Monoclonal antibody YB5.B8 identifies the human c-kit protein product

Abstract

The c-kit proto-oncogene encodes a 145- to 160-Kd transmembrane tyrosine kinase, which is a member of the platelet-derived growth factor receptor family and is allelic with the murine white spotting locus (W). W mutations affect several aspects of hematopoiesis, most notably erythroid progenitors and mast cells. A monoclonal antibody, YB5.B8, had been raised against the leukemic blasts of a patient with M1-type acute myelocytic leukemia (AML) and it precipitates a 150-Kd cell surface glycoprotein from leukemic cells. The YB5.B8 epitope is expressed on mast cells, on up to 3% of normal mononuclear bone marrow cells, and it identifies a sub-group of AML patients with a poor prognosis. In view of similarities noted between the cell surface antigen identified by YB5.B8 and the c-kit protein product, we performed experiments to determine whether they are identical. c-kit RNA expression in the cell lines HEL (human erythroleukemia) and A172 (glioblastoma) was shown to parallel the expression of the YB5.B8 epitope in these lines as measured by flow cytometry. Immunoprecipitation analysis with anti-kit serum and YB5.B8 antibody indicated that the two antibodies identified proteins of identical size in HEL (155 Kd) and A172 (145 Kd) cells, and sequential immunoprecipitations with the kit and the YB5.B8 antibodies demonstrated that the two antibodies recognize the same molecule. The proteins identified by both the anti-kit and YB5.B8 antibodies displayed in vitro autophosphorylation activity in immune complex kinase assays. In addition, YB5.B8 was able to inhibit the binding of the kit ligand to HEL cells. These studies provide evidence that the YB5.B8 antigen and the c-kit protein product are identical and raise certain hypotheses regarding the role of c-kit in AML.

VEGF‐R blockade causes endothelial cell apoptosis, expansion of surviving CD34+precursor cells and transdifferentiation to smooth muscle‐like and neuronal‐like cells

“…CD117 (c-kit) recognizes a 145 kDa cell-surface glycoprotein with tyrosine kinase activity. CD117 is present on hematopoietic progenitor cell subsets, thymocytes, mast cells, hepatocytes, and histiocytes (43)(44)(45)(46). To test whether the mature HPMVEC used in our experiments include hematopoietic progenitor cells, they were assessed by flow cytometry for both CD34 and/or c-kit.…”

Functional and phenotypic studies of two variants of a human mast cell line with a distinct set of mutations in the c‐kit proto‐oncogene

“…2 Monoclonal antibodies against the following cell surface markers were used: CD 13 (WM-15), CD 32 (IV.3), CD 44 (F-10-44-2), CD 54 (My13) from the 4th International Workshop of Human Leukocyte. The monoclonal antibody (mAb) against the a-chain of the FcERI receptor (29C6) 39 was kindly provided by Drs R. Chizzonite and F. Riske (Hoffmann-La Roche, Nutley, NJ); the antibody against Kit (YB5.B8) 40 was a kind gift from Dr L. Ashman (Hanson Centre for Cancer Research, Adelaide, Australia), the mAbs against CD 49b (P1H5) and CD 49c (P1B5) were kindly provided by Prof K. Rubin (Uppsala University, Uppsala, Sweden), and the antibody against CD 18 (IB4) was a kind gift from Dr C. Lundberg (Pharmacia, Uppsala, Sweden). As negative controls, isotype-matched irrelevant antibodies were used (Dako Glostrup, Denmark).…”

Nerve growth factor‐β induces mast‐cell marker expression during in vitro culture of human umbilical cord blood cells

“…The monoclonal antibody against mast cell tryptase (AA1) 45 was kindly provided by A. Walls, Southampton, UK, that against the a-chain of the FceRI (29C6) by J. Hakimi, Nutley, NJ, 46 that against CD117 (c-Kit) (YB5.B8) by L. Ashman, Adelaide, Australia, 47 and the basophil speci®c antibody (2D7) by L.B. Schwartz, Richmond, VA. 40 The antibody against NGFR L was purchased from Boehringer Mannheim, that against NGFR H from Santa Cruz Biotechnology (Santa Cruz, CA), and that against desmin (D33) from Dianova, Hamburg, Germany.…”