title: Immobilized anti-KIT monoclonal antibody induces ligand-independent dimerization and activation of Steel factor receptor: biologic similarity with membrane-bound form of Steel factor rather than its soluble form.
abstract: Interaction of a tyrosine kinase type receptor and its ligand induces receptor-dimerization or -oligomerization followed by transphosphorylation and activation of its intrinsic kinase, which leads to a series of intracellular signals. We have previously reported that the membrane-bound form of Steel factor (SLF) induces more persistent tyrosine kinase activation and longer life span of c-kit encoded protein (KIT) than its soluble form (Miyazawa et al, Blood 85:641, 1995). In this study, we used YB5.B8 monoclonal antibody (MoAb) that recognizes the extracellular domain of KIT to investigate whether immobilized anti-KIT MoAb can substitute for SLF as a potent activator of KIT by cross-linking receptors and further compared its effect with each SLF isoform in a factor-dependent cell line M07e. YB5.B8 MoAb in a soluble state suppressed SLF-induced M07e cell proliferation in a dose-dependent manner. By contrast, once this antibody was immobilized on the goat-antimouse MoAb (GAM)-coated culture plates, it supported the growth of M07e cells in the absence of any growth factors, whereas culture the cells in GAM alone or YB5.B8 without GAM-coated plates resulted in rapid cell-death within 24 hours. As with the natural ligand SLF, immobilized YB5.B8 MoAb synergized with granulocyte-macrophage colony-stimulating factor (GM-CSF) in inducing cell proliferation compared with either YB5.B8 MoAb or GM-CSF alone. Immunoblotting with antiphosphotyrosine MoAb showed that interaction of M07e cells with immobilized YB5.B8 induced tyrosine phosphorylation of a series of intracellular proteins including KIT (145 kD). In addition, cross-linking studies using a water-soluble cross linking reagent bis-sulfosuccinimidyl-suberate showed that immobilized YB5.B8 MoAb induced dimerization and activation of KIT. However, as with stimulation by the membrane-bound form of SLF, the kinetics of KIT activation with YB5.B8 MoAb was more prolonged compared with the cells treated with recombinant soluble SLF. Flow cytometry showed that, unlike the cells treated with soluble SLF, no downmodulation of cell-surface KIT expression was observed in M07e cells cultured with immobilzed YB5.B8 MoAb. These data suggest that immobilized antibodies against hematopoietic receptors may replace their ligand-stimulators; however, their activities may resemble the membrane-bound form rather than the soluble form of natural ligands.
authors.affiliation: The First Department of Internal Medicine (Hematology/Oncology), Tokyo Medical College, Tokyo Japan.
authors.forename: K
authors.initials: K
authors.lastname: Kurosawa
authors.forename: K
authors.initials: K
authors.lastname: Miyazawa
authors.forename: A
authors.initials: A
authors.lastname: Gotoh
authors.forename: T
authors.initials: T
authors.lastname: Katagiri
authors.forename: J
authors.initials: J
authors.lastname: Nishimaki
authors.forename: L K
authors.initials: LK
authors.lastname: Ashman
authors.forename: K
authors.initials: K
authors.lastname: Toyama
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology / Q000494:pharmacology*; D002460:Cell Line; D006041:Goats; D006412:Hematopoietic Stem Cells / Q000187:drug effects; D006801:Humans; D007947:Leukemia, Megakaryoblastic, Acute / Q000473:pathology; D008024:Ligands; D008565:Membrane Proteins / Q000737:chemistry / Q000187:drug effects* / Q000276:immunology / Q000378:metabolism; D051379:Mice; D018716:Molecular Mimicry; D008969:Molecular Sequence Data; D014411:Neoplastic Stem Cells / Q000187:drug effects; D010446:Peptide Fragments / Q000276:immunology; D011485:Protein Binding; D011487:Protein Conformation / Q000187:drug effects; D019009:Proto-Oncogene Proteins c-kit / Q000737:chemistry / Q000187:drug effects* / Q000276:immunology / Q000378:metabolism; D012995:Solubility; D019089:Stem Cell Factor / Q000737:chemistry*
chemical_list: D000911:Antibodies, Monoclonal; D008024:Ligands; D008565:Membrane Proteins; D010446:Peptide Fragments; D019089:Stem Cell Factor; D019009:Proto-Oncogene Proteins c-kit