Proteomics of Progression in MCF10 Xenograft Model

MCF10异种移植模型由来自单个患者的许多变体组成，其中包括正常永生化的乳腺上皮细胞、雌激素反应性癌前变体、形成快速生长但浸润性导管癌的原位癌的变体、以及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在共同的遗传背景下，代表了乳房疾病中的多种事件，将用于比较研究，以确定随着疾病进展而发生的基因变化。我们推测，在癌前阶段(E2加速MCF10AT1异种移植瘤的进展)，不依赖于E2的恶性细胞株可能结构性地表达被E2改变的蛋白质。因此，当务之急将是确定在恶性变异体中结构性表达的蛋白质，这些变异体也是由E2诱导的癌前MCF10AT1细胞。有人建议研究蛋白质的相对水平，而不是核酸。用于监测蛋白质表达的方法包括使用无孔反相高效液相色谱分离，这种分离方法可以从整个细胞裂解物中快速分离大量蛋白质，并在液体中有效地回收这些蛋白质供进一步分析。该方法将与使用液体等电聚焦(IEF)的第一维分离相结合，以根据等电点对分离前蛋白质进行分离。其结果是一张类似于二维凝胶电泳法的二维蛋白质图，其中数百种蛋白质的表达可以被成像和监测。基质辅助激光解吸/电离飞行时间质谱仪将对每个目标蛋白质进行质量测定，并用胰酶或Glu-C消化将生成肽图。分子量和肽图可以用来识别每种蛋白质，电喷雾电离质谱仪将用来精确定位修饰，如错义突变和翻译后事件，如磷酸化。MCF10系统提供了一种直接的手段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，在特定的进展阶段抑制基因表达或在细胞中引入错义突变，并监测这些变化对进展的影响，这取决于异种移植形成的疾病阶段。

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.