Quantitative Proteomic Analysis of the Secretory Pattern of Senescent Cells

描述(申请人提供)：培养细胞的增殖衰老是研究体外衰老的一个有用的模型系统。在体内，衰老细胞聚集在衰老组织和与年龄相关的病变部位，如动脉粥样硬化性病变和癌前病变，增加了衰老细胞可能促进衰老表型或与年龄相关的病理的可能性。事实上，衰老的细胞可以分泌蛋白质，这些蛋白质可以改变周围组织的功能和结构。除了正常细胞外，肿瘤细胞在接受化疗药物治疗后也会经历增殖性衰老。重要的是，在衰老的肿瘤细胞中诱导的许多基因编码具有促肿瘤和抑瘤活性的分泌性蛋白，这些蛋白可以影响肿瘤患者的预后。因此，新的假设是，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，重要的是要注意到，只有少数人对衰老细胞分泌的蛋白质进行了研究，而且我们仍然缺乏对正常细胞和正在衰老的肿瘤细胞的分泌模式的全面了解。利用一种新的定量蛋白质组学技术--ICAT(同位素编码亲和标签)，我们建立了一种系统地鉴定和定量培养上清液中蛋白质分泌的方法，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成纤维细胞的分泌模式，2)比较衰老的成纤维细胞和衰老的内皮细胞的分泌模式，以及3)分析化疗药物诱导的肿瘤细胞的分泌模式来扩展这一分析。通过这一探索性项目确定的衰老细胞的分泌模式将为未来研究衰老过程的基本原理以及更有效地治疗和预防与年龄相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.