Stem cell factor protects c-kit+ human primary erythroid cells from apoptosis

Objective. It has been reported that stem cell factor (SCF) promotes cell survival in primary cultured human erythroid colony-forming cells (ECFC), Given the heterogeneous nature of ECFC, which may affect interpretation of the data, we purified c-kit(+) ECFC and investigated the specificity and mechanisms of the anti-apoptotic effects of SCF on these cells. Materials and Methods. Glycophorin A(+) (GPA(+)) c-kit(+) cells were purified from primary cultured ECFC derived from purified human CD34(+) cells. The GPA(+)c-kit(-) and nonerythroid cells were generated from the same CD34(+) cells. Apoptosis of ECFC was investigated in the absence or presence of SCF and erythropoietin (EPO) in serum-free medium. DNA fragmentation was measured with enzyme linked immunosorbent assay for oligonucleosome-sized DNA, gel electrophoresis, and annexin V labeling. Characterization of expanded cells and enriched cells was performed using multiparameter flow cytometry, For Akt assay, cells were lysed and the cleared lysates subjected to SDS-PAGE followed by Western blotting. Results. In GPA(+)c-kit(+) cells, deprivation of cytokine caused rapid DNA fragmentation within 4 hours that reached a maximum at 6 hours. This was partially but clearly prevented by SCF or EPO, In contrast, no significant DNA fragmentation was seen in GPA(+)c-kit(-) and nonerythroid cells within 24 hours. PP2, a specific Src family kinase inhibitor, but not its inactive analogue PP3, reversed the anti-apoptotic effects of SCF, PP2 also inhibited SCF-induced phosphorylation of Akt, Conclusions. These data indicate that SCF protects purified human GPA(+)c-kit(+) cells from apoptosis and suggest that kit-mediated Src kinase activation is involved in Akt activation and cell survival. (C) 2001 International Society for Experimental Hematology. Published by Elsevier Science Inc.