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.


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.
Endo
T
T
Department of Internal Medicine II, Hokkaido University School of Medicine, Sapporo, Japan.
Odb
A
A
Satoh
I
I
Haseyama
Y
Y
Nishio
M
M
Koizumi
K
K
Takashima
H
H
Fujimoto
K
K
Amasaki
Y
Y
Fujita
H
H
Koike
T
T
Sawada
K
K
D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D002478:Cells, Cultured; D004912:Erythrocytes / Q000473:pathology* / Q000502:physiology*; D006801:Humans; D019009:Proto-Oncogene Proteins c-kit / Q000502:physiology*; D015398:Signal Transduction / Q000187:drug effects; D019089:Stem Cell Factor / Q000494:pharmacology*; D019061:src-Family Kinases / Q000502:physiology
D019089:Stem Cell Factor; D019009:Proto-Oncogene Proteins c-kit; D019061:src-Family Kinases