title: Immunoreactivity of Stat5 phosphorylated on tyrosine as a cell-based measure of Bcr/Abl kinase activity.
abstract: BACKGROUND
  Stat5(1) (Signal Transducer and Activator of Transcription 5) is normally phosphorylated and activated by Janus kinases. In cells transformed with BCR/ABL, Stat5 is constitutively activated by promiscuous phosphorylation. Cytometry of intracellular antigens can be used to evaluate cell treatments affecting gene expression, because it precisely provides the fraction of affected cells and the quantitative change in expression. Here, we asked whether we could measure a phosphorylated epitope on Stat5 by cytometry, and whether that measurement would respond to Bcr/Abl inhibition.
  
  
  METHODS
  Chronic myelogenous leukemia (CML) cell lines or control Bcr/Abl-negative cells were treated or not with imatinib mesylate, fixed and permeabilized with formaldehyde followed by methanol; reacted with rabbit polyclonal and mouse monoclonal antibodies against an epitope including tyrosine 694 of Stat5a (pSTAT5); reacted with antibodies that mark mitotic cells; counterstained with secondary fluorescent antibodies and 4',6-diamidino-2-phenylindole (DAPI); and then subjected to flow cytometry. Western blotting was performed with pSTAT5 and Stat5 antibodies.
  
  
  RESULTS
  Optimal fixation and staining parameters were established for pSTAT5 antibodies with K562 cells. These cells displayed high levels of immunoreactivity with pSTAT5 probes that could be inhibited uniformly with imatinib mesylate in a dose-response and time-dependent manner. The IC50 for downregulation of pSTAT5 immunoreactivity for K562 cells by cytometry was approximately 70 nM. The inhibition half-time was approximately 1 min. At micromolar doses this reactivity remained minimal for up to 7 days. Cultured cells also displayed a population of negative cells that increased under conditions related to cessation of cell growth (media nutrient depletion). This study also showed quantitatively that a rabbit polyclonal antibody that cross-reacted with an additional epitope could be used successfully as a measure of Bcr/Abl activity.
  
  
  CONCLUSIONS
  We have developed a sensitive cytometric assay for Bcr/Abl kinase activity in human hematopoietic cell lines.
authors.affiliation: Comprehensive Cancer Center, Case Western Reserve University, Cleveland, Ohio 44106-4944, USA. jwj@cwru.edu
authors.forename: James W
authors.initials: JW
authors.lastname: Jacobberger
authors.forename: R Michael
authors.initials: RM
authors.lastname: Sramkoski
authors.forename: Phyllis S
authors.initials: PS
authors.lastname: Frisa
authors.forename: Peggy Peng
authors.initials: PP
authors.lastname: Ye
authors.forename: Megan A
authors.initials: MA
authors.lastname: Gottlieb
authors.forename: David W
authors.initials: DW
authors.lastname: Hedley
authors.forename: T Vincent
authors.initials: TV
authors.lastname: Shankey
authors.forename: Bradley L
authors.initials: BL
authors.lastname: Smith
authors.forename: Mary
authors.initials: M
authors.lastname: Paniagua
authors.forename: Charles L
authors.initials: CL
authors.lastname: Goolsby
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry; D001549:Benzamides; D015153:Blotting, Western; D002460:Cell Line; D045744:Cell Line, Tumor; D004247:DNA / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D000939:Epitopes / Q000737:chemistry; D005434:Flow Cytometry / Q000379:methods*; D016044:Fusion Proteins, bcr-abl / Q000737:chemistry / Q000378:metabolism*; D006412:Hematopoietic Stem Cells / Q000166:cytology; D006801:Humans; D000068877:Imatinib Mesylate; D015151:Immunoblotting; D007211:Indoles / Q000494:pharmacology; D020128:Inhibitory Concentration 50; D007376:Interleukin-2 / Q000096:biosynthesis; D015847:Interleukin-4 / Q000096:biosynthesis; D020014:K562 Cells; D007700:Kinetics; D000432:Methanol / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D008894:Milk Proteins; D010766:Phosphorylation; D010879:Piperazines / Q000494:pharmacology; D011485:Protein Binding; D011743:Pyrimidines / Q000494:pharmacology; D050799:STAT5 Transcription Factor; D015398:Signal Transduction; D013050:Spectrometry, Fluorescence; D013997:Time Factors; D015534:Trans-Activators / Q000378:metabolism*; D025521:Tumor Suppressor Proteins; D014443:Tyrosine / Q000737:chemistry
chemical_list: D000911:Antibodies, Monoclonal; D001549:Benzamides; D004268:DNA-Binding Proteins; D000939:Epitopes; D007211:Indoles; D007376:Interleukin-2; D008894:Milk Proteins; D010879:Piperazines; D011743:Pyrimidines; D050799:STAT5 Transcription Factor; C494092:STAT5A protein, human; D015534:Trans-Activators; D025521:Tumor Suppressor Proteins; D015847:Interleukin-4; D014443:Tyrosine; C007293:DAPI; D000068877:Imatinib Mesylate; D004247:DNA; D016044:Fusion Proteins, bcr-abl; D000432:Methanol