Telomere dysfunction, p53 and tumorigenesis

描述(申请人提供)：端粒是覆盖真核染色体末端的核蛋白结构，由TTAGGG重复序列组成，末端为3‘-单链富含G的突出部分。端粒被一系列复杂的蛋白质结合在一起，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关键作用。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，双着丝粒染色体启动断裂-融合-桥(BFB)循环，从而引发致癌基因。我们观察到，在体外培养的小鼠胚胎成纤维细胞(MEF)中，关键的端粒结合蛋白POT1(端粒保护)的条件性缺失导致了端粒功能障碍、DNA损伤反应(DDR)的激活和P53依赖的复制性衰老。此外，POT1的缺失与P53的缺失协同在体内启动了肿瘤的发生。本研究的目的是建立一种条件性POT1基因敲除小鼠模型，以：1)研究POT1诱导的P53依赖的复制衰老的分子决定因素；2)确定P53在体内丢失POT1启动的肿瘤发生中的生物学功能；3)对肿瘤标本中的染色体异常进行细胞遗传学表征，并研究这些异常在人类肿瘤的发生和发展中的作用。为了实现这些目标，POT1缺陷小鼠将与细胞衰老缺失的小鼠(p21基因敲除小鼠)或凋亡成分缺失的小鼠(p53R172P敲门小鼠)杂交，以比较端粒功能异常的小鼠队列中肿瘤发展的潜伏期和肿瘤光谱。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将被用于识别染色体异常，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。这项研究的长期目标是确定在端粒功能障碍的背景下参与人类癌症启动或/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.