PROTEOMIC ANALYSIS OF THE HMEC MITOGENIC RESPONSE

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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.