Stem cell factor influences the proliferation and erythroid differentiation of the MB-02 human erythroleukemia cell line by binding to a high-affinity c-kit receptor

Abstract

Abstract Stem cell factor (SCF) acts in synergy with other growth factors such as erythropoietin (Epo), granulocyte-macrophage colony-stimulating factor (GM-CSF), or interleukin-3 (IL-3), to stimulate the growth of primitive hematopoietic cells. Because of the prominent role of CSF in the maintenance of normal erythropoiesis in vivo, we examined the effects of SCF on the Epo-inducible human erythroleukemia cell line MB- 02, and characterized the c-kit receptor in these cells. MB-02 cells cultured in serum-containing media do not survive in the absence of exogenous growth factors, but the addition of SCF, Epo, or IL-3 as a single factor enhanced MB-02 survival. Furthermore, in the presence of Epo, SCF (5 to 25 ng/mL) enhanced MB-02 proliferation in a dose- dependent manner, and increased the relative and absolute number of benzidine-positive cells generated. SCF also enhanced cell proliferation in the presence of either IL-3 or low concentrations of GM-CSF. A neutralizing anti-c-kit receptor monoclonal antibody (SR-1) blocked binding of 125I-SCF to MB-02 cells by 98%, and the effect of SCF on MB-02 growth, c-kit receptor-binding parameters were quantitated by equilibrium-binding experiments with 125I-SCF. MB-02 cells display a single class of high-affinity (50 pmol/L) c-kit receptors, with approximately 8,000 receptors per cell. The molecular weight of the c- kit receptor was determined by affinity cross-linking 125I-SCF to MB-02 cells. 125I-SCF-c-kit receptor complexes of approximately 155,000 and approximately 310,000 daltons were found, likely representing the monomeric and dimeric forms of the c-kit receptor. The binding affinity and molecular weight of the c-kit receptor on MB-02 cells are similar to those of normal human marrow cells. These results suggest that SCF synergizes with Epo to influence not only the proliferation but the erythroid differentiation of MB-02 cells. Thus, the MB-02 cell line may be a useful model in which to investigate the molecular mechanisms of SCF action.