title: Alteration of c-mpl-mediated signal transduction in CD34(+) cells from patients with myelodysplastic syndromes.
abstract: OBJECTIVE
  Megakaryocytic differentiation is frequently defective in patients with myelodysplastic syndromes (MDS). As underlying mechanisms, deregulated thrombopoietin receptor (c-mpl)-mediated signaling pathways have been suggested. This study therefore examined whether the impaired signaling in MDS and AML cells includes alterations of c-mpl itself or postsignaling events.
  
  
  METHODS
  Bone marrow-derived CD34(+) cells from healthy donors, patients with MDS (RA, RAEB-T), and patients with AML after MDS were isolated by MACS. Expression of c-mpl cDNA was studied by RT-PCR. Thrombopoietin dependent activation of STAT proteins and MAP Kinase p42(erk-2)/44(erk-1) was analyzed by Western blot.
  
  
  RESULTS
  Both splicing isoforms of c-mpl (c-mpl-p and c-mpl-k) were expressed in all of the CD34(+) cells examined. Analysis of the c-mpl cDNA revealed no sequence abnormality. We show c-mpl dependent activation of the transcription factors STAT3 and STAT5 as well as MAP Kinase p42(erk-2)/44(erk-1) in CD34(+) cells from healthy individuals. Cells derived from RA patients revealed low basal levels of phosphorylated STAT3 and STAT5 molecules. This phosphorylation was enhanced by stimulation with recombinant thrombopoietin (PEG-rHuMGDF). STAT1 failed to be activated by PEG-rHuMGDF in CD34(+) cells from healthy donors as well as from patients with MDS. In RAEB-T and AML M7 the constitutive expression levels of STAT1, 3, 5, and MAPK were markedly upregulated, resulting in a strong activation of STAT3 and 5 by PEG-rHuMGDF. Despite its high expression, the level of MAPK phosphorylation was not increased in RA or RAEB-T compared to the normal control, and was completely undetectable in AML M7.
  
  
  CONCLUSIONS
  These results suggest that the defective megakaryopoiesis in MDS is not caused by a lack of c-mpl and that STAT3 and STAT5 may contribute to the malignant phenotype of the leukemic cells.
authors.affiliation: Department of Hematology/Oncology, Johann Wolfgang Goethe University Hospital, 60590, Frankfurt/Main, Germany. Kalina@em.uni-frankfurt.de
authors.forename: U
authors.initials: U
authors.lastname: Kalina
authors.forename: W K
authors.initials: WK
authors.lastname: Hofmann
authors.forename: S
authors.initials: S
authors.lastname: Koschmieder
authors.forename: S
authors.initials: S
authors.lastname: Wagner
authors.forename: D
authors.initials: D
authors.lastname: Kauschat
authors.forename: D
authors.initials: D
authors.lastname: Hoelzer
authors.forename: O G
authors.initials: OG
authors.lastname: Ottmann
mesh_terms: D000753:Anemia, Refractory / Q000378:metabolism / Q000473:pathology; D000754:Anemia, Refractory, with Excess of Blasts / Q000378:metabolism / Q000473:pathology; D018952:Antigens, CD34 / Q000032:analysis*; D001854:Bone Marrow Cells / Q000276:immunology / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D015870:Gene Expression; D006412:Hematopoietic Stem Cells / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D015470:Leukemia, Myeloid, Acute / Q000378:metabolism / Q000473:pathology; D008894:Milk Proteins; D009190:Myelodysplastic Syndromes / Q000378:metabolism* / Q000473:pathology; D009363:Neoplasm Proteins; D010766:Phosphorylation; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000502:physiology*; D012333:RNA, Messenger / Q000032:analysis; D018121:Receptors, Cytokine; D053628:Receptors, Thrombopoietin; D011994:Recombinant Proteins / Q000494:pharmacology; D050796:STAT3 Transcription Factor; D050799:STAT5 Transcription Factor; D015398:Signal Transduction; D013926:Thrombopoietin / Q000494:pharmacology; D015534:Trans-Activators / Q000378:metabolism
chemical_list: D018952:Antigens, CD34; D004268:DNA-Binding Proteins; D008894:Milk Proteins; D009363:Neoplasm Proteins; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D018121:Receptors, Cytokine; D053628:Receptors, Thrombopoietin; D011994:Recombinant Proteins; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human; D050799:STAT5 Transcription Factor; D015534:Trans-Activators; C082218:MPL protein, human; D013926:Thrombopoietin