title: High-resolution kinetics of cytokine signaling in human CD34/CD117-positive cells in unfractionated bone marrow.
abstract: Cytokine-mediated phosphorylation of Erk (pErk), ribosomal S6 (pS6), and Stat5 (pStat5) in CD34(+)/CD117(+) blast cells in normal bone marrow from 9 healthy adult donors were analyzed over 60 minutes. Treatment with stem cell factor (SCF), Flt3-ligand (FL), IL-3, and GM-CSF and measurement by multiparametric flow cytometry yielded distinctive, highly uniform phosphoprotein kinetic profiles despite a diverse sample population. The correlated responses for SCF- and FL-stimulated pErk and pS6 were similar. Half the population phosphorylated Erk in response to SCF between 0.9 and 1.2 minutes, and S6 phosphorylation followed approximately a minute later (t&frac12;(pS6 rise) = 2.2-2.7 minutes). The FL response was equally fast but more variable (t&frac12;(pErk rise) = 0.9-1.3 minutes; t&frac12;(pS6 rise) = 2.5-3.5 minutes). Stat5 was not activated in 97% of the cells by either cytokine. IL-3 and GM-CSF were similar to each other with half of blast cells phosphorylating Stat5 and 15% to 20% responding through Erk and S6. Limited comparison with leukemic blasts confirmed universal abnormal signaling in AML that is significantly different from normal bone marrow blasts. These differences included sustained signals, a larger fraction of responding cells, and amplification of phosphorylation levels for at least one phosphoprotein. These data support the eventual use of this approach for disease diagnosis and monitoring.
authors.affiliation: Case Comprehensive Cancer Center, Case Western Reserve University, Cleveland, OH, USA.
authors.forename: Philip G
authors.initials: PG
authors.lastname: Woost
authors.forename: Luis A
authors.initials: LA
authors.lastname: Solchaga
authors.forename: Howard J
authors.initials: HJ
authors.lastname: Meyerson
authors.forename: T Vincent
authors.initials: TV
authors.lastname: Shankey
authors.forename: Charles L
authors.initials: CL
authors.lastname: Goolsby
authors.forename: James W
authors.initials: JW
authors.lastname: Jacobberger
mesh_terms: D000328:Adult; D018952:Antigens, CD34 / Q000378:metabolism*; D015415:Biomarkers / Q000378:metabolism; D001853:Bone Marrow / Q000378:metabolism*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005260:Female; D005434:Flow Cytometry; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000378:metabolism / Q000494:pharmacology; D006410:Hematopoiesis / Q000502:physiology*; D006801:Humans; D007377:Interleukin-3 / Q000378:metabolism / Q000494:pharmacology; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism / Q000494:pharmacology; D008875:Middle Aged; D010750:Phosphoproteins / Q000378:metabolism; D019009:Proto-Oncogene Proteins c-kit / Q000378:metabolism*; D038744:Ribosomal Protein S6 Kinases, 90-kDa / Q000378:metabolism; D050799:STAT5 Transcription Factor / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D019089:Stem Cell Factor / Q000378:metabolism / Q000494:pharmacology
chemical_list: D018952:Antigens, CD34; D015415:Biomarkers; C508596:IL3 protein, human; D007377:Interleukin-3; D008565:Membrane Proteins; D010750:Phosphoproteins; D050799:STAT5 Transcription Factor; D019089:Stem Cell Factor; C084881:flt3 ligand protein; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D019009:Proto-Oncogene Proteins c-kit; C069329:RPS6KA1 protein, human; D038744:Ribosomal Protein S6 Kinases, 90-kDa; D048049:Extracellular Signal-Regulated MAP Kinases