title: Inhibition of glutamate regulated calcium entry into leukemic megakaryoblasts reduces cell proliferation and supports differentiation.
abstract: Human megakaryocytes release glutamate and express glutamate-gated Ca(2+)-permeable N-methyl-D-aspartate receptors (NMDARs) that support megakaryocytic maturation. While deregulated glutamate pathways impact oncogenicity in some cancers, the role of glutamate and NMDARs in megakaryocytic malignancies remains unknown. The aim of this study was to determine if NMDARs participate in Ca(2+) responses in leukemic megakaryoblasts and if so, whether modulating NMDAR activity could influence cell growth. Three human cell lines, Meg-01, Set-2 and K-562 were used as models of leukemic megakaryoblasts. NMDAR components were examined in leukemic cells and human bone marrow, including in megakaryocytic disease. Well-established NMDAR modulators (agonists and antagonists) were employed to determine NMDAR effects on Ca(2+) flux, cell viability, proliferation and differentiation. Leukemic megakaryoblasts contained combinations of NMDAR subunits that differed from normal bone marrow and the brain. NMDAR agonists facilitated Ca(2+) entry into Meg-01 cells, amplified Ca(2+) responses to adenosine diphosphate (ADP) and promoted growth of Meg-01, Set-2 and K-562 cells. Low concentrations of NMDAR inhibitors (riluzole, memantine, MK-801 and AP5; 5-100μM) were weakly cytotoxic but mainly reduced cell numbers by suppressing proliferation. The use-dependent NMDAR inhibitor, memantine (100μM), reduced numbers and proliferation of Meg-01 cells to less than 20% of controls (IC50 20μM and 36μM, respectively). In the presence of NMDAR inhibitors cells acquired morphologic and immunophenotypic features of megakaryocytic differentiation. In conclusion, NMDARs provide a novel pathway for Ca(2+) entry into leukemic megakaryoblasts that supports cell proliferation but not differentiation. NMDAR inhibitors counteract these effects, suggesting a novel opportunity to modulate growth of leukemic megakaryoblasts.
authors.affiliation: Department of Molecular Medicine and Pathology, University of Auckland, Auckland, New Zealand.
authors.forename: Tania
authors.initials: T
authors.lastname: Kamal
authors.affiliation: Department of Molecular Medicine and Pathology, University of Auckland, Auckland, New Zealand.
authors.forename: Taryn N
authors.initials: TN
authors.lastname: Green
authors.affiliation: Department of Haematology and Transfusion Medicine, Royal North Shore Hospital, Sydney, Australia; Northern Blood Research Centre, Kolling Institute of Medical Research, The University of Sydney, Australia.
authors.forename: Marie-Christine
authors.initials: MC
authors.lastname: Morel-Kopp
authors.affiliation: Department of Haematology and Transfusion Medicine, Royal North Shore Hospital, Sydney, Australia; Northern Blood Research Centre, Kolling Institute of Medical Research, The University of Sydney, Australia.
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Ward
authors.affiliation: School of Pharmacy and Centre for Brain Research, University of Auckland, Auckland, New Zealand.
authors.forename: Ailsa L
authors.initials: AL
authors.lastname: McGregor
authors.affiliation: Department of Anatomy with Radiology, University of Auckland, Auckland, New Zealand.
authors.forename: Susan R
authors.initials: SR
authors.lastname: McGlashan
authors.affiliation: Department of Molecular Medicine and Pathology, University of Auckland, Auckland, New Zealand.
authors.forename: Stefan K
authors.initials: SK
authors.lastname: Bohlander
authors.affiliation: Department of Molecular Medicine and Pathology, University of Auckland, Auckland, New Zealand; Department of Haematology, Auckland City Hospital, Auckland, New Zealand.
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Browett
authors.affiliation: Department of Paediatric Haematology and Oncology, Starship Children's Health, Auckland, New Zealand.
authors.forename: Lochie
authors.initials: L
authors.lastname: Teague
authors.affiliation: Department of Molecular Medicine and Pathology, University of Auckland, Auckland, New Zealand; Cancer Genetics and Neuroscience Program, Department of Molecular Virology, Immunology and Medical Genetics, The Ohio State University, Columbus, OH 43210, United States; the Comprehensive Cancer Center, The Ohio State University, Columbus, OH 43210, United States.
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: During
authors.affiliation: Centre for Integrated Research into Musculoskeletal Ageing, Department of Human Metabolism, University of Sheffield, Sheffield, United Kingdom.
authors.forename: Timothy M
authors.initials: TM
authors.lastname: Skerry
authors.affiliation: The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, VIC 3052 Australia; University of Melbourne, Department of Medical Biology, 1G Royal Parade, VIC 3052 Australia.
authors.forename: Emma C
authors.initials: EC
authors.lastname: Josefsson
authors.affiliation: Department of Molecular Medicine and Pathology, University of Auckland, Auckland, New Zealand; LabPlus Haematology, Auckland District Health Board, Auckland, New Zealand. Electronic address: m.kalev@auckland.ac.nz.
authors.forename: Maggie L
authors.initials: ML
authors.lastname: Kalev-Zylinska
mesh_terms: D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling; D002454:Cell Differentiation; D049109:Cell Proliferation; D005260:Female; D018698:Glutamic Acid / Q000378:metabolism*; D006801:Humans; D020014:K562 Cells; D007947:Leukemia, Megakaryoblastic, Acute / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D008297:Male; D016194:Receptors, N-Methyl-D-Aspartate / Q000235:genetics / Q000378:metabolism
chemical_list: D016194:Receptors, N-Methyl-D-Aspartate; D018698:Glutamic Acid; D002118:Calcium
keywords: Blood; Cancer; Leukemia; Megakaryocyte; Memantine; N-methyl-D-aspartate receptor