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: Joanna Stanicka, Eileen G Russell, John F Woolley, Thomas G Cotter
• Journal: The Journal of biological chemistry (J Biol Chem) Vol. 290 Issue 15 Pg. 9348-61 (Apr 10 2015) ISSN: 1083-351X [Electronic] United States
• PMID: 25697362 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Chemical References

• Protein Kinase Inhibitors
• Hydrogen Peroxide
• NADPH Oxidase 4
• NADPH Oxidases
• NOX4 protein, human
• CYBA protein, human
• FLT3 protein, human
• fms-Like Tyrosine Kinase 3
• Staurosporine
• midostaurin

Topics

• Acute Disease
• Animals
• Blotting, Western
• Cell Line
• DNA Damage
• Genomic Instability (drug effects, genetics)
• HL-60 Cells
• Humans
• Hydrogen Peroxide (metabolism)
• Leukemia, Myeloid (genetics, metabolism)
• Mice
• Microscopy, Confocal
• Mutation
• NADPH Oxidase 4
• NADPH Oxidases (genetics, metabolism)
• Protein Kinase Inhibitors (pharmacology)
• RNA Interference
• Signal Transduction (drug effects, genetics)
• Staurosporine (analogs & derivatives, pharmacology)
• Tandem Repeat Sequences (genetics)
• fms-Like Tyrosine Kinase 3 (genetics)