Cyclin D3 and DNA double strand breaks: genome stability and lymphomagenesis

描述(申请人提供)：本申请的目的是阐明发育中的淋巴细胞协调DNA双链断裂(DSB)修复和细胞周期进展的机制。这些过程的适当协调是维持细胞活力和防止可能导致癌症的基因组不稳定所必需的。淋巴细胞在V(D)J重组和类开关重组(CSR)过程中必须诱导和修复多个DSB，因此淋巴细胞是研究细胞对DSB生理水平反应的良好模型系统。在B细胞中，IgH基因上的V(D)J完全重组，而T细胞中的TCR？基因位点上调了细胞周期蛋白D3，从而导致了细胞的快速增殖。成熟的B细胞暴露在次级淋巴器官中，类似地诱导D3的表达，增殖，并开始CSR的过程。在IR诱导的DSB诱导下，原代小鼠淋巴细胞D3蛋白和mRNA的表达下调，这是通过一种需要肿瘤抑制因子ATM和p53以及microRNA(MiR)加工酶Disher的机制实现的。初步数据表明，D3的表达主要是通过增加mRNA周转率来抑制的。这与ATM和P53调节参与DSB反应的miR表达的几个已知途径是一致的。目的1试图揭示依赖ATM和P53的机制，DSB通过这些机制下调D3的表达，特别强调miR介导的途径。为了实现这一目标，对3‘UTR的突变分析将与miR过表达和基因敲除试验相结合，以确定针对D3mRNA的特定miR，并确认这些miRs的功能活性。对于被发现针对D3的miRs，生化研究将 研究ATM和P53如何促进miR的生物发生。目的2研究DSB诱导的D3抑制在体内的相关性，方法是通过骨髓重建来产生其造血系统表达突变的D3基因的小鼠，该突变的D3基因不能被BREAKS正常调节。利用几种可用于研究淋巴细胞发育、基因组稳定性和恶性程度的技术，拟议的实验将检验这样一种假设，即具有未修复断裂的发育中的淋巴细胞必须下调D3的表达，以避免进入S期，以防止可能导致细胞凋亡或淋巴瘤的基因组不稳定。流式细胞术将研究D3异常是否扰乱正常淋巴细胞的发育，基于聚合酶链式反应的分析将确定在存在异常D3的情况下，未修复的断裂是否异常地进入S期。最后，嵌合体将老化以监测肿瘤的发展，并对由此产生的肿瘤进行分子特征分析。由于DSB修复和细胞周期进展的协调缺陷是许多人类免疫缺陷和淋巴瘤的基础，拟议的工作将大大有助于我们对这些病理的病因和治疗的了解。

DESCRIPTION (provided by applicant): The goal of this application is to elucidate mechanisms by which developing lymphocytes coordinate DNA double strand break (DSB) repair and cell cycle progression. Proper coordination of these processes is required for maintenance of cellular viability and prevention of genomic instability that can cause cancer. Lymphocytes are an excellent model system to study the response of cells to physiologic levels of DSBs, since developing lymphocytes must induce and repair several DSBs during the processes of V(D)J recombination and class switch recombination (CSR). Complete V(D)J recombination at the IgH locus in B cells, and the TCR¿ locus in T cells upregulates cyclin D3, which then induces a burst of rapid proliferation. Mature B cells exposed to antigen in secondary lymphoid organs similarly induce D3 expression, proliferate, and begin the process of CSR. D3 protein and mRNA expression is downregulated in primary mouse lymphocytes in response to DSBs induced by IR, through a mechanism that requires the tumor suppressors ATM and p53, and the microRNA (miR) processing enzyme Dicer. Preliminary data suggest that D3 expression is repressed primarily through increased mRNA turnover. This is consistent with several known pathways by which ATM and p53 regulate the expression of miRs involved in the DSB response. Aim 1 seeks to uncover the ATM and p53-dependent mechanisms through which DSBs downregulate D3 expression, with a particular emphasis on miR-mediated pathways. To achieve this goal, mutational analysis of the 3'UTR will be combined with miR overexpression and knockdown assays to identify specific miRs that target the D3 mRNA, and to confirm functional activity of these miRs. For miRs found to target D3, biochemical studies will investigate how ATM and p53 may enhance miR biogenesis. Aim 2 addresses the in vivo relevance of DSB-induced D3 repression by using bone marrow reconstitution to generate mice whose hematopoietic systems express a mutant D3 cDNA that cannot be normally regulated by breaks. Taking advantage of several techniques available to study lymphocyte development, genome stability, and malignancy, the proposed experiments will test the hypothesis that developing lymphocytes with unrepaired breaks must downregulate D3 expression to avoid S phase entry, in order to prevent genome instability that would cause apoptosis or lymphoma. Flow cytometry will address whether D3 dysregulation disrupts normal lymphocyte development, and PCR-based assays will determine whether unrepaired breaks pass into S phase aberrantly in the presence of dysregulated D3. Finally, chimeras will be aged to monitor for tumor development, and resulting tumors will be molecularly characterized. Because defective coordination of DSB repair and cell cycle progression underlie many human immunodeficiencies and lymphomas, the proposed work will contribute substantially to our knowledge of the etiology and treatment of these pathologies.