Phenotyping breast cancer cell lines EM-G3, HCC1937, MCF7 and MDA-MB-231 using 2-D electrophoresis and affinity chromatography for glutathione-binding proteins

Abstract

Background: Transformed phenotypes are common to cell lines derived from various cancers. Proteome profiling is a valuable tool that may reveal uncharacteristic cell phenotypes in transformed cells. Changes in expression of glutathione S-transferases (GSTs) and other proteins interacting with glutathione (GSH) in model cell lines could be of particular interest.

Methods: We compared the phenotypes of breast cell lines EM-G3, HCC1937, MCF7 and MDA-MB-231 using 2-D electrophoresis (2-DE). We further separated GSH-binding proteins from the cell lines using affinity chromatography with GSH-Sepharose 4B, performed 2-DE analysis and identified the main protein spots.

Results: Correlation coefficients among 2-DE gels from the cell lines were lower than 0.65, pointing to dissimilarity among the cell lines. Differences in primary constituents of the cytoskeleton were shown by the 2-D protein maps and western blots. The spot patterns in gels of GSH-binding fractions from primary carcinoma-derived cell lines HCC1937 and EM-G3 were similar to each other, and they differed from the spot patterns of cell lines MCF7 and MDA-MB-231 that were derived from pleural effusions of metastatic mammary carcinoma patients. Major differences in the expression of GST P1-1 and carbonyl reductase [NADPH] 1 were observed among the cell lines, indicating differential abilities of the cell lines to metabolize xenobiotics.

Conclusions: Our results confirmed the applicability of targeted affinity chromatography to proteome profiling and allowed us to characterize the phenotypes of four breast cancer cell lines.

Figure 1

Main constituents of cytoskeletons from the breast cell lines. Sections of the 2-DE gels (Mr 38-60, pI 4.6-5.7) from breast cell lines showing the main constituents of the cytoskeleton. (A) The identities of spots are shown for cell line EM-G3. Spots corresponding to CKs are framed. Simple CKs are in green, basal CKs are in blue and CKs atypical for mammary glands are in yellow. The data were subtracted from Selicharova et al. [23]. Protein names are abbreviated as 40S rib.p.SA = 40S ribosomal protein SA, CK = cytokeratin, HSP = heat shock protein and PDI = protein disulfide isomerase. (B) Estimated positions of CKs are shown for the cell lines HCC1937, MCF7 and MDA-MB-231. (C) Immunoblot analysis. Proteins from each cell lysate were separated by 1-D SDS-PAGE and detected with rabbit anti-actin antibody and mouse monoclonal anti-vimentin, anti-CK19 and anti-CK13 antibodies.

Figure 2

Specific activities of GST. Specific activities of GST in four primary cultures (black columns) of NME cells (NME23, NME35, MNE36 and NME37) and in permanent breast cell lines (white columns).

Figure 3

GSH-binding proteins from the breast cell lines. Sections of the 2-DE gels (Mr 20-52, pI 4.3-9.3) for GSH-binding proteins from EM-G3, HCC1937, MCF7 and MDA-MB-231 cell lines. Desalted and concentrated fractions from the affinity chromatography were focused on 7 cm IPG strips pH 3-10 and separated in 12% SDS-PAGE gels. The proteins identified with numbered spots are listed in Table 3.