In human immature BFU-E tumor necrosis factor-α 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-α (TNF-α) 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-α, 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-α. Apoptosis was observed in single-cell plasma clot assays. TUNEL assays showed 20% ± 6% apoptotic cells with TNF-α while controls had 2.8% ± 2.2%. Caspases 3 and 8 were strongly activated while the amount of CDK6 was reduced by TNF-α. When SCF, a potent stimulator of cell growth, was added with TNF-α, 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-α downregulates CDK6 but also directly produces apoptosis which is prevented by SCF.