Conceived and designed the experiments: JFW TGC RN BCD CJC. Performed the experiments: JFW RN DRG JS LB. Analyzed the data: JFW RN TGC JS LB DRG BCD CJC. Contributed reagents/materials/analysis tools: BCD CJC. Wrote the paper: JFW TGC.
The authors have declared that no competing interests exist.
The internal tandem duplication (ITD) of the juxtamembrane region of the FLT3 receptor has been associated with increased reactive oxygen species (ROS) generation in acute myeloid leukemia (AML). How this elevated level of ROS contributes to the leukemic phenotype, however, remains poorly understood. In this work we show that ROS in the FLT3-ITD expressing AML cell line MV4-11 is reduced by treatment with PKC412, an inhibitor of FLT3, DPI, a flavoprotein inhibitor, and VAS2870, a Nox specific inhibitor, suggesting that ROS production is both FLT3 and NADPH oxidase dependent. The majority of these ROS co-localize to the endoplasmic reticulum (ER), as determined with the H2O2-specific aryl-boronate dye Peroxyorange 1, which also corresponds to co-localization of p22phox. Moreover, knocking down p22phox dramatically reduces H2O2 after 24 hours in the ER, without affecting mitochondrial ROS. Significantly, the FLT3 inhibitor PKC412 reduces H2O2 in FLT3-ITD expressing cell lines (MV4-11, MOLM-13) through reduction of p22phox over 24 hours. Reduced p22phox is achieved by proteasomal degradation and is prevented upon GSK3-β inhibition. Knockdown of p22phox resulted in reduced STAT5 signalling and reduced Pim-1 levels in the cells after 24 hours. Thus, we have shown that FLT3 driven H2O2 production in AML cells is mediated by p22phox and is critical for STAT5 signalling.
Aberrant signalling through receptor tyrosine kinases (RTKs) is known to be a significant pathway in tumour development and perpetuation
ROS were traditionally thought of as an unwanted by-product of cellular respiration, but in recent years there has been in a renaissance of research into their role as mediators of intracellular signalling
Similar to other tumours, increased ROS has been seen in a number of myeloid diseases. Haematopoietic growth factor signalling is mediated through ROS
(a) Flow cytometric analysis of intracellular ROS as measured by DCF fluorescence in MV4-11, MOLM-13, HL-60 untreated leukemic cell lines (Unt) and following treatment with PKC412 (250nM) or VAS2870 (10μM) or DPI (5μM) for 1 hours. Mean fluorescence of untreated cells (black lines) is overlaid with that of PKC412 or DPI treated cells (green lines). Bar chart shows relative mean DCF fluorescence of PKC412, VAS2870 or DPI treated cells as a percentage of control untreated cells. All results are expressed as mean ± SD and are representative of three independent experiments. Statistical analysis was performed using ANOVA (*p<0.005). (b) MV4-11 cells were plated on the poly-D-lysine coated glass bottomed dishes and stained with Peroxyorange 1 (PO1). Pseudo-coloured image on the left represents intensity distribution (from highest intensity indicated by white to the lowest designated by black) (c) Cells were incubated with ER tracker, Peroxy Orange and where indicated with PKC412 (250nM;) or (d) VAS-2870 (10μM) for an hour at 37°C/5% CO2 followed by multi-tracking imaging using Zeiss LSM510 META confocal microscope. ER tracker and PO1 probes were excited at 488nm and 543nm respectively and their emissions were collected at 505–530nm and 560–615nm respectively (e) Cells were incubated with MitoPY1 and where indicated with PKC412 (250nM) or VAS-2870 (10μM) for an hour at 37°C/5% CO2 followed by imaging using Zeiss LSM510 META confocal microscope. MitoPY1 was excited at 514nm with a subsequent collection of emission between 505–550nm.
Nox proteins were originally identified in phagocytes, where Nox2 was shown to generate ROS as part of the innate immune system, but numerous homologues have since been identified outside of phagocytes
(a) Western blot analysis of Nox protein expression in untreated MV4-11 cells (Unt), vehicle controls (Veh) and following treatment with PKC412 (250nM) over 24 hours using β-Actin as loading control. Histograms represent the ratio of the intensity of target bands quantified by densitometry factored by the densitometric measurement of loading control band. The data are expressed as percentage of control, where the ratio in the control was defined as 1. Values are the mean ± SD and are representative of three independent experiments. Western blot analysis of p22phox protein expression: (b) in untreated MOLM-13 (Unt), vehicle controls (Veh) and following treatment with PKC412 (250nM); (c) in untreated MV4-11(Unt), vehicle controls (Veh) and following treatment with CEP-701 (50nM); (d) in untreated HL-60 (Unt), vehicle controls (Veh) and following treatment with PKC412 (250nM) over 24 hours using β-Actin as loading control.(e) MV4-11 cells were cultured for 16 hours in Poly-D-lysine coated glass bottomed dishes. Cells were fixed in 3% PFA/PBS prior to staining with KDEL and P22phox antibodies. Images are represented as a single slice from a Z stack projection. Brightness and contrast adjustments have been made for convenience, with identical parameters applied across images. Relative measures of co-localization are included, values are the mean ± SD and are representative of three independent experiments. Digital images were analyzed with Meta-Morph software. Scale bar represents 10 μm. (f) Silencing p22phox reduces ROS generation. MV4-11cells were electroporated with either control siRNA or p22phox siRNA and plated in Poly-D-lysine coated glass bottomed dishes and stained with peroxyorange 1 (PO1), then imaged with a confocal laser scanning microscope. The same plates were then fixed and stained with the p22phox antibody for immunofluorescence. Brightness and contrast adjustments have been made for convenience, with identical parameters applied across images. The scale bar represents 10µm.
In this study we used a number of leukemic cell lines, expressing wild-type and mutated FLT3, to investigate elevated endogenous-ROS associated with FLT3-ITD. We show that the vast majority of H2O2 in these cells localize to the enodoplasmic reticulum (ER). Inhibition of FLT3 signalling by means of PKC412, which is currently in phase II clinical trials for AML
(a) Products of quantitative PCR for p22phox mRNA in untreated MV4-11 cells (Unt) and cells treated with PKC412 (250nM) for 24 hours were visualised by agarose gel separation, loading control is β-Actin. Histogram shows relative expression of p22phox mRNA in untreated MV4-11 cells and cells treated with PKC412 (250nM) for 24 hours as determined by quantitative PCR ΔΔCt-method. Results are expressed as mean ± SD and are representative of three independent experiments. Statistical analysis was performed using student t-test (*p<0.005) (b) Western blot analysis of p22phox protein expression in untreated MV4-11 cells (UT), vehicle controls (Veh), and cells treated with PKC412 (250nM) and lactacystin (5μM; Lac) for 8 hours using β-Actin as a loading control. Histograms represent the ratio of the intensity of target bands quantified by densitometry factored by the densitometric measurement of loading control band. The data are expressed as percentage of control, where the ratio in the control was defined as 1. Values are the mean ± SD and are representative of three independent experiments (c) Immunoprecipitation of p22phox from whole-cell lysates of untreated MV4-11 cells (Unt), cells treated with PKC412 (250nM) and lactacystin (5 μM; Lac) for 8 hours. Nitrocellulose membranes were probed for the presence of ubiquitin. Values are the mean ± SD and are representative of three independent experiments.
The human leukemic cell lines MV4-11 and MOLM-13, (homozygous and heterozygous for the FLT3-ITD mutation, respectively) as well as HL-60, were maintained in RPMI 1640 medium supplemented with 10% fetal calf serum, 2mM L-glutamine and 1% penicillin/streptomycin (all from Sigma Aldrich, Dublin, Ireland) in a humidified incubator at 37°C with 5% CO2. Inhibition of FLT3-ITD signalling was achieved using PKC412 (250nM; Tocris Biosciences, Bristol, UK) for up to 24 hours. Nox inhibition was via flavoprotein inhibitor diphenyleneiodonium (5μM DPI; Sigma) or VAS2870 (10 μM; Enzo Life Sciences, Exeter, UK) for up to 1 hour. Inhibition of the 20S proteasome was via lactacystin (5μM; Sigma) for up to 8 hours. GSK3-β inhibition was via lithium chloride (10μM; Sigma) or SB 216763 (10μM; Tocris) for up to 8 hours.
(a) Western blot analysis of GSK3-β signalling in untreated MV4-11 and MOLM-13 cells (Unt), vehicle controls (Veh), and after treatment with PKC412 (250nM) for 16 hours or DPI (5μM) for 1 hour with GAPDH as a loading control (b) Western blot analysis of p22phox expression in untreated MV4-11 cells (Unt) and upon treatment with PKC412 (250nM), SB 216763 (10μM; SB), Lithium Chloride (10μM; LiCl) for 8 hours using β-Actin as a loading control. Histograms represent the ratio of the intensity of target bands quantified by densitometry factored by the densitometric measurement of loading control band. The data are expressed as percentage of control, where the ratio in the control was defined as 1. Values are the mean ± SD and are representative of three independent experiments.
Following treatments, ROS levels were determined using the cell-permeable fluorogenic probe 2, 7-dichlorodihydrofluorescin diacetate (DCF, Invitrogen Biosciences, Dun Laoghaire, Ireland). Briefly, DCF (50μM) was added to cells in suspension 30 min, and incubated in the dark, before analysis on a FACScalibur (Becton and Dickinson, Oxford, UK) with excitation and emission spectra set at 488 and 530 nm, and using CellQuest software. H2O2 and O2− production was calculated by the increase in mean fluorescence.
(a) Western blot analysis of signalling pathways in untreated MV4-11 cells (Unt) and 24 hours post knockdown of p22phox protein levels via siRNA (A, B). Cells were transfected with either of two siRNA oligomers for p22phox knockdown or a negative control oligomer (Scr) and compared to untreated cells 24 hours post-transfection. Histograms represent the ratio of the intensity of target bands quantified by densitometry factored by the densitometric measurement of loading control band. The data are expressed as percentage of control, where the ratio in the control was defined as 1. Values are the mean ± SD and are representative of three independent experiments (b) Western blot analysis of Pim-1 protein expression in untreated MV4-11 cells (Unt), vehicle controls (Veh) and following treatment with VAS2870 (10μM) over 4 hours using β-Actin as loading control. Histograms represent the ratio of the intensity of target bands quantified by densitometry factored by the densitometric measurement of loading control band. The data are expressed as percentage of control, where the ratio in the control was defined as 1. Values are the mean ± SD and are representative of three independent experiments.
Primary antibodies used for immonoblotting or immunoprecipitation were Akt (#9272), phospho-Akt (Thr308; #9276S), ERK (#9102), phospho-ERK (Thr202/204; #9275S), GSK3-β (#9315), phospho- GSK3-β (Ser9; #9336S), Pim-1 (#2907S; all from Cell Signalling Technology, Boston, MA, USA), Nox1 (#SC25545), Duox (sc-48858), p22phox (#SC20781; all from Santa Cruz Biotechnology, Santa Cruz, CA, USA), GAPDH (#RGM2-500; Advanced Immunochemicals, Long Beach, CA, USA), β-Actin (#A5441; Sigma), STAT5 (#610191; BD Biosciences, Oxford, UK), phospho-STAT5 (Tyr694/699; #04-886), ubiquitin (#MAB1510), Nox2 (#07-024; all from Millipore, Cork, Ireland), KDEL (#ab12223; Abcam, Cambridge, UK). Nox4 antibody was a kind gift from Dr JD Lambeth (Emory University School of Medicine, Atlanta, GA, USA). Nox5 antibody was a kind gift from Dr WM Nauseef (Department of Medicine, University of Iowa and the Veterans' Administration Medical Center, Iowa City, IA, USA). All secondary antibodies for western blotting were peroxidase conjugated (Dako, Glostrup, Denmark).
p22phox is required for the production of H2O2 in AML cell lines at the Endoplasmic reticulum. Inhibition of FLT3 signalling in these cells leads to degradation of p22phox by the proteasome, which requires active GSK3-β. Removal of p22phox from this system halts STAT5 signalling to Pim-1.
Confocal fluorescence live imaging studies were performed with a Zeiss LSM510 META confocal microscope fitted with a 63×1.4 plan apochromat lens. Excitation of PO1 at 543nm was carried out with Ar laser and emission was collected between 560–615nm. Excitation of MitoPY11 at 514nm was carried out using Ar laser and collected between 505–550nm. Excitation of ER tracker blue at 405nm was carried out using HeNe laser and emission was collected between 420–480nm. Excitation of ER tracker green at 488nm was carried out using Ar laser and emission was collected between 505–530nm. The multi-tracking mode of scanning was applied for acquisition of the images. Image analysis was performed in MetaMorph Offline and Carl Zeiss Zen 2009 Light Edition.
Aprroximately 4–5 hours (for PKC412 and VAS-2870 treatments) or 24 hrs (after siRNA transfection) before imaging MV-411 cells were plated on poly-D-lysine (#P4707; Sigma) coated glass bottomed dishes (#P35G-1.5-14-C; MatTek Corporation, Ashland,). 1hr before imaging, ER tracker dye (1µM; Molecular probes), Peroxy Orange (PO1; 15µM), Mito PY1 (8µM) were added. Where indicated, cells were treated with PKC412 (200nM) or VAS-2870 (10µM) for 1hr before imaging. After treatment, cells were washed twice with PBS buffer and incubated in fresh medium during imaging.
Briefly, for siRNA experiments MV4-11 cells were cultured for 24 hours before the experiment in Poly-D-lysine coated glass bottomed dishes and imaged as previously described
Cells were lysed with RIPA buffer [Tris–HCl (50mM; pH 7.4), 1% NP-40, 0.25% sodium deoxycholate, NaCl (150mM), EGTA (1mM), sodium orthovanadate (1mM), sodium fluoride (1mM), cocktail protease inhibitors (Roche, Welwyn, Hertforshire, UK) and 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride (200mM)] for 20 min on ice followed by centrifugation at 14 000 g for 15 min to remove cell debris. Equivalent amounts of protein, as determined by the Bio-Rad Protein Assay (Bio-Rad, Hemel Hempstead, UK) were resolved using SDS–polyacrylamide gel electrophoresis followed by transfer to nitrocellulose membrane (Schleicher and Schuell, Dassel, Germany) and incubated overnight with the appropriate antibodies. Membrane development was achieved using enhanced chemiluminescence (GE Healthcare, Buckinghamshire, UK).
Briefly, approximately 1×107 cells were lysed, as described above, and reconstituted at a concentrated of 1mg/mL with RIPA buffer. Total protein was incubated with anti-p22phox antibody (1/100 dilution) for 2 hours with rocking at 4°C. 20μL of Protein A/G PLUS –Agarose (Santa Cruz Biotechnology) was then added and incubated overnight at 4°C. This solution was then spun down at maximum speed in a microcentrifuge for 1 minute and the supernatant was removed. The beads were washed 5 times with PBS prior to resuspension in loading dye with Dithiothreitol (DTT; Sigma) and boiling at 90°C for 5 mins. This solution was then spun down at maximum speed in a microcentrifuge for 1 minute and the total supernatant resolved using SDS–polyacrylamide gel electrophoresis, as described above.
RNA interference mediated by duplexes of 21-nucleotide RNA was performed in MV4-11 cells. Different Ambion Silencer Select predesigned siRNA (Applied Biosystems, Warrington, UK) were used for silencing. For p22phox, the siRNA used were siRNA ID s3786 and s194371 (Ambion). For the negative control, the siRNA used were Silencer Select Negative Control #1 siRNA (Ambion). The sequences are available from the manufacturer website. The transfection of siRNA used the Amaxa Nucleofactor technology with the Amaxa cell optimization kit L (Amaxa, Cologne, Germany) and followed the Amaxa guidelines using program Q-001.
Quantitative PCR was performed on oligo-dT generated cDNA using the MJ Research Opticon 2 detection system in combination with the Quantitect SYBR Green PCR Master Mix (Qiagen, Crawley, UK). The primers for p22phox and β-Actin were purchased as Quantitect Primer Assays (Qiagen). The following PCR parameters were used for each primer set: denaturing at 95°C for 15 min, followed by 45 cycles of 94°C for 15 seconds, annealing temperature of 56°C for 30 seconds and extension at 72°C for 30 seconds. RNA sample was analyzed in triplicate, and p22phox expression relative to β-2-microglobulin was obtained by the equation &2circ;(Ct-Control)/&2circ;(Ct-Target). Data were represented as the mean relative expression ± standard deviation (SD). Statistical significance was evaluated by Student's t-test for comparisons between groups. PCR products were then visualised by separation on a 2% agarose gel and staining with SYBR Safe gel stain (Invitrogen).
Data are given as mean ± SD. Statistical significance was evaluated by Student's t-test for comparisons between groups, and analysis of variance (ANOVA).
MV4-11 and MOLM-13 are established IL-3 independent myelomonocytic leukemia cell lines homozygous and heterozygous for FLT3-ITD, respectively. Both of these cell lines have been used as models of FLT3-ITD AML in numerous studies to date and display a higher endogenous ROS level compared to cells with non-mutated receptors
Recently FLT3 was shown to affect signalling outcomes in the endoplasmic reticulum (ER)
Given that both DPI and PKC412 treatment result in a large drop in ROS, we investigated whether Nox proteins changed in expression upon FLT3 inhibition, to determine if this reduction was mediated through a decrease in Nox protein expression (
Given that p22phox protein levels are downregulated relatively rapidly post-treatment with PKC412 (
Mutated FLT3 signalling is associated with constitutively activated signalling pathways
It is known that one of the major pathways activated downstream of FLT3 in AML cells is STAT5, whose constitutive activation is associated with protection from apoptosis in mutant FLT3 cells
A number of studies have examined the role of ROS, and specifically Nox-derived ROS, in leukemia
Nox proteins have been seen localized to the ER in various cell line types
Nox family members have been associated with numerous signalling pathways relevant to survival, apoptosis, migration and transformation in AML cells
Given that the levels of p22phox drop relatively soon after FLT3 inhibition, the question was raised as to how this was mediated. We have shown that the level of mRNA is unaffected by PKC412 and that proteasomal inhibition prevents protein degradation after ubiquitination. To our knowledge, treatment with PKC412 has not been reported to induce the proteasomal degradation of any proteins in AML cells and that this occurs to p22phox is a significant finding. Confirming that this was not a general stress response due to removal of FLT3 signalling we have seen that chaperone proteins Hsp70 and Hsp90, as well as a marker of the unfolded-stress response Grp78, are all unaffected by PKC412 treatment (data not shown). Interestingly, it is known that the von Hippel-Lindau tumor suppressor gene (VHL) is responsible for downregulating p22phox in renal carcinoma cells via ubiquitination and subsequent proteasomal degradation
Inhibition of FLT3 signalling was associated with an increase in activated GSK3-β, through a reduction in its deactivated phosphorylated-form. We have demonstrated that this activation of GSK3-β is essential for the reduction of p22phox levels (
The dramatic reduction in p22phox and ROS upon PKC412 treatment would likely have consequences for downstream signalling relevant to AML. Indeed, targeted knockdown of p22phox resulted in a large reduction in the active, phosphorylated STAT5 and one of its target genes Pim-1 (
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