Quantitative Proteomic Analysis of the Secretory Pattern of Senescent Cells

描述（申请人提供）：培养细胞的增殖性衰老是体外研究衰老的一个有用的模型系统。在体内，衰老细胞在衰老组织和年龄相关病变（如动脉粥样硬化病变和肿瘤前病变）的部位积累，这提高了衰老细胞可能促进衰老表型或年龄相关病变的可能性。事实上，衰老细胞分泌的蛋白质可以改变周围组织的功能和结构。除正常细胞外，肿瘤细胞在化疗药物的作用下也会发生增殖性衰老。重要的是，在衰老肿瘤细胞中诱导的许多基因编码具有促瘤和抑瘤活性的分泌蛋白，这可以影响肿瘤患者的预后。因此，新出现的假设是，衰老细胞的蛋白质分泌介导了体内衰老过程和与年龄相关的病理，以及肿瘤对治疗的反应。在这种情况下，重要的是要注意，只有少数的研究已经进行了从衰老细胞分泌的蛋白质，我们仍然缺乏全面的知识，正常以及肿瘤细胞的分泌模式经历衰老。我们采用一种新的定量蛋白质组学技术，ICAT（同位素编码亲和标签），建立了一种系统地鉴定和定量培养上清中分泌的蛋白质的方法，并利用该方法分析了人成纤维细胞在复制衰老过程中的分泌模式。我们建议通过1)比较不同机制诱导衰老的衰老成纤维细胞的分泌模式，2)比较衰老成纤维细胞和衰老内皮细胞的分泌模式，3)分析化疗药物治疗诱导衰老的肿瘤细胞的分泌模式来扩展这一分析。通过这个探索性项目确定的衰老细胞的分泌模式将为未来研究控制衰老过程的基本原理以及更有效地治疗和预防与年龄有关的疾病和癌症提供基础。

DESCRIPTION (provided by applicant): Proliferative senescence of cultured cells is a useful model system to study aging in vitro. In vivo, senescent cells accumulate in aging tissues and at sites of age-related pathologies such as atherosclerotic lesions and preneoplastic lesions, raising the possibility that senescent cells may promote the aging phenotype or age-related pathologies. Indeed, senescent cells were shown to secrete proteins that can alter the function and architecture of the surrounding tissues. In addition to normal cells, tumor cells also undergo proliferative senescence upon treatment with chemotherapeutic drugs. Importantly, many genes that are induced in senescent tumor cells encode secreted proteins with both tumor-promoting and tumor-suppressing activities, which can affect the prognosis of tumor patients. Therefore, the emerging hypothesis is that protein secretion from senescent cells mediates the in vivo aging process and age-related pathologies, as well as the tumor response to therapy. In this context, it is important to note that only a small number of studies have been carried out on proteins secreted from senescent cells and that we still lack comprehensive knowledge of the secretory patterns of normal as well as tumor cells undergoing senescence. Employing a novel quantitative proteomics technology, ICAT (isotope-coded affinity tag), we have developed a method to systematically identify and quantify proteins secreted in culture supernatant, and using this method, we analyzed the secretory pattern of human fibroblasts undergoing replicative senescence. We propose to extend this analysis by 1) comparing the secretory patterns of senescent fibroblasts induced to senesce by different mechanisms, by 2) comparing the secretory patterns of senescent fibroblasts and senescent endothelial cells, and by 3) analyzing the secretory patterns of tumor cells induced to senesce by chemotherapeutic drug treatment. The secretory patterns of senescent cells identified through this exploratory project will provide a basis for future investigations on the fundamental principles governing aging processes as well as more effective treatment and prevention of age-related diseases and cancer.