c-kit protein (stem cell factor receptor) expression on cells with erythroid characteristics and on normal human bone marrow without the use of monoclonal antibodies.
Stem cell factor (SCF) is a determining and crucial element in the development of early hematopoietic cells. The SCF receptor protein has been identified as the product of the protooncogene c-kit and has been detected using monoclonal antibodies (MAbs) on a broad selection of erythroid, myeloid, and lymphoid cell lines as well as on bone marrow mononuclear cells (BMMNC). SCF is known to increase both the number and size of burst-forming unit-erythroid (BFU-E) colonies in normal human BM culture in a dose-dependent fashion. A detailed study of the involvement of SCF and its receptor c-kit in normal erythropoiesis will help elucidate intrinsic irregularities of anemias such as Diamond Blackfan Anemia, an aregenerative congenital anemia. Abnormalities of this heterogeneous disorder are confined to the red cell lineage and are thought to arise through a defect at the stem/progenitor cell level. Our in vitro studies suggest that SCF therapy will influence BFU-E production in at least a portion of these patients, although in another group, SCF response is limited or absent. Additionally, further investigations have shown a possible c-kit signaling defect that clearly necessitates further c-kit characterization. To parallel this, we, therefore, attempted to study the relationship of c-kit with its ligand. This report describes a nonradioactive method for detecting SCF receptors that varies from conventional assays in that the fluorescent label conjugated to the SCF/c-kit complex is connected via an extended-ester linkage that reduces steric influence and promotes full normal structural ligand binding of the SCF to its receptor.(ABSTRACT TRUNCATED AT 250 WORDS)
McGuckin
C P
CP
Division of Haematology, St. George's Hospital Medical School, University of London, United Kingdom.
Uhr
M R
MR
Gordon-Smith
E C
EC
D000911:Antibodies, Monoclonal; D001853:Bone Marrow / Q000737:chemistry*; D002460:Cell Line; D004912:Erythrocytes / Q000737:chemistry*; D005434:Flow Cytometry; D016650:Fluorescein-5-isothiocyanate; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000737:chemistry; D011518:Proto-Oncogene Proteins / Q000032:analysis* / Q000378:metabolism; D019009:Proto-Oncogene Proteins c-kit; D020794:Receptor Protein-Tyrosine Kinases / Q000032:analysis* / Q000378:metabolism; D016184:Receptors, Colony-Stimulating Factor / Q000032:analysis* / Q000378:metabolism
D000911:Antibodies, Monoclonal; D011518:Proto-Oncogene Proteins; D016184:Receptors, Colony-Stimulating Factor; D019009:Proto-Oncogene Proteins c-kit; D020794:Receptor Protein-Tyrosine Kinases; D016650:Fluorescein-5-isothiocyanate