PROTEOMIC ANALYSIS OF THE HMEC MITOGENIC RESPONSE

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。我们和其他实验室最近的研究表明，表皮生长因子受体(EGFR)信号通路在整合多种有丝分裂生长因子的信号方面发挥着重要作用。特别是，细胞对细胞因子(肿瘤坏死因子)的反应以及一系列不同的G蛋白偶联受体(GPCR)通路已被证明涉及EGFR的次级激活(反式激活)。在乳腺上皮细胞中，EGFR通路在控制细胞有丝分裂和细胞分化中起着关键作用，是乳腺癌治疗的重要靶点。因此，我们研究的目标是了解EGFR反式激活在调节乳腺细胞对不同信号级联反应中的作用。为了解决这个问题，我们正在开发一个全面的清单，在同步化的人乳腺上皮细胞(HMEC)中表达的基因和蛋白在由表皮生长因子添加启动的G1-S转变过程中。在抑制EGFR信号后，HMEC将细胞周期停滞在G1期。EGFR信号的重新刺激允许高度可重复性的有丝分裂反应，其中信号和蛋白质表达事件的时间顺序可以被识别。我们已经大规模地进行了这些实验，为全基因组微阵列分析RNA表达提供了足够的样本材料，也为高通量蛋白质组分析提供了足够的样本。此外，全细胞蛋白质裂解产物样本还用于高通量蛋白质组分析(Powerblot、BD Biosciences)，其中包括对8个时间点中的每个时间点进行多达1000种不同抗体的定量分析。LC-FTICR分析将提供一个独特的人类乳腺细胞有丝分裂反应的全球数据集，这应该会引起乳腺癌研究界的普遍兴趣。结合我们的RNA和Western印迹分析，我们预计这些实验将提供一个独特的机会来比较全球蛋白质组和基因组平台的结果。这些结果还将提供一个高度稳健的数据集，供对生成正常人类细胞类型的信号转导路径的网络模型感兴趣的生物建模人员免费使用，这对NIH资助的广泛研究具有重要意义。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Recent studies from our and others' laboratories have demonstrated that the epidermal growth factor receptor (EGFR) signaling pathway plays an important role in integrating signaling from a diverse set of mitogenic growth factors. In particular, cellular responses to cytokines (tumor necrosis factor) as well as a diverse array of G-protein coupled receptor (GPCR) pathways have been shown to involve secondary activation (transactivation) of EGFR. In mammary epithelial cells, the EGFR pathway plays a critical role in control of mitogenesis and cell differentiation, and is an important therapeutic target for mammary cancer. Thus, the goal of our research is to understand the role of EGFR transactivation in regulating the mammary cell response to diverse signaling cascades. To address this issue, we are developing a comprehensive inventory of the genes and proteins expressed in synchronized human mammary epithelial cells (HMEC) during the G1-S transition initiated by EGF addition. After inhibition of EGFR signaling, HMEC arrest in G1 of the cell cycle. Restimulation of EGFR signaling allows for a highly reproducible mitogenic response, where the temporal ordering of signaling and protein expression events can be identified. We have conducted these experiments at a large scale, allowing for isolation of sufficient sample material for analysis of RNA expression by whole-genome microarray as well as obtaining sufficient sample for high-throughput proteomic analysis. In addition, whole cell protein lysate samples are also being used for proteome analysis by a high-throughput Western blot approach (Powerblot, BD Biosciences), which include quantitative analysis for up to 1000 different antibodies for each of the 8 time points. LC-FTICR analysis will provide a unique global data set of the mitogenic response of human mammary cells, which should be of general interest to the breast cancer research community. Combined with our RNA and Western blot analysis, we anticipate these experiments will provide a unique opportunity to compare results across global proteomic and genomic platforms. The results will also provide a highly robust dataset which will be made freely available to biological modelers interested in generating network models of signal transduction pathways in a normal human cell type of importance to a broad range of NIH-funded research.