Abstract
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 H(2)O(2)-specific aryl-boronate dye Peroxyorange 1, which also corresponds to co-localization of p22phox. Moreover, knocking down p22phox dramatically reduces H(2)O(2) after 24 hours in the ER, without affecting mitochondrial ROS. Significantly, the FLT3 inhibitor PKC412 reduces H(2)O(2) 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 H(2)O(2) production in AML cells is mediated by p22phox and is critical for STAT5 signalling.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
MeSH terms
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Benzoxazoles / pharmacology
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Cell Line, Tumor
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Endoplasmic Reticulum / drug effects
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Endoplasmic Reticulum / metabolism
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Fluorescent Dyes
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Gene Expression Regulation, Neoplastic / drug effects*
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Gene Knockdown Techniques
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Glycogen Synthase Kinase 3 / antagonists & inhibitors
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Glycogen Synthase Kinase 3 / genetics
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Glycogen Synthase Kinase 3 / metabolism
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Glycogen Synthase Kinase 3 beta
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Humans
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Hydrogen Peroxide / metabolism*
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Imidazoles / pharmacology
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Leukemia, Myeloid, Acute / genetics*
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Leukemia, Myeloid, Acute / metabolism
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Leukemia, Myeloid, Acute / pathology
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Mitochondria / drug effects
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Mitochondria / metabolism
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Mutation
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NADPH Oxidases / antagonists & inhibitors
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NADPH Oxidases / genetics
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NADPH Oxidases / metabolism*
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Proteasome Endopeptidase Complex / drug effects
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Proteasome Endopeptidase Complex / metabolism
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Protein Kinase Inhibitors / pharmacology
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Protein Structure, Tertiary
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Proto-Oncogene Proteins c-pim-1 / antagonists & inhibitors
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Proto-Oncogene Proteins c-pim-1 / genetics
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Proto-Oncogene Proteins c-pim-1 / metabolism
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Pyrroles / pharmacology
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RNA, Small Interfering / genetics
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STAT5 Transcription Factor / antagonists & inhibitors
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STAT5 Transcription Factor / genetics
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STAT5 Transcription Factor / metabolism*
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Signal Transduction / drug effects
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Staurosporine / analogs & derivatives
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Staurosporine / pharmacology
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Triazoles / pharmacology
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fms-Like Tyrosine Kinase 3 / antagonists & inhibitors
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fms-Like Tyrosine Kinase 3 / genetics
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fms-Like Tyrosine Kinase 3 / metabolism*
Substances
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3-benzyl-7-(2-benzoxazolyl)thio-1,2,3-triazolo(4,5-d)pyrimidine
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6,7-dihydro-5H-pyrrolo(1,2-a)imidazole
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Benzoxazoles
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Fluorescent Dyes
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Imidazoles
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Protein Kinase Inhibitors
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Pyrroles
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RNA, Small Interfering
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STAT5 Transcription Factor
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Triazoles
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Hydrogen Peroxide
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NADPH Oxidases
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CYBA protein, human
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FLT3 protein, human
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fms-Like Tyrosine Kinase 3
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Glycogen Synthase Kinase 3 beta
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PIM1 protein, human
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Proto-Oncogene Proteins c-pim-1
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Glycogen Synthase Kinase 3
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Proteasome Endopeptidase Complex
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Staurosporine
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midostaurin