title: Activation of cyclic AMP pathway prevents CD34(+) cell apoptosis.
abstract: OBJECTIVE
  Although cAMP is involved in a number of physiologic functions, its role in hematopoietic cell fate decision remains poorly understood. We have recently demonstrated that in CD34(+)-derived megakaryocytes, cAMP-related agents prevent apoptosis. In this study we addressed the question of whether cAMP also regulates survival of their precursors, CD34(+) cells.
  
  
  METHODS
  Apoptosis was evaluated by fluorescence microscopy, and detection of hypodiploid or annexin V(+) cells by flow cytometry. Mitochondrial membrane potential and bcl-xL or caspase-3 expression were assessed by flow cytometry. Colony-forming units were studied by clonogenic assays in methylcellulose.
  
  
  RESULTS
  We found that two different cAMP analogs such as Dibutiril-cAMP and sp-5,6-DCl-BIMPS (BIMPS) promoted survival of human umbilical cord-derived CD34(+) cells by suppressing apoptosis induced by either nitric oxide (NO) or serum deprivation. Involvement of PKA and PI3K pathway was demonstrated by the ability of their specific inhibitors Rp-cAMP and Wortmannin or LY294002 respectively to reverse the antiapoptotic effect of BIMPS. Treatment of CD34(+) cell with BIMPS not only restrained the bcl-xL downregulation but also suppressed the loss of mitochondrial membrane potential and caspase-3 activation induced by serum starvation. While thrombopoietin (TPO), granulocyte colony-stimulating factor (G-CSF) or stem cell factor (SCF) were not able to increase cAMP levels, the antiapoptotic activity exerted by these growth factors was blocked by inhibition of the adenylate cyclase and synergized by BIMPS. Cyclic AMP analogs suppressed the decreased colony formation in cells exposed to NO or serum deprivation.
  
  
  CONCLUSIONS
  Altogether, our results strongly suggest that cAMP appears to be not only a key pathway controlling CD34(+) survival, but also a mediator of the TPO-, G-CSF- and SCF-mediated cytoprotection.
authors.affiliation: Hematological Research Institute, National Academy of Medicine, National Research Council (CONICET), Buenos Aires, Argentina.
authors.forename: Soledad
authors.initials: S
authors.lastname: Negrotto
authors.forename: Natalia
authors.initials: N
authors.lastname: Pacienza
authors.forename: Lina Paola
authors.initials: LP
authors.lastname: D'Atri
authors.forename: Roberto Gabriel
authors.initials: RG
authors.lastname: Pozner
authors.forename: Elisa
authors.initials: E
authors.lastname: Malaver
authors.forename: Oscar
authors.initials: O
authors.lastname: Torres
authors.forename: Maria Angela
authors.initials: MA
authors.lastname: Lazzari
authors.forename: Ricardo Martín
authors.initials: RM
authors.lastname: Gómez
authors.forename: Mirta
authors.initials: M
authors.lastname: Schattner
mesh_terms: D018952:Antigens, CD34; D017209:Apoptosis / Q000187:drug effects*; D003994:Bucladesine / Q000378:metabolism / Q000494:pharmacology*; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002478:Cells, Cultured; D002867:Chromones / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004004:Dichlororibofuranosylbenzimidazole / Q000031:analogs & derivatives* / Q000494:pharmacology; D015536:Down-Regulation / Q000187:drug effects / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005312:Fetal Blood / Q000166:cytology / Q000378:metabolism; D006133:Growth Substances / Q000378:metabolism; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D008533:Megakaryocytes / Q000166:cytology / Q000378:metabolism*; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D008928:Mitochondria / Q000378:metabolism; D009025:Morpholines / Q000494:pharmacology; D009569:Nitric Oxide / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects* / Q000502:physiology; D013873:Thionucleotides / Q000494:pharmacology*; D051020:bcl-X Protein / Q000096:biosynthesis
chemical_list: D018952:Antigens, CD34; C494811:BCL2L1 protein, human; D002867:Chromones; D004791:Enzyme Inhibitors; D006133:Growth Substances; D009025:Morpholines; D013873:Thionucleotides; D051020:bcl-X Protein; C071644:5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole-3',5'-monophosphorothioate; D009569:Nitric Oxide; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D004004:Dichlororibofuranosylbenzimidazole; D003994:Bucladesine; D019869:Phosphatidylinositol 3-Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases