Proteomic Analysis of the HMEC Mitogenic Response
EMSL Project ID
10198
Abstract
This EMSL User Proposal is for a collaborative effort with the PNNL Proteomic National Center for Research Resources.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.
We have recently established an experimental model using normal human mammary epithelial cells (HMEC) to examine the temporal events during EGFR activation. After inhibition of EGFR signaling, HMEC arrest in G0/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. Using this approach, we are currently analyzing the RNA expression patterns of the EGFR mitogenic response across 8 time points, ranging from 15 min to 24 h after cell stimulation, using microarray analysis. In addition, whole cell protein lysate samples have been prepared for proteome analysis by a high-throughput Western blot approach (Powerblot, BD Biosciences), which will include quantitative analysis for up to 1000 different antibodies for each of the 8 time points. Using the same protein lysate, we propose to conduct quantitative proteomic analysis of protein expression changes by LC-FTICR, using the Proteomic Resource at PNNL. The proposed time points to be analyzed include 0 hr, 15 min, 1 h, 4 h, 8 h, 13 h, 18 h, and 24 h after EGFR stimulation. We have available approximately 5 mg of total cell lysate protein for this analysis.
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.
Project Details
Project type
Exploratory Research
Start Date
2004-07-28
End Date
2007-06-28
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members
Related Publications
Rogers S, M Girolami, W Kolch, KM Waters, T Liu, BD Thrall, and HS Wiley. 2008. "Investigating the Correspondence Between Transcriptomic and Proteomic Expression Profiles Using Coupled Cluster Models." Bioinformatics 24(24):2894-2900. doi:10.1093/bioinformatics/btn553