title: Characterization of a mast cell line that lacks the extracellular domain of membrane c-kit.
abstract: Expression of the c-kit proto-oncogene receptor on mast cells is essential for their normal proliferation and maturation as well as for several biological responses such as chemotaxis and attachment. In the present study we report that the interleukin-3 (IL-3)-dependent mast cell line CFTL-15 lacks the extracellular domain of the c-kit receptor. This observation was made after noting that the c-kit ligand stem cell factor (SCF) could not prevent IL-3 deprivation-induced mast cell apoptosis and that CFTL-15 cells did not proliferate in response to SCF. Flow cytometric analysis employing monoclonal anti-c-kit antibodies, and immunogold labelling with analysis by electron microscopy, subsequently showed a diminished expression of c-kit on CFTL-15 cells. There was no identifiable message for the extracellular domain of c-kit in these cells, as determined by reverse transcriptase-polymerase chain reaction (RT-PCR). These previously unrecognized properties of the CFTL-15 mast cell line allowed the examination of other biological consequences of the lack of c-kit on mast cells. Analysing the ability of these cells to adhere to surface-bound fibronectin, it was found that addition of SCF did not increase their adhesion to this substrate, in opposition to what is reported with other mast cells. Similarly, CFTL-15 mast cells did not adhere to fibroblasts, which is known to require c-kit expression. Also, there was no protein tyrosine phosphorylation in these cells in response to SCF. CFTL-15 cells underwent apoptosis on removal of IL-3 coincident with a decrease in endogenous Bcl-2 mRNA. Overexpression of Bcl-2 cDNA prolonged survival of Bcl-2-transfected CFTL-15 cells upon withdrawal of IL-3. Thus, the CFTL-15 cell line that lacks surface c-kit is not able to proliferate in response to SCF, undergoes apoptosis in the presence of SCF, and does not adhere to fibroblasts. These results confirm earlier studies on the functional consequences of c-kit and provide a novel experimental model for further investigation.
authors.affiliation: Laboratory of Allergic Diseases, National Institute of Allergy and Infectious Diseases and National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-1881, USA.
authors.forename: Y A
authors.initials: YA
authors.lastname: Mekori
authors.forename: C K
authors.initials: CK
authors.lastname: Oh
authors.forename: J
authors.initials: J
authors.lastname: Dastych
authors.forename: J P
authors.initials: JP
authors.lastname: Goff
authors.forename: S
authors.initials: S
authors.lastname: Adachi
authors.forename: P J
authors.initials: PJ
authors.lastname: Bianchine
authors.forename: A
authors.initials: A
authors.lastname: Worobec
authors.forename: T
authors.initials: T
authors.lastname: Semere
authors.forename: J H
authors.initials: JH
authors.lastname: Pierce
authors.forename: D D
authors.initials: DD
authors.lastname: Metcalfe
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000276:immunology; D002448:Cell Adhesion / Q000276:immunology; D002450:Cell Communication / Q000276:immunology; D002455:Cell Division / Q000276:immunology; D002460:Cell Line; D002470:Cell Survival / Q000276:immunology; D007377:Interleukin-3 / Q000276:immunology; D008407:Mast Cells / Q000276:immunology* / Q000648:ultrastructure; D051379:Mice; D008807:Mice, Inbred BALB C; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D019009:Proto-Oncogene Proteins c-kit / Q000378:metabolism; D020794:Receptor Protein-Tyrosine Kinases / Q000172:deficiency* / Q000502:physiology; D019089:Stem Cell Factor / Q000276:immunology
chemical_list: D007377:Interleukin-3; D019253:Proto-Oncogene Proteins c-bcl-2; D019089:Stem Cell Factor; D019009:Proto-Oncogene Proteins c-kit; D020794:Receptor Protein-Tyrosine Kinases