Evaluation of Two-dimensional Differential Gel Electrophoresis for Proteomic Expression Analysis of a Model Breast Cancer Cell System

Abstract

The technique of fluorescent two-dimensional (2D) difference gel electrophoresis for differential protein expression analysis has been evaluated using a model breast cancer cell system of ErbB-2 overexpression. Labeling of paired cell lysate samples with N-hydroxy succinimidyl ester-derivatives of fluorescent Cy3 and Cy5 dyes for separation on the same 2D gel enabled quantitative, sensitive, and reproducible differential expression analysis of the cell lines. SyproRuby staining was shown to be a highly sensitive and 2D difference gel electrophoresis-compatible method for post-electrophoretic visualization of proteins, which could then be picked and identified by matrixassisted laser-desorption ionization mass spectroscopy. Indeed, from these experiments, we have identified multiple proteins that are likely to be involved in ErbB-2-mediated transformation. A triple dye labeling methodology was used to identify proteins differentially expressed in the cell system over a time course of growth factor stimulation. A Cy2-labeled pool of samples was used as a standard with all Cy3-and Cy5-labeled sample pairs to facilitate cross-gel quantitative analysis. DeCyder (Amersham Biosciences, Inc.) software was used to distinguish clear statistical differences in protein expression over time and between the cell lines. Molecular & Cellular Proteomics 1:91-98, 2002.The ability to determine statistically significant alterations in protein expression that correlate with disease or occur consequent to experimentally induced changes in cells is fundamental to the exploitation of proteomics. Previous studies from our laboratory have used two-dimensional (2D) 1 gel analysis of immunomagnetic affinity-sorted primary human breast cells to establish the protein expression profiles of luminal and myoepithelial cells (1). These differential expression studies were carried out using replicate 2D gels of each sample and post-staining with a fluorescent protein dye. The subsequent detailed curation and correlation of images was used to derive data sets for statistical analysis. This approach was attractive, because the fluorescent protein stain has a wide linear range of detection, thus giving improved quantification of both high and low abundance proteins. However, in these experiments, many pairs of gels were required to establish statistically significant differences in protein expression as each gel contains inherent experimental variations that limit image superimposition.The introduction of fluorescent 2D differential gel electrophoresis (DIGE) by Unlu et al. (2) has now made it possible to detect and quantitate differences between experimental pairs of samples resolved on the same 2D gel. The basis of the technique is the use of two mass-and charge-matched Nhydroxy succinimidyl ester derivatives of the fluorescent cyanine dyes Cy3 and Cy5, which possess distinct excitation and emission spectra. These are used to differentially label lysine residues of two protein samples for comparative analysis of the mixed sample on one gel. The ab...

Copine-III interacts with ErbB2 and promotes tumor cell migration

“…Copine-III was expressed to varying degrees in the breast cancer cells lines, with the highest levels in SKBr3 and BT474, both having ERBB2 amplification (Figure 8a). These data correlate with previous publications showing Copine-III upregulation in response to ErbB2 overexpression (Gharbi et al, 2002;White et al, 2004). We also examined Copine-III RNA expression in 49 primary breast tumors (Farmer et al, 2005).…”

“…In primary human tumors, there was some evidence for a functional relationship between Copine-III and ErbB2, because Copine-III mRNA and protein levels were shown to correlate with ErbB2 expression (Gharbi et al, 2002;White et al, 2004). We have extended these studies by examining a panel of 49 breast tumors (Farmer et al, 2005) and showing that there is a significant correlation between CPNE3 RNA levels, and ERBB2 gene amplification and expression.…”

“…As a control for equal loading, fractions of the input cell lysate were also loaded on the gel (input). (Gharbi et al, 2002;White et al, 2004). Therefore, we examined Copine-III in more detail using the breast cancer cell lines T47D, with moderate ErbB2 levels, and SKBr3, which has ERBB2 amplification and very high levels of the receptor (Lane et al, 2000).…”

“…Interestingly, Copine-III was shown to be upregulated in tumor cells in response to ErbB2 overexpression (Gharbi et al, 2002;White et al, 2004). We examined the role of Copine-III downstream of ErbB2 receptor signaling and show here that Copine-III is a Ca 2 þ -dependent, phospholipid-binding protein that interacts with activated ErbB2 at the plasma membrane (PM) of tumor cells.…”

Proteomics in human cancer research

“…It demonstrates some relationship triggering both kinds of breast carcinomas. 2-D DIGE technology was also successfully applied in breast cells and epithelial cells with ErbB-2 overexpression [101,102]. Li et al [103] used the SELDI-TOF in combination with bioinformatics tests to identify three serum biomarkers with high sensitivity and specificity for detection of breast cancer.…”