Analysis of differential protein expression in normal and neoplastic human breast epithelial cell lines

High‐resolution two‐dimensional gel electrophoresis (2‐DE) and database analysis was used to establish protein expression patterns for cultured normal human mammary epithelial cells and thirteen breast cancer cell lines. The Human Breast Epithelial Cell database contains the 2‐DE protein patterns, including relative protein abundances, for each cell line, plus a composite pattern that contains all the common and specifically expressed proteins from all the cell lines. Significant differences in protein expression, both qualitative and quantitative, were observed not only between normal cells and tumor cells, but also among the tumor cell lines. Eight percent (56/727) of the consistently detected proteins were found in significantly (P < 0.001) variable levels among the cell lines. Eight proteins present in normal cultured breast epithelial cells were not detected in any of the tumor cell lines. We identified a subset of the differentially expressed proteins using a combination of immunostaining, protein sequencing, comigration, and subcellular fractionation. These identified proteins include the intermediate filament components vimentin and cytokeratins. The cell lines can be classified into four distinct groups based on their intermediate filament protein profile. We also identified heat shock proteins; hsp27 and hsp60 varied in abundance and in some cases in the relative phosphorylation levels among the cell lines. Many of the differentially expressed proteins we identified have roles in cellular proliferation and differentiation, including annexin V, elongation initiation factor 5A, Rho GDP dissociation inhibitor, and prohibitin. We identified inosine‐5‐monophosphate dehydrogenase in each of the cell lines, and found the levels of this enzyme in the tumor cell lines elevated 2‐ to 20‐fold relative to the levels in normal cells. These results expand the human breast epithelial cell protein database (http://www.anl.gov/CMB/PMG) which is being built to assist researchers with the identification of abnormal patterns of expression and pathways associated with malignancy.