Functional Analysis of metastasis-Associated Proteins

描述（由申请人提供）：本项目的总体目标是获得关于肿瘤转移分子机制的新信息，以有助于理解该过程的发病机制并获得临床有用的数据。所提出的方法旨在从识别和验证转移相关蛋白和转录物，到在匹配的转移性和非转移性克隆细胞系中对特定基因进行遗传操作，并使用存档的人类肿瘤组织进行临床相关性研究。为了促进乳腺肿瘤转移的分子机制的研究，我们已经获得了一系列的单克隆乳腺肿瘤细胞系中的候选转移相关基因可以筛选和功能测试。具有相反转移倾向的单克隆肿瘤细胞系来源于多克隆乳腺癌细胞系MDA-MB-435和MDA-MB-231。在这个建议中，我们将进行一系列的乳腺肿瘤细胞的蛋白质谱的详细分析，使用新开发的2D液体分离/质谱蛋白质组学方法。该方法使用等电聚焦和无孔二氧化硅反相高效液相色谱法来创建类似于2D凝胶电泳的pI与Mr的图像。将根据MALDI-TOF-MS肽图谱和完整Mr鉴别蛋白质，并在细胞系裂解物样品之间进行定量比较。将选择经鉴定和验证为在转移表型方面差异表达的蛋白质进行遗传操作，并使用体外和体内试验检测所得细胞系的行为。最后，将在存档的正常和患病人类乳腺组织样本中研究候选蛋白/基因的表达。与临床病理信息的相关性将确定具有最有希望的诊断和预后标记物效用的那些候选乳腺抗原。

DESCRIPTION (provided by applicant): The overall goal of this project is to obtain novel information on the molecular mechanisms of tumor metastasis, in order to contribute to understanding of the pathogenesis of this process and to obtain data that would be clinically useful. The proposed approach is designed to progress from identifying and verifying metastasis-associated proteins and transcripts, to genetic manipulation of specific genes in matched metastatic and non-metastatic clonal cell lines, and to lead into clinical correlation studies using archived human tumor tissues. In order to facilitate research investigations into the molecular mechanisms of breast tumor metastasis, we have derived a series of monoclonal breast tumor cell lines in which candidate metastasis-associated genes can be screened and functionally tested. Monoclonal tumor cell lines of opposite metastatic propensity were derived from the polyclonal breast carcinoma cell lines MDA-MB-435 and MDA-MB-231. Within this proposal, we will conduct a detailed analysis of the protein profile of the series of breast tumor cells using a newly developed 2D liquid separation/mass-mapping proteomic approach. This method uses isoelectric focusing and non-porous silica reversed-phase high-performance liquid chromatography to create an image of pI vs Mr analogous to 2D gel electrophoresis. Proteins will be identified based upon MALDI-TOF-MS peptide mapping and intact Mr, and quantitative comparisons will be performed between cell line lysate samples. Proteins identified and validated as being differentially expressed with regard to metastatic phenotype will be selected for genetic manipulation and resulting cell lines will be tested for behavior using in vitro and in vivo assays. Finally, the expression of candidate proteins/genes will be studied in archived normal and diseased human breast tissue samples. Correlation with clinicopathologic information will identify those candidate breast antigens with the most promising diagnostic and prognostic marker utility.