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，其中包括正常的永生化乳腺上皮细胞，雌激素反应性癌前变体，形成快速生长但侵袭性原位导管癌的变体，以及免疫缺陷小鼠中雌激素独立的恶性变体。这组人类乳腺细胞系具有共同的遗传背景，代表了乳腺疾病的多种事件，将用于比较研究，以确定随着进展而发生的遗传改变。我们假设不依赖于E2的恶性细胞系可能在癌前阶段组成性地表达被E2改变的蛋白（E2加速MCF10AT1异种移植物的进展）。因此，首要任务将是鉴定在恶性变异体中组成性表达的蛋白质，这些变异体也在恶性前MCF10AT1细胞中由E2诱导。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，它可以从整个细胞裂解物中快速分离大量蛋白质，并为液相中进一步分析提供有效的蛋白质回收。该方法将与液相等电聚焦（IEF）的一维分离相结合，根据pI预分离蛋白质。结果是类似于二维凝胶电泳的二维蛋白质图谱，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个目标蛋白进行质量测定，胰蛋白酶或葡萄糖- c消化将生成肽图。分子量和肽图可用于识别每个蛋白质，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。MCF10系统为检测到的变化与恶性表型之间的因果关系提供了一种直接的手段。我们将使用基因转换的新技术来抑制基因表达或在细胞的特定进展阶段引入错义突变，并根据异种移植物形成的疾病阶段来监测这些改变对进展的影响。

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.