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

描述（由申请人提供）：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）修复和细胞周期进程的机制。这些过程的适当协调是维持细胞活力和预防可能导致癌症的基因组不稳定性所必需的。淋巴细胞是研究细胞对生理水平DSB反应的极好模型系统，因为发育中的淋巴细胞必须在V（D）J重组和类别转换重组（CSR）过程中诱导和修复若干DSB。在B细胞中IgH基因座和T细胞中TCR基因座的完全V（D）J重组上调细胞周期蛋白D3，然后诱导快速增殖的爆发。在次级淋巴器官中暴露于抗原的成熟B细胞类似地诱导D3表达、增殖并开始CSR过程。D3蛋白和mRNA的表达在原代小鼠淋巴细胞中下调，以响应IR诱导的DSB，通过一种需要肿瘤抑制因子ATM和p53以及microRNA（miR）加工酶Dicer的机制。初步数据表明，D3的表达主要是通过增加mRNA周转抑制。这与ATM和p53调节参与DSB应答的miR表达的几种已知途径一致。目的1旨在揭示ATM和p53依赖的机制，通过DSB下调D3的表达，特别强调miR介导的途径。为了实现这一目标，3'UTR的突变分析将与miR过表达和敲低测定相结合，以鉴定靶向D3 mRNA的特异性miR，并确认这些miR的功能活性。对于发现靶向D3的miR，生物化学研究将 研究ATM和p53如何增强miR生物合成。目的2通过使用骨髓重建来产生造血系统表达不能正常受断裂调控的突变型D3 cDNA的小鼠，来解决DSB诱导的D3抑制的体内相关性。利用几种可用于研究淋巴细胞发育，基因组稳定性和恶性肿瘤的技术，拟议的实验将测试这一假设，即发育中的淋巴细胞与未修复的断裂必须下调D3的表达，以避免S期进入，以防止基因组不稳定，将导致细胞凋亡或淋巴瘤。流式细胞术将解决D3失调是否会破坏正常的淋巴细胞发育，基于PCR的测定将确定在D3失调的情况下未修复的断裂是否异常地进入S期。最后，将对嵌合体进行老化以监测肿瘤的发展，并对产生的肿瘤进行分子表征。由于DSB修复和细胞周期进程的协调缺陷是许多人类免疫缺陷和淋巴瘤的基础，因此拟议的工作将大大有助于我们了解这些病理的病因和治疗。

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.