Regulation of c-Kit Expression In Human Mast Cells Inhibition of SCF-fnduced c-Kit Downregulation by STI 571 (Gleevec) in LAD 2 and HMC-1 Cells

摘要

Background: c-Kit is of fundamental relevance to mast cell (MC) development and maintenance. However, little is known about the regulation of Kit cell surface levels in normal tissue-derived human MC. Methods: Terminally differentiated MC were isolated from human skin and compared with the less mature MC lines LAD2 and HMC-1. Cells were exposed to stem cell factor (+-STI571), cycloheximide, actinomycin D, and combinations thereof for 20 min to 16 h, before determination of Kit and other cell surface receptors. Results: Ligand-induced Kit internalizalion was a universal mechanism and detectable in HMC-1, LAD2, and skin MC. STI571, (Gleevec, Novartis, Basel, Switzerland) an inhibitor of the intrinsic Kit kinasc, was able to significantly delay Kit internali/ation in LAD2 and also in HMC-1 cells that carry the D816V mutation and had been considered unresponsive to STI571. Investigations into the natural turnover of Kit expression in the three types of MC revealed that Kit is rapidly affected by the inhibition of fundamental cellular processes, even in nonproliferating skin MC. Only a minor decrease of other cell surface receptors (ICAM-1, 1CAM-3, beta1 integrin chain) was noticed in the same time frame. On combined treatment, cycloheximide, actinomycin D, and stem cell factor (SCF) displayed additive effects, resulting in an almost complete disappearance of Kit from the cell surface. Conclusions: Kit represents a rapidly cycling cell surface receptor. Not only is it rapidly internalized upon binding of its ligand, but it is also greatly affected by inhibition of de-novo-protein synthesis or transcription when viewed against the background of other receptors.