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A combination of proteomics, principal component analysis and transcriptomics is a powerful tool for the identification of biomarkers for macrophage maturation in the U937 cell line

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Author: Verhoeckx, K.C.M. · Bijlsma, S. · Groene, E.M. de · Witkamp, R.F. · Greef, J. van der · Rodenburg, R.J.T.
Type:article
Date:2004
Institution: TNO Voeding
Source:Proteomics, 4, 4, 1014-1028
Identifier: 237706
doi: doi:10.1002/pmic.200300669
Keywords: Pharmacology · Analytical research · Differentiation markers · Macrophage maturation · Oligonucleotide microarray · Principal component analysis · Two-dimensional gel electrophoresis · complementary DNA · oligonucleotide · peptide · protein · RNA · analytic method · article · cell differentiation · cell strain U937 · genetic transcription · human · human cell · immunoblotting · lymphoma cell · macrophage · mass spectrometry · matrix assisted laser desorption ionization time of flight mass spectrometry · monocyte · nucleotide sequence · peptide analysis · polyacrylamide gel electrophoresis · polymerase chain reaction · principal component analysis · priority journal · proteomics · two dimensional gel electrophoresis · Cell Differentiation · Electrophoresis, Gel, Two-Dimensional · Gene Expression Profiling · Gene Expression Regulation · Humans · Macrophages · Monocytes · Principal Component Analysis · Proteome · Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization · Statistics · Tetradecanoylphorbol Acetate · U937 Cells

Abstract

The monocyte-like human histiocytic lymphoma cell line U937 can be induced by phorbol 12-myristate 13-acetate (PMA) to undergo differentiation into a macrophage-like phenotype. We have used two-dimensional gel electrophoresis (2-DE), oligonucleotide microarrays and principal component analysis (PCA) to characterize the U937 cell line as a model system for the differentiation of monocytes into macrophages. A total of 226 differentially expressed proteins were found, of which 41 were selected by PCA for identification using matrix-assisted laser desorption/ionization tandem mass spectrometry. Based on the PCA results, three marker proteins were selected for confirmation of differential expression using Western blot and quantitative real time-PCR. The selected marker proteins were: gamma interferon inducible lysosomal thiol reductase, cathepsin D and adipocyte-fatty acid binding protein. All three proved to be good differentiation markers for macrophage maturation of U937 cells as well as peripheral blood-derived macrophages. The transcriptomics data revealed a large number of additional putative differentiation markers in U937 macrophages, many of which are known to be expressed in peripheral blood-derived macrophages. These include osteospontin, matrix metalloproteinase 9, and HC-gp39. Our results show that the characteristics of U937 macrophages resemble those of inflammatory (exudate) macrophages, exemplified by the down-regulation of 5′ nucleotidase and the up-regulation of leucine aminopeptidase mRNAs. In conclusion, using the powerful combination of transcriptomics, 2-DE and PCA, our results show that U937 cells differentiated by PMA treatment are an excellent model system for monocyte derived macrophage generation from blood.