title: Paradoxical MAPK-activation in response to treatment with tyrosine kinase inhibitors in CML: flow cytometry loses track.
abstract: BACKGROUND
  Paradoxical activation of the MAP-kinases, ERK1, and ERK2 (ERK1/2) is observed in CML cell lines and primary CML patient cells treated with tyrosine kinase inhibitors (TKI) in vitro. The commonly accepted assumption is that activated ERK1/2 is key regulators of survival of leukemic cells treated with kinase inhibitors. Hence, paradoxical ERK1/2-activation may trigger resistance in vivo, which yet has to be shown. We therefore sought to establish a flow cytometric assay that enables us to measure paradoxical TKI-induced ERK1/2-activation on a single cell basis in primary CML cells.
  
  
  METHODS
  Side-by-side Western blot and intracellular flow cytometry (FCM) after in vitro exposure of cell lines and primary cells to nilotinib were performed. Detailed analysis of pre-analytical factors and the issue of compartmentalization of phosphorylated ERK1/2 by confocal laser scanning microscopy were performed.
  
  
  RESULTS
  Results were conflicting in that pERK-activation was robustly detected in Western blot assays, but not when cells were analyzed by FCM despite well functioning positive and negative controls. This is in contrast to experiments on other targets such as phospho-CrkL, where also in our hands TKI-dependent inhibition of phosphorylation is trackable by both Western blot and FCM assays.
  
  
  CONCLUSIONS
  To our knowledge this is the first report of discordant results in phospho-protein analysis in TKI-treated cells analyzed by Western blot vs. FCM. We speculate that a substance specific interaction interferes with fluorescence dependent methods seeking to track phosphorylated ERK1/2 in TKI-treated cells.
authors.affiliation: Klinik für Innere Medizin II, Abteilung für Hämatologie und Internistische Onkologie, Universitätsklinikum Jena, Erlanger Allee 101, 07740 Jena, Germany.
authors.forename: Ulf
authors.initials: U
authors.lastname: Schnetzke
authors.forename: Mike
authors.initials: M
authors.lastname: Fischer
authors.forename: Jochen J
authors.initials: JJ
authors.lastname: Frietsch
authors.forename: Andreas
authors.initials: A
authors.lastname: Finkensieper
authors.forename: Joachim H
authors.initials: JH
authors.lastname: Clement
authors.forename: Andreas
authors.initials: A
authors.lastname: Hochhaus
authors.forename: Paul
authors.initials: P
authors.lastname: La Rosée
mesh_terms: D000818:Animals; D018952:Antigens, CD34 / Q000378:metabolism; D002461:Cell Line, Transformed; D004789:Enzyme Activation / Q000187:drug effects; D005434:Flow Cytometry; D006801:Humans; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000188:drug therapy* / Q000201:enzymology* / Q000378:metabolism; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism*; D014411:Neoplastic Stem Cells / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
chemical_list: D018952:Antigens, CD34; D047428:Protein Kinase Inhibitors; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3
keywords: CML; MAPK; flow cytometry; phospho-staining