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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
K Kurosawa, K Miyazawa, A Gotoh, T Katagiri, J Nishimaki, LK Ashman and K Toyama
The First Department of Internal Medicine (Hematology/Oncology), Tokyo
Medical College, Tokyo Japan.
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.
Volume 87,
Issue 6,
pp. 2235-2243,
03/15/1996
Copyright © 1996 by The American Society of Hematology
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