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Effects of stem cell factor on hypoxia-inducible factor 1 alpha accumulation in human acute myeloid leukaemia and LAD2 mast cells.

Gibbs BF, Yasinska IM, Oniku AE, Sumbayev VV - PLoS ONE (2011)

Bottom Line: However, the mechanisms of this pathophysiological effect remain unclear.We demonstrated that LAD2 cells have a more robust glutathione (GSH)-dependent antioxidative system compared to THP-1 cells and are therefore protected against the actions of ROS generated in an SCF-dependent manner.BSO-induced GSH depletion led to a significant decrease in HIF-1α prolyl hydroxylase (PHD) activity in THP-1 cells and to near attenuation of it in LAD2 cells.

View Article: PubMed Central - PubMed

Affiliation: Medway School of Pharmacy, University of Kent, Kent, United Kingdom. B.F.Gibbs@kent.ac.uk

ABSTRACT
Stem cell factor (SCF) is a hematopoietic growth factor that exerts its activity by signalling through the tyrosine kinase receptor known as Kit or CD117. SCF-Kit signalling is crucial for the survival, proliferation and differentiation of hematopoietic cells of myeloid lineage. Furthermore, since myeloid leukaemia cells express the Kit receptor, SCF may play an important role in myeloid leukaemia progression too. However, the mechanisms of this pathophysiological effect remain unclear. Recent evidence shows that SCF triggers accumulation of the inducible alpha subunit of hypoxia-inducible factor 1 (HIF-1) in hematopoietic cells--a transcription complex that plays a pivotal role in cellular adaptation to low oxygen availability. However, it is unknown how SCF impacts on HIF-1α accumulation in human myeloid leukaemia and mast cells. Here we show that SCF induces HIF-1α accumulation in THP-1 human myeloid leukaemia cells but not in LAD2 mast cells. We demonstrated that LAD2 cells have a more robust glutathione (GSH)-dependent antioxidative system compared to THP-1 cells and are therefore protected against the actions of ROS generated in an SCF-dependent manner. BSO-induced GSH depletion led to a significant decrease in HIF-1α prolyl hydroxylase (PHD) activity in THP-1 cells and to near attenuation of it in LAD2 cells. In THP-1 cells, SCF-induced HIF-1α accumulation is controlled via ERK, PI3 kinase/PKC-δ/mTOR-dependent and to a certain extent by redox-dependent mechanisms. These results demonstrate for the first time an important cross-talk of signalling pathways associated with HIF-1 activation--an important stage of the myeloid leukaemia cell life cycle.

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GSH depletion leads to a reduction of SCF-induced HIF-1α PHD activity in THP-1 cells.THP-1 human myeloid leukaemia cells were cultured (in fresh medium) for 24 h in the absence or presence of 100 ng/ml SCF ± 500 µM BSO. (A) HIF-1α accumulation/PHD activity and (B) annexin V/DAPI staining were performed as outlined in Materials and methods. Quantitative data are mean values ± S.D. of at least three individual experiments. *P<0.01 vs control. All Western blot and imaging data are from one representative experiment out of three that gave similar results.
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pone-0022502-g003: GSH depletion leads to a reduction of SCF-induced HIF-1α PHD activity in THP-1 cells.THP-1 human myeloid leukaemia cells were cultured (in fresh medium) for 24 h in the absence or presence of 100 ng/ml SCF ± 500 µM BSO. (A) HIF-1α accumulation/PHD activity and (B) annexin V/DAPI staining were performed as outlined in Materials and methods. Quantitative data are mean values ± S.D. of at least three individual experiments. *P<0.01 vs control. All Western blot and imaging data are from one representative experiment out of three that gave similar results.

Mentions: Since ROS are likely to be produced in response to treatment with SCF [8], [9], we therefore investigated the role of the GSH-dependent antioxidative system in both THP-1 and LAD2 cells. THP-1 cells were stimulated for 24 h with 100 ng/ml SCF in the absence or presence of 500 µM buthionine sulphoximine (BSO) for 24 h. For control purposes, SCF-untreated cells also were exposed to 500 µM BSO for 24 h. We observed that, in THP-1 cells, the presence of BSO but in the absence of SCF led to decreased HIF-1α PHD activity (Figure 3A). In the presence of SCF, BSO also reduced PHD activity in THP-1 cells (Figure 3A). In parallel, HIF-1α accumulation in THP-1 cells was decreased by BSO in the absence of SCF. However, following 24 h exposure to SCF, HIF-1α accumulation was not affected by this inhibitor. The number of cells stainable with annexin V was significantly higher compared to the number of DAPI stainable cells in cells exposed to BSO independently of the presence or absence of SCF (Figure 3B).


Effects of stem cell factor on hypoxia-inducible factor 1 alpha accumulation in human acute myeloid leukaemia and LAD2 mast cells.

Gibbs BF, Yasinska IM, Oniku AE, Sumbayev VV - PLoS ONE (2011)

GSH depletion leads to a reduction of SCF-induced HIF-1α PHD activity in THP-1 cells.THP-1 human myeloid leukaemia cells were cultured (in fresh medium) for 24 h in the absence or presence of 100 ng/ml SCF ± 500 µM BSO. (A) HIF-1α accumulation/PHD activity and (B) annexin V/DAPI staining were performed as outlined in Materials and methods. Quantitative data are mean values ± S.D. of at least three individual experiments. *P<0.01 vs control. All Western blot and imaging data are from one representative experiment out of three that gave similar results.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3140519&req=5

pone-0022502-g003: GSH depletion leads to a reduction of SCF-induced HIF-1α PHD activity in THP-1 cells.THP-1 human myeloid leukaemia cells were cultured (in fresh medium) for 24 h in the absence or presence of 100 ng/ml SCF ± 500 µM BSO. (A) HIF-1α accumulation/PHD activity and (B) annexin V/DAPI staining were performed as outlined in Materials and methods. Quantitative data are mean values ± S.D. of at least three individual experiments. *P<0.01 vs control. All Western blot and imaging data are from one representative experiment out of three that gave similar results.
Mentions: Since ROS are likely to be produced in response to treatment with SCF [8], [9], we therefore investigated the role of the GSH-dependent antioxidative system in both THP-1 and LAD2 cells. THP-1 cells were stimulated for 24 h with 100 ng/ml SCF in the absence or presence of 500 µM buthionine sulphoximine (BSO) for 24 h. For control purposes, SCF-untreated cells also were exposed to 500 µM BSO for 24 h. We observed that, in THP-1 cells, the presence of BSO but in the absence of SCF led to decreased HIF-1α PHD activity (Figure 3A). In the presence of SCF, BSO also reduced PHD activity in THP-1 cells (Figure 3A). In parallel, HIF-1α accumulation in THP-1 cells was decreased by BSO in the absence of SCF. However, following 24 h exposure to SCF, HIF-1α accumulation was not affected by this inhibitor. The number of cells stainable with annexin V was significantly higher compared to the number of DAPI stainable cells in cells exposed to BSO independently of the presence or absence of SCF (Figure 3B).

Bottom Line: However, the mechanisms of this pathophysiological effect remain unclear.We demonstrated that LAD2 cells have a more robust glutathione (GSH)-dependent antioxidative system compared to THP-1 cells and are therefore protected against the actions of ROS generated in an SCF-dependent manner.BSO-induced GSH depletion led to a significant decrease in HIF-1α prolyl hydroxylase (PHD) activity in THP-1 cells and to near attenuation of it in LAD2 cells.

View Article: PubMed Central - PubMed

Affiliation: Medway School of Pharmacy, University of Kent, Kent, United Kingdom. B.F.Gibbs@kent.ac.uk

ABSTRACT
Stem cell factor (SCF) is a hematopoietic growth factor that exerts its activity by signalling through the tyrosine kinase receptor known as Kit or CD117. SCF-Kit signalling is crucial for the survival, proliferation and differentiation of hematopoietic cells of myeloid lineage. Furthermore, since myeloid leukaemia cells express the Kit receptor, SCF may play an important role in myeloid leukaemia progression too. However, the mechanisms of this pathophysiological effect remain unclear. Recent evidence shows that SCF triggers accumulation of the inducible alpha subunit of hypoxia-inducible factor 1 (HIF-1) in hematopoietic cells--a transcription complex that plays a pivotal role in cellular adaptation to low oxygen availability. However, it is unknown how SCF impacts on HIF-1α accumulation in human myeloid leukaemia and mast cells. Here we show that SCF induces HIF-1α accumulation in THP-1 human myeloid leukaemia cells but not in LAD2 mast cells. We demonstrated that LAD2 cells have a more robust glutathione (GSH)-dependent antioxidative system compared to THP-1 cells and are therefore protected against the actions of ROS generated in an SCF-dependent manner. BSO-induced GSH depletion led to a significant decrease in HIF-1α prolyl hydroxylase (PHD) activity in THP-1 cells and to near attenuation of it in LAD2 cells. In THP-1 cells, SCF-induced HIF-1α accumulation is controlled via ERK, PI3 kinase/PKC-δ/mTOR-dependent and to a certain extent by redox-dependent mechanisms. These results demonstrate for the first time an important cross-talk of signalling pathways associated with HIF-1 activation--an important stage of the myeloid leukaemia cell life cycle.

Show MeSH
Related in: MedlinePlus