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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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The impact of SCF on HIF-1α accumulation and the GSH-dependent antioxidative system in human myeloid leukaemia cells.(A) THP-1 human myeloid leukaemia cells were stimulated for 24 h with 100 ng/ml SCF. HIF-1α accumulation, GSH and GSSG levels, GPx/GR activities and the quantity of the TBRS were then measured as outlined in Materials and methods. (B) THP-1 human myeloid leukaemia cells were cultured in the presence or absence of 100 ng/ml SCF for 24 h. HIF-1 DNA-binding activity was analysed using 10 mg/ml BSA as a negative control. (C) THP-1 cells were exposed for 4 h to 25, 50 and 100 ng/ml SCF. HIF-1α accumulation was then measured as described in Materials and methods. Quantitative data are mean values ± S.D. of at least three individual experiments. *P<0.01 vs control. Western blot data are shown from one representative experiment out of three that gave similar results.
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pone-0022502-g001: The impact of SCF on HIF-1α accumulation and the GSH-dependent antioxidative system in human myeloid leukaemia cells.(A) THP-1 human myeloid leukaemia cells were stimulated for 24 h with 100 ng/ml SCF. HIF-1α accumulation, GSH and GSSG levels, GPx/GR activities and the quantity of the TBRS were then measured as outlined in Materials and methods. (B) THP-1 human myeloid leukaemia cells were cultured in the presence or absence of 100 ng/ml SCF for 24 h. HIF-1 DNA-binding activity was analysed using 10 mg/ml BSA as a negative control. (C) THP-1 cells were exposed for 4 h to 25, 50 and 100 ng/ml SCF. HIF-1α accumulation was then measured as described in Materials and methods. Quantitative data are mean values ± S.D. of at least three individual experiments. *P<0.01 vs control. Western blot data are shown from one representative experiment out of three that gave similar results.

Mentions: Firstly, we established whether SCF is able to induce HIF-1α accumulation and, at the same time, affects the GSH-dependent antioxidative system in THP-1 human myeloid leukaemia cells. We observed that SCF significantly upregulated HIF-1α accumulation in THP-1 cells following exposure to 100 ng/ml SCF for 24 h (Figure 1A). THP-1 cells also responded by a decrease in GSH production and a reduced GSH/GSSG ratio (Figure 1A). The presence of SCF reduced GPx activity, while no significant changes were observed in GR activity. TBRS levels were moderately increased in the presence of SCF (Figure 1A). Analysis of HIF-1 DNA-binding activity showed that the protein is transcriptionally active and significantly increased by 24 h incubation of THP-1 cells with SCF (Figure 1B). Furthermore, we observed that this SCF-mediated increase in HIF-1α accumulation was concentration dependent (Figure 1C).


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)

The impact of SCF on HIF-1α accumulation and the GSH-dependent antioxidative system in human myeloid leukaemia cells.(A) THP-1 human myeloid leukaemia cells were stimulated for 24 h with 100 ng/ml SCF. HIF-1α accumulation, GSH and GSSG levels, GPx/GR activities and the quantity of the TBRS were then measured as outlined in Materials and methods. (B) THP-1 human myeloid leukaemia cells were cultured in the presence or absence of 100 ng/ml SCF for 24 h. HIF-1 DNA-binding activity was analysed using 10 mg/ml BSA as a negative control. (C) THP-1 cells were exposed for 4 h to 25, 50 and 100 ng/ml SCF. HIF-1α accumulation was then measured as described in Materials and methods. Quantitative data are mean values ± S.D. of at least three individual experiments. *P<0.01 vs control. Western blot data are shown 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-g001: The impact of SCF on HIF-1α accumulation and the GSH-dependent antioxidative system in human myeloid leukaemia cells.(A) THP-1 human myeloid leukaemia cells were stimulated for 24 h with 100 ng/ml SCF. HIF-1α accumulation, GSH and GSSG levels, GPx/GR activities and the quantity of the TBRS were then measured as outlined in Materials and methods. (B) THP-1 human myeloid leukaemia cells were cultured in the presence or absence of 100 ng/ml SCF for 24 h. HIF-1 DNA-binding activity was analysed using 10 mg/ml BSA as a negative control. (C) THP-1 cells were exposed for 4 h to 25, 50 and 100 ng/ml SCF. HIF-1α accumulation was then measured as described in Materials and methods. Quantitative data are mean values ± S.D. of at least three individual experiments. *P<0.01 vs control. Western blot data are shown from one representative experiment out of three that gave similar results.
Mentions: Firstly, we established whether SCF is able to induce HIF-1α accumulation and, at the same time, affects the GSH-dependent antioxidative system in THP-1 human myeloid leukaemia cells. We observed that SCF significantly upregulated HIF-1α accumulation in THP-1 cells following exposure to 100 ng/ml SCF for 24 h (Figure 1A). THP-1 cells also responded by a decrease in GSH production and a reduced GSH/GSSG ratio (Figure 1A). The presence of SCF reduced GPx activity, while no significant changes were observed in GR activity. TBRS levels were moderately increased in the presence of SCF (Figure 1A). Analysis of HIF-1 DNA-binding activity showed that the protein is transcriptionally active and significantly increased by 24 h incubation of THP-1 cells with SCF (Figure 1B). Furthermore, we observed that this SCF-mediated increase in HIF-1α accumulation was concentration dependent (Figure 1C).

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