Telomere dysfunction, p53 and tumorigenesis

描述（由申请人提供）：端粒，覆盖真核生物染色体末端的核蛋白结构，由TTAGGG重复序列组成，终止于3'单链富含g的悬垂。端粒由一组复杂的蛋白质结合，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增加有关。端粒功能障碍导致双中心染色体的产生，启动断裂-融合桥（BFB）循环，引发促进癌症的基因型。我们观察到，小鼠胚胎成纤维细胞（MEFs）中关键端粒结合蛋白Pot1（保护端粒）的条件缺失导致端粒功能障碍、DNA损伤反应（DDR）激活和p53依赖性复制性衰老。此外，在体内，Pot1的缺失与p53的缺失共同启动了肿瘤的发生。本研究项目的目的是建立一个条件敲除po1小鼠模型，以：1)表征po1诱导的p53依赖性复制性衰老的分子决定因素；2)确定p53在体内丢失Pot1引发的肿瘤发生中的生物学功能；3)对肿瘤样本的染色体畸变进行细胞遗传学表征，并探讨这些畸变在人类肿瘤发生和发展中的作用。为了实现这一目标，将pot1缺陷小鼠与细胞衰老缺失的小鼠（p21敲除小鼠）或细胞凋亡成分缺失的小鼠（p53R172P敲除小鼠）杂交，比较端粒功能障碍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（SKY）、阵列-比较基因组杂交（aCGH）和基于慢病毒的siRNA，将用于鉴定染色体畸变，并验证在条件缺失的Pot1小鼠队列中参与肿瘤发生的潜在分子靶点。本研究的长期目标是在端粒功能障碍的情况下确定参与人类癌症起始或/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.