In human immature BFU-E tumor necrosis factor-alpha not only downregulates CDK6 but also directly produces apoptosis which is prevented by stem cell factor.
OBJECTIVE
The aim of this study was to reveal the mechanisms by which tumor necrosis factor-alpha (TNF-alpha) inhibits immature human day-4 burst-forming units-erythroid (BFU-E) and the effect of stem cell factor (SCF) on this process.


METHODS
Sequential density-gradient centrifugation, depletion of lymphocytes, removal of adherent cells, and negative selection with CD2, CD11b, CD16, and CD45 monoclonal antibodies were used to purify day-1 BFU-E, which were then incubated for 3 days to generate day-4 cells. The day-4 cells were incubated with TNF-alpha, and/or SCF, and the extent of apoptosis was gauged by morphologic observations, TUNEL assays, and Western blots.


RESULTS
The cell number and the number and size of erythroid colonies were significantly reduced when day-4 cells were incubated with TNF-alpha. Apoptosis was observed in single-cell plasma clot assays. TUNEL assays showed 20% +/- 6% apoptotic cells with TNF-alpha while controls had 2.8% +/- 2.2%. Caspases 3 and 8 were strongly activated while the amount of CDK6 was reduced by TNF-alpha. When SCF, a potent stimulator of cell growth, was added with TNF-alpha, cell growth inhibition was reduced and the apoptotic cells decreased to 0.9% +/- 1.2%. The activations of caspase 3 and caspase 8 were almost completely blocked by SCF while CDK6 and the FLICE-inhibitory protein (FLIP) were increased.


CONCLUSIONS
Our results indicate that in immature human BFU-E, TNF-alpha downregulates CDK6 but also directly produces apoptosis which is prevented by SCF.
Dai
Chunhua
C
Hematology/Oncology Division, Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN 37232-6307, USA.
Chung
Ik-Joo
IJ
Krantz
Sanford B
SB
D017209:Apoptosis / Q000187:drug effects*; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein; D020169:Caspases / Q000378:metabolism; D002452:Cell Count; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D051361:Cyclin-Dependent Kinase 6; D018844:Cyclin-Dependent Kinases / Q000187:drug effects* / Q000235:genetics; D015536:Down-Regulation / Q000187:drug effects; D015672:Erythroid Precursor Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000032:analysis; D019089:Stem Cell Factor / Q000494:pharmacology*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D053446:CASP8 and FADD-Like Apoptosis Regulating Protein; C506386:CFLAR protein, human; D047908:Intracellular Signaling Peptides and Proteins; D019089:Stem Cell Factor; D014409:Tumor Necrosis Factor-alpha; C495911:CDK6 protein, human; D051361:Cyclin-Dependent Kinase 6; D018844:Cyclin-Dependent Kinases; D020169:Caspases