PROTEOMIC ANALYSIS OF THE HMEC MITOGENIC RESPONSE

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得主要资金， 因此可以在其他CRISP条目中表示。列出的机构是 中心，不一定是研究者的机构。 我们和其他实验室最近的研究表明，表皮生长因子受体（EGFR）信号通路在整合来自不同促有丝分裂生长因子的信号传导中起着重要作用。 特别是，对细胞因子（肿瘤坏死因子）的细胞应答以及多种G蛋白偶联受体（GPCR）途径已显示涉及EGFR的次级激活（反式激活）。 在乳腺上皮细胞中，EGFR通路在控制有丝分裂和细胞分化中起关键作用，并且是乳腺癌的重要治疗靶点。 因此，我们的研究目标是了解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库存的基因和蛋白质表达的同步人乳腺上皮细胞（HMEC）在G1-S过渡启动EGF添加。 抑制EGFR信号后，HMEC阻滞在细胞周期的G1期。 EGFR信号传导的再刺激允许高度可再现的促有丝分裂应答，其中可以鉴定信号传导和蛋白质表达事件的时间顺序。 我们已经进行了大规模的这些实验，允许分离足够的样品材料，用于通过全基因组微阵列分析RNA表达，以及获得足够的样品用于高通量蛋白质组学分析。 此外，全细胞蛋白裂解物样品也用于通过高通量蛋白质印迹方法（Powerblot，BD Biosciences）进行蛋白质组分析，其包括对8个时间点中的每个时间点的多达1000种不同抗体的定量分析。 LC-FTICR分析将提供人类乳腺细胞促有丝分裂反应的独特全球数据集，这应该是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，在全球蛋白质组学和基因组平台上比较结果。 研究结果还将提供一个高度稳健的数据集，该数据集将免费提供给有兴趣在正常人类细胞类型中生成信号转导途径网络模型的生物建模者，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.