Chk1 regulation not only provides insights the DNA damage response, but may also lead to the development of novel strategies in cancer therapy. Recent studies from this laboratory and others revealed an exciting spatiotemporal regulation model of Chk1, in which DNA damage induces phosphorylation and a rapid release of Chk1 from chromatin into the nucleus, and subsequently into the cytoplasm. Failure to undergo chromatin release of Chk1 impairs cell cycle checkpoints. On the other hand, cellular accumulation of active Chk1 proteins leads to the resistance of cancer cells to a clinically used chemotherapeutic drug, camptothecin (CPT). This is probably because Chk1-profient cells are better equipped to handle DNA damage induced by CPT. Thus, a general hypothesis is that the dynamic mobilization of Chk1 from one cellular compartment to another is crucial for the activation of cell cycle checkpoints in response to DNA damage, as well as for the chemotherapy response. In order to test this hypothesis, we first wish to understand the detailed molecular mechanisms underlying this chromatin->nucleus->cytoplasm mobilization of Chk1 in cells. We will first determine the factors that regulate chromatin association and disassociation of Chk1, a non-DNA binding protein (Aim 1). Second, we will define how Chk1 is exported from the nucleus to the cytoplasm (Aim 2). Further, we will interrogate the functional significance of the spatiotemporal regulation of Chk1. To do so, we will utilize a novel in vitro system established by this laboratory that highly recapitulates cell cycle checkpoints, as well as unique cell lines we generated with Chk1 mutant proteins specifically localized in a particular cellular compartment (Aim 2). Lastly, we will expand this study from a pure biochemical and molecular research into something that will offer novel insights into cancer etiology and chemotherapy through testing two hypotheses: one is that blocking the spatiotemporal mobilization of Chk1 sensitizes cancer cells to chemotherapeutic drugs, and the other is that Chk1 is a predictive marker for the therapy response of human tumors to chemotherapy (Aim 3).