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A novel regulator of cell cycle and cell death

细胞周期和细胞死亡的新型调节剂

基本信息

批准号：
7406757
负责人：
HONGLIN LI
金额：
$ 17.72万
依托单位：
LURIE CHILDREN'S HOSPITAL OF CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-04-15 至 2009-09-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7406757
关键词：
Affect Age Animals Apoptosis Apoptotic Attenuated Binding Proteins Biochemical Biological Assay CDC2 Protein Kinase Cdc25B protein Cell Cycle Cell Cycle Progression Cell Cycle Regulation Cell Cycle Stage Cell Death Cell division Cells Centrosome Checkpoint kinase 1 Chromosome Segregation Complex Cytokinesis DNA Damage DNA Repair DNA biosynthesis DNA damage checkpoint Data Defect Development Docking Ensure Event Foundations G2/M Transition Genetic Materials Genome Stability Genomic Instability Genotoxic Stress Goals Homeostasis Immunofluorescence Immunologic Lead Localized Malignant Neoplasms Mediating Microtubule-Organizing Center Mitosis Mitotic Mutagens Normal Cell Normal tissue morphology Organelles Pathogenesis Phase Phosphotransferases Play Principal Investigator Process Protein Overexpression Protein-Serine-Threonine Kinases Proteins Regulation Resistance Role Signal Pathway Signal Transduction Solid Testing Trimethoprim-Sulfamethoxazole aurora-A kinase base cancer cell cancer therapy chemotherapeutic agent cyclin B1 daughter cell driving force human PLK1 protein human disease insight novel novel therapeutics prevent programs promoter repaired response therapeutic target transmission process tumorigenesis

项目摘要

DESCRIPTION (provided by applicant): Our long-term goal is to elucidate the roles of a novel protein C53 in regulation of cell cycle progression and DNA damage response, and to explore potential exploitation of this protein as a novel therapeutic approach for cancer treatment. Regulation of cell cycle and cellular response to DNA damage is critical for animal development and ageing, and the pathogenesis of human diseases like cancer. Defects in cell cycle regulation and DNA damage response may lead to genome instability and oncogenesis. Cells utilize the so-called checkpoint mechanisms to ensure the completion of a particular phase before the next phase is initiated, thereby maintaining truthful transmission of genetic material. One of the key checkpoints is so-called the G2/M checkpoint that regulates mitotic entry and activation of cyclin-dependent kinase 1 (Cdk1), a driving force for progression of cell division. We have recently identified Cdk5 activator p35 binding protein C53 as a novel regulator of cell cycle and DNA damage response. Our study demonstrated that C53 depletion leads to defective cell cycle and resistance to apoptosis induced by chemotherapeutic agents. Additionally, C53 overexpression abolished the G2/M checkpoint-mediated Cdk1 inactivation, thereby sensitizing cancer cells to various DNA damage agents. We further found that C53 depletion results in inactivation of Cdk1 and delayed mitotic entry, while C53 overexpression antagonizes Chk1-mediated Cdk1 inactivation. Importantly, we found that a fraction of endogenous C53 protein localizes at the centrosome, an organelle that is emerging as a pivotal multi- functional platform that integrates many important signal pathways. Based upon our preliminary study, we hypothesize that C53 plays a crucial role in unperturbed cell cycle progression. By antagonizing the activity of centrosome-associated Chk1, C53 acts as a positive promoter of initial activation of cyclin B1/Cdk1 kinase at the centrosome. To test our hypothesis, we aim: 1) To Investigate the centrosomal association of C53. In this aim, by using GFP-centrin stable cells and immunofluorescence and biochemical analyses, we will investigate the centrosomal association of C53 and its docking mechanism. Moreover, We will make a centrosome-targeted C53 to test whether C53 centrosomal localization is important for its function. 2) To characterize the role of C53 in initial activation of Cdk1/cyclin B1 during the G2/M transition. In this aim, we will use synchronized cells and immunofluorescence assays to examine the effect of C53 on initial activation of Cdk1/cyclin B1 at the centrosome. Furthermore, we will test whether C53 exerts its function through affecting the activity of centrosomal Chk1 and other centrosome-associated mitotic regulators. This study will provide insight on a novel regulatory mechanism for cell cycle control and checkpoint response. It will lay a foundation for exploitation of this protein as a novel therapeutic target in cancer treatment.

描述（由申请人提供）：我们的长期目标是阐明新型蛋白C53在调节细胞周期进程和DNA损伤反应中的作用，并探索该蛋白作为癌症治疗的新型治疗方法的潜在开发。细胞周期和细胞对DNA损伤的反应的调节对于动物的发育和衰老以及癌症等人类疾病的发病机制至关重要。细胞周期调控和DNA损伤反应的缺陷可能导致基因组不稳定和肿瘤发生。细胞利用所谓的检查点机制来确保在下一阶段开始之前完成特定阶段，从而保持遗传物质的真实传输。其中一个关键检查点是所谓的G2/M检查点，它调节有丝分裂进入和细胞周期蛋白依赖性激酶1（Cdk 1）的激活，这是细胞分裂进程的驱动力。我们最近发现Cdk 5激活剂p35结合蛋白C53作为一种新的调节细胞周期和DNA损伤反应。我们的研究表明，C53缺失导致细胞周期缺陷和对化疗药物诱导的凋亡的抵抗。此外，C53过表达消除了G2/M检查点介导的Cdk 1失活，从而使癌细胞对各种DNA损伤剂敏感。我们进一步发现，C53耗竭导致Cdk 1失活和延迟有丝分裂进入，而C53过表达拮抗Chk 1介导的Cdk 1失活。重要的是，我们发现一部分内源性C53蛋白定位于中心体，中心体是一种正在成为整合许多重要信号通路的关键多功能平台的细胞器。基于我们的初步研究，我们假设C53在未受干扰的细胞周期进程中起着至关重要的作用。通过拮抗中心体相关的Chk 1的活性，C53作为一个积极的启动子，在中心体的细胞周期蛋白B1/Cdk 1激酶的初始激活。为了验证我们的假设，我们的目标是：1）调查C53的中心体关联。因此，我们将利用GFP-centrin稳定细胞，通过免疫荧光和生化分析，研究C53的中心体结合及其对接机制。此外，我们还将制备一个以中心体为靶点的C53，以检测C53的中心体定位是否对其功能有重要作用。2)探讨C53在G2/M期细胞周期蛋白Cdk 1/cyclin B1初始激活中的作用。在这个目标中，我们将使用同步化细胞和免疫荧光检测C53对Cdk 1/细胞周期蛋白B1在中心体的初始激活的影响。此外，我们将测试C53是否通过影响中心体Chk 1和其他中心体相关有丝分裂调节因子的活性来发挥其功能。这项研究将为细胞周期控制和检查点反应提供一种新的调控机制。为进一步开发该蛋白作为肿瘤治疗的新靶点奠定了基础。