Telomere dysfunction, p53 and tumorigenesis

描述（由申请人提供）：端粒是覆盖真核染色体末端的核蛋白结构，由终止于3'单链富G突出端的TTAGGG重复序列组成。端粒由一系列复杂的蛋白质结合，这些蛋白质有助于稳定端粒的形成，并在防止DNA损伤检查点激活方面发挥重要作用。最近的证据表明，端粒功能障碍与人类癌症发病率的增加有关。端粒功能障碍导致双着丝粒染色体的产生，其启动断裂-融合-桥（BFB）循环以引发癌症促进基因型。我们观察到，条件性删除Pot 1（端粒的保护），一个关键的端粒结合蛋白，在小鼠胚胎成纤维细胞（MEFs）导致端粒功能障碍，激活DNA损伤反应（DDR），和p53依赖性复制衰老在体外。此外，Pot 1的缺失与p53缺陷协同启动体内肿瘤发生。本研究的目的是建立条件性Pot 1基因敲除小鼠模型，以：1）表征Pot 1诱导的p53依赖性复制衰老的分子决定因素; 2）确定p53在Pot 1缺失引发的体内肿瘤发生中的生物学功能;以及3）对肿瘤样品中的染色体畸变进行细胞遗传学表征，并研究这些畸变在人类肿瘤发生和发展中的功能。为了实现目标，Pot 1缺陷小鼠将与细胞衰老丧失（p21敲除小鼠）或细胞凋亡组分丧失（p53 R172 P敲入小鼠）的小鼠杂交，以比较端粒功能障碍的小鼠队列中肿瘤发展的潜伏期和肿瘤谱。最后，分子细胞遗传学技术，如光谱核型分析（SKY），阵列比较基因组杂交（aCGH）和基于慢病毒的siRNA将被用来确定染色体畸变和验证潜在的分子靶点参与肿瘤发生的条件性缺失的Pot 1小鼠队列。本研究的长期目标是确定参与端粒功能障碍背景下人类癌症起始或/和进展的分子靶点。

DESCRIPTION (provided by applicant): Telomeres, the nucleoprotein structures that cap the ends of eukaryotic chromosomes, are composed of TTAGGG repetitive sequences that terminate in a 3' single-stranded G-rich overhang. Telomeres are bound by a complex array of proteins that help stabilize its formation and serve essential roles in preventing DNA damage checkpoint activation. Recent evidence suggests that telomere dysfunction is associated with increased human cancer incidence. Telomere dysfunction leads to the generation of dicentric chromosomes that initiate the breakage-fusion-bridge (BFB) cycle to elicit a cancer promoting genotype. We observed that conditional deletion of Pot1 (protection of telomere), a critical telomere binding protein, in mouse embryo fibroblasts (MEFs) resulted in telomere dysfunction, activation of DNA damage response (DDR), and p53-dependent replicative senescence in vitro. In addition, loss of Pot1 cooperated with p53 deficiency to initiate tumorigenesis in vivo. The objective of this research project is to generate a conditional Pot1 knockout mouse model to: 1) characterize the molecular determinants of Pot1-induced p53-dependent replicative senescence; 2) to determine the biological function of p53 in loss of Pot1 initiated tumorigenesis in vivo; and 3) to cytogenetically characterize chromosomal aberrations in tumor samples, and investigate the function of these aberrations in human tumor initiation and progression. To achieve the objectives, Pot1-deficient mice will be crossed with mice bearing loss of cellular senescence (p21 knockout mouse) or apoptosis components (p53R172P knockin mice) to compare the latency of tumor development and tumor spectrum in mouse cohorts with dysfunctional telomeres. Finally, molecular cytogenetic techniques such as spectral karyotyping (SKY), array-comparative genomic hybridization (aCGH) and lentivirus-based siRNA will be used to identify chromosomal aberrations and validate potential molecular targets involved in tumorigenesis in conditionally deleted Pot1 mouse cohorts. The long-term goal of this study is to identify molecular targets involved in human cancer initiation or/and progression in the setting of telomere dysfunction.