Removal of stem cell factor or addition of monoclonal anti-c-KIT antibody induces apoptosis in murine melanocyte precursors

Previous findings indicate that the protein c-KIT and its Ligand, stem cell factor (SCF) play a crucial role in the development of melanocytes from th eir precursors in the embryonic neural crest cells. Using a monoclonal anti -c-KIT antibody, ACK2, which is an antagonistic blocker of c-KIT function, we and others demonstrated that mouse melanocytes disappeared with the inje ction of ACK2 during certain periods of embryonic and postnatal life. The p recise mechanisms of this disappearance, however, remain unclear. Because m elanocytes disappeared without any inflammation in these in vivo studies, w e suspect that apoptosis was a main cause of their disappearance. In this s tudy, to clarify the underlying mechanism, we studied whether ACK2 induces apoptosis in c-KIT-positive melanoblasts, which appear in mouse neural cres t cells cultured with SCF from 9.5 d old mouse embryos,With an in situ apop tosis detection kit, a significant increase in apoptosis was detected after the removal of SCF, which further increased with the addition of ACK2 duri ng SCF-dependent periods. The occurrence of apoptosis in the cultured cells was also demonstrated by a DNA analysis and electron microscopy, Immunohis tochemical double staining confirmed that the apoptotic cells were c-KIT po sitive, and the electron microscopy showed that these apoptotic cells were melanocyte precursors. It was therefore demonstrated that apoptosis was ind uced in the SCF-dependent c-KIT-positive melanocytes in vitro when the SCF/ c-KIT interaction was obstructed. These findings elucidate the mechanism of the regulation of melanocyte development, and the survival and proliferati on of these precursor cells, by SCF/c-KIT interaction.