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.
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.
Verhoeckx
Kitty C M
KC
TNO Pharma, Zeist, The Netherlands. Verhoeckx@pharma.tno.nl
Bijlsma
Sabina
S
de Groene
Els M
EM
Witkamp
Renger F
RF
van der Greef
Jan
J
Rodenburg
Richard J T
RJ
D002454:Cell Differentiation / Q000187:drug effects* / Q000502:physiology; D015180:Electrophoresis, Gel, Two-Dimensional; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology; D006801:Humans; D008264:Macrophages / Q000166:cytology* / Q000378:metabolism; D009000:Monocytes / Q000166:cytology* / Q000378:metabolism; D025341:Principal Component Analysis; D020543:Proteome; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013223:Statistics as Topic; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D020298:U937 Cells
D020543:Proteome; D013755:Tetradecanoylphorbol Acetate