CHARACTERIZATION OF A MAST-CELL LINE THAT LACKS THE EXTRACELLULAR DOMAIN OF MEMBRANE C-KIT

Expression of the c-kit proto-oncogene receptor on mast cells is essen tial for their normal proliferation and maturation as well as for seve ral biological responses such as chemotaxis and attachment. In the pre sent study we report that the interleukin-3 (IL-3)-dependent mast cell line CFTL-15 lacks the extracellular domain of the c-kit receptor. Th is observation was made after noting that the c-kit ligand stem cell f actor (SCF) could not prevent IL-3 deprivation-induced mast cell apopt osis and that CFTL-15 cells did not proliferate in response to SCF. Fl ow cytometric analysis employing monoclonal anti-c-kit antibodies, and immunogold labelling with analysis by electron microscopy, subsequent ly showed a diminished expression of c-kit on CFTL-15 cells. There was no identifiable message for the extracellular domain of c-kit in thes e cells, as determined by reverse transcriptase-polymerase chain react ion (RT-PCR). These previously unrecognized properties of the CFTL-15 mast cell line allowed the examination of other biological consequence s of the lack of c-kit on mast cells. Analysing the ability of these c ells to adhere to surface-bound fibronectin, it was found that additio n of SCF did not increase their adhesion to this substrate, in opposit ion 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 the se cells in response to SCF. CFTL-15 cells underwent apoptosis on remo val of IL-3 coincident with a decrease in endogenous Bcl-2 mRNA. Overe xpression of Bcl-2 cDNA prolonged survival of Bcl-2-transfected CFTL-1 5 cells upon withdrawal of IL-3. Thus, the CFTL-15 cell line that lack s surface c-kit is not able to proliferate in response to SCF, undergo es apoptosis in the presence of SCF, and does not adhere to fibroblast s. These results confirm earlier studies on the functional consequence s of c-kit and provide a novel experimental model for further investig ation.