Proteomics of Progression in MCF10 Xenograft Model

MCF10 异种移植模型进展的蛋白质组学

• 批准号: 6698074
• 负责人: FRED Raymond MILLER
• 金额: $ 23.35万
• 依托单位: WAYNE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-02-20 至 2006-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6698074
• 关键词: biotechnology; breast neoplasms; cell line; electrospray ionization mass spectrometry; estrogens; gel electrophoresis; high performance liquid chromatography; laboratory mouse; matrix assisted laser desorption ionization; molecular weight; oncoproteins; point mutation; preneoplastic state; protein localization; protein sequence; protein structure function; proteomics; tissue /cell culture

The MCF10 xenograft model consists of a number of variants derived from a single patient which include normal immortalized breast epithelial cells, estrogen responsive premalignant variants, a variant which forms rapidly growing but preinvasive ductal carcinoma in situ, and estrogen independent malignant variants in immune deficient mice. This panel of human breast cell lines, on a common genetic background, representing multiple events in breast disease will be used for comparative studies to identify genetic alterations which occur with progression. We hypothesize that E2 independent malignant lines may constitutively express proteins which are altered by E2 in the premalignant stages (E2 accelerates progression of MCF10AT1 xenografts). Thus, first priority will be to identify proteins expressed constitutively in malignant variants which are also induced by E2 in premalignant MCF10AT1 cells. It is proposed to look at relative levels of proteins rather than nucleic acids. The methods to be used to monitor protein expression involve the use of nonporous reversed phase HPLC separations which can rapidly separate large numbers of proteins from whole cell lysates and provide efficient recovery of those proteins in the liquid phase for further analysis. The method will be combined with a first dimension separation using liquid phase isoelectric focusing (IEF) to prefractionate proteins according to pI. The result is a 2-D protein map analagous to 2-D gel electrophoresis where the expression of hundreds of proteins can be imaged and monitored. Matrix-assisted Laser Desorption/Ionization time-of- flight mass spectrometry will mass size each target protein and digestion by trypsin or Glu-C will generate a peptide map. The molecular weight and peptide maps can be used to identify each protein and electrospray ionization mass spectometry will be used to pinpoint modifications such as missense mutations and post- translational events such as phosphorylation. The MCF10 system provides a direct means to test the cause and effect relationship of the detected change to the malignant phenotype. We will use the novel technique of gene conversion to inhibit gene expression or introduce missense mutations in cells at defined stages of progression and to monitor the effect of these alterations on progression as determined by stage of disease formed in xenografts.

MCF 10异种移植模型由来自单个患者的许多变体组成，包括正常永生化乳腺上皮细胞、雌激素应答性癌前变体、形成快速生长但浸润前原位导管癌的变体和免疫缺陷小鼠中的雌激素非依赖性恶性变体。 这组人类乳腺细胞系具有共同的遗传背景，代表了乳腺疾病中的多种事件，将用于比较研究，以确定随着疾病进展而发生的遗传改变。 我们假设E2非依赖性恶性细胞系可能组成性表达在癌前阶段被E2改变的蛋白质（E2加速MCF 10AT 1异种移植物的进展）。 因此，第一优先将是鉴定在恶性变体中组成型表达的蛋白质，所述恶性变体也由E2在癌前MCF 10AT 1细胞中诱导。 有人建议研究蛋白质而不是核酸的相对水平。用于监测蛋白质表达的方法包括使用无孔反相HPLC分离，其可以从全细胞裂解物中快速分离大量蛋白质，并在液相中有效回收这些蛋白质用于进一步分析。 该方法将与使用液相等电聚焦（IEF）的第一维分离相结合，以根据pI预分离蛋白质。 其结果是一个2-D蛋白质图谱类似于2-D凝胶电泳，其中数百种蛋白质的表达可以成像和监测。 基质辅助激光解吸/电离飞行时间质谱法将对每种靶蛋白进行质量测定，并通过胰蛋白酶或Glu-C消化产生肽图谱。 分子量和肽图谱可用于鉴定每种蛋白质，电喷雾电离质谱法将用于查明修饰，如错义突变和翻译后事件，如磷酸化。 MCF 10系统提供了一种直接的方法来测试检测到的变化与恶性表型的因果关系。 我们将使用基因转换的新技术来抑制基因表达或在确定的进展阶段在细胞中引入错义突变，并监测这些改变对异种移植物中形成的疾病阶段所确定的进展的影响。