title: NADPH oxidase-generated hydrogen peroxide induces DNA damage in mutant FLT3-expressing leukemia cells.
abstract: Internal tandem duplication of the FMS-like tyrosine kinase (FLT3-ITD) receptor is present in 20% of acute myeloid leukemia (AML) patients and it has been associated with an aggressive AML phenotype. FLT3-ITD expressing cell lines have been shown to generate increased levels of reactive oxygen species (ROS) and DNA double strand breaks (DSBs). However, the molecular basis of how FLT3-ITD-driven ROS leads to the aggressive form of AML is not clearly understood. Our group has previously reported that inhibition of FLT3-ITD signaling results in post-translational down-regulation of p22(phox), a small membrane-bound subunit of the NADPH oxidase (NOX) complex. Here we demonstrated that 32D cells, a myeloblast-like cell line transfected with FLT3-ITD, have a higher protein level of p22(phox) and p22(phox)-interacting NOX isoforms than 32D cells transfected with the wild type FLT3 receptor (FLT3-WT). The inhibition of NOX proteins, p22(phox), and NOX protein knockdowns caused a reduction in ROS, as measured with a hydrogen peroxide (H2O2)-specific dye, peroxy orange 1 (PO1), and nuclear H2O2, as measured with nuclear peroxy emerald 1 (NucPE1). These reductions in the level of H2O2 following the NOX knockdowns were accompanied by a decrease in the number of DNA DSBs. We showed that 32D cells that express FLT3-ITD have a higher level of both oxidized DNA and DNA DSBs than their wild type counterparts. We also observed that NOX4 and p22(phox) localize to the nuclear membrane in MV4-11 cells expressing FLT3-ITD. Taken together these data indicate that NOX and p22(phox) mediate the ROS production from FLT3-ITD that signal to the nucleus causing genomic instability.
authors.affiliation: From the Tumour Biology Laboratory, School of Biochemistry and Cell Biology, Bioscience Research Institute, University College Cork, Cork, Ireland.
authors.forename: Joanna
authors.initials: J
authors.lastname: Stanicka
authors.affiliation: From the Tumour Biology Laboratory, School of Biochemistry and Cell Biology, Bioscience Research Institute, University College Cork, Cork, Ireland.
authors.forename: Eileen G
authors.initials: EG
authors.lastname: Russell
authors.affiliation: From the Tumour Biology Laboratory, School of Biochemistry and Cell Biology, Bioscience Research Institute, University College Cork, Cork, Ireland.
authors.forename: John F
authors.initials: JF
authors.lastname: Woolley
authors.affiliation: From the Tumour Biology Laboratory, School of Biochemistry and Cell Biology, Bioscience Research Institute, University College Cork, Cork, Ireland t.cotter@ucc.ie.
authors.forename: Thomas G
authors.initials: TG
authors.lastname: Cotter
mesh_terms: D000208:Acute Disease; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D004249:DNA Damage; D042822:Genomic Instability / Q000187:drug effects / Q000235:genetics; D018922:HL-60 Cells; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism*; D007951:Leukemia, Myeloid / Q000235:genetics / Q000378:metabolism; D051379:Mice; D018613:Microscopy, Confocal; D009154:Mutation; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D034622:RNA Interference; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics; D019311:Staurosporine / Q000031:analogs & derivatives / Q000494:pharmacology; D020080:Tandem Repeat Sequences / Q000235:genetics; D051941:fms-Like Tyrosine Kinase 3 / Q000235:genetics*
chemical_list: D047428:Protein Kinase Inhibitors; D006861:Hydrogen Peroxide; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C498910:NOX4 protein, human; C086986:CYBA protein, human; C497970:FLT3 protein, human; D051941:fms-Like Tyrosine Kinase 3; D019311:Staurosporine; C059539:midostaurin
keywords: DNA Damage; Genomic Instability; Leukemia; NADPH Oxidase; Oncogene; Reactive Oxygen Species (ROS); Redox