Peroxy Orange 1

Subcellular localization of the FLT3-ITD oncogene plays a significant role in the production of NOX- and p22(phox)-derived reactive oxygen species in acute myeloid leukemia.[Pubmed:27870947]

Leuk Res. 2017 Jan;52:34-42.

Internal tandem duplication of the juxtamembrane domain of FMS-like tyrosine kinase 3 (FLT3-ITD) receptor is the most prevalent FLT3 mutation accounting for 20% of acute myeloid leukemia (AML) patients. FLT3-ITD mutation results in ligand-independent constitutive activation of the receptor at the plasma membrane and 'impaired trafficking' of the receptor in compartments of the endomembrane system, such as the endoplasmic reticulum (ER). FLT3-ITD expressing cells have been shown to generate increased levels of reactive oxygen species (ROS), in particular NADPH oxidase (NOX)-generated ROS which act as pro-survival signals. The purpose of this study is to investigate FLT3-ITD production of ROS at the plasma membrane and ER in the FLT3-ITD expressing AML cell line MV4-11. Receptor trafficking inhibitors; Tunicamycin and Brefeldin A induce ER retention of FLT3-ITD, resulting in a decrease in protein expression of NOX4 and its partner protein p22(phox), thus demonstrating the critical importance of FLT3-ITD localization for the generation of pro-survival ROS. NOX-generated ROS contribute to total endogenous hydrogen peroxide (H2O2) in AML as quantified by flow cytometry using the cell-permeable H2O2-probe Peroxy Orange 1 (PO1). We found that PI3K/AKT signaling only occurs when FLT3-ITD is expressed at the plasma membrane and is required for the production of NOX-generated ROS. ER retention of FLT3-ITD resulted in NOX4 deglycosylation and p22(phox) protein degradation.

An oxidative fluctuation hypothesis of aging generated by imaging H(2)O(2) levels in live Caenorhabditis elegans with altered lifespans.[Pubmed:25701790]

Biochem Biophys Res Commun. 2015 Mar 20;458(4):896-900.

Reactive oxygen species (ROS) are important factors mediating aging according to the free radical theory of aging. Few studies have systematically measured ROS levels in relationship to aging, partly due to the lack of tools for detection of specific ROS in live animals. By using the H(2)O(2)-specific fluorescence probe Peroxy Orange 1, we assayed the H(2)O(2) levels of live Caenorhabditis elegans with 41 aging-related genes being individually knocked down by RNAi. Knockdown of 14 genes extends the lifespan but increases H(2)O(2) level or shortens the lifespan but decreases H(2)O(2) level, contradicting the free radical theory of aging. Strikingly, a significant inverse correlation between lifespan and the normalized standard deviation of H(2)O(2) levels was observed (p < 0.0001). Such inverse correlation was also observed in worms cultured under heat shock conditions. An oxidative fluctuation hypothesis of aging is thus proposed and suggests that the ability of animals to homeostatically maintain the ROS levels within a narrow range is more important for lifespan extension than just minimizing the ROS levels though the latter still being crucial.

A palette of fluorescent probes with varying emission colors for imaging hydrogen peroxide signaling in living cells.[Pubmed:20361787]

J Am Chem Soc. 2010 Apr 28;132(16):5906-15.

We present a new family of fluorescent probes with varying emission colors for selectively imaging hydrogen peroxide (H(2)O(2)) generated at physiological cell signaling levels. This structurally homologous series of fluorescein- and rhodol-based reporters relies on a chemospecific boronate-to-phenol switch to respond to H(2)O(2) over a panel of biologically relevant reactive oxygen species (ROS) with tunable excitation and emission maxima and sensitivity to endogenously produced H(2)O(2) signals, as shown by studies in RAW264.7 macrophages during the phagocytic respiratory burst and A431 cells in response to EGF stimulation. We further demonstrate the utility of these reagents in multicolor imaging experiments by using one of the new H(2)O(2)-specific probes, Peroxy Orange 1 (PO1), in conjunction with the green-fluorescent highly reactive oxygen species (hROS) probe, APF. This dual-probe approach allows for selective discrimination between changes in H(2)O(2) and hypochlorous acid (HOCl) levels in live RAW264.7 macrophages. Moreover, when macrophages labeled with both PO1 and APF were stimulated to induce an immune response, we discovered three distinct types of phagosomes: those that generated mainly hROS, those that produced mainly H(2)O(2), and those that possessed both types of ROS. The ability to monitor multiple ROS fluxes simultaneously using a palette of different colored fluorescent probes opens new opportunities to disentangle the complex contributions of oxidation biology to living systems by molecular imaging.

H2O2 production downstream of FLT3 is mediated by p22phox in the endoplasmic reticulum and is required for STAT5 signalling.[Pubmed:22807997]

PLoS One. 2012;7(7):e34050.

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-beta 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.

Chemistry and biology of reactive oxygen species in signaling or stress responses.[Pubmed:21769097]

Nat Chem Biol. 2011 Jul 18;7(8):504-11.

Reactive oxygen species (ROS) are a family of molecules that are continuously generated, transformed and consumed in all living organisms as a consequence of aerobic life. The traditional view of these reactive oxygen metabolites is one of oxidative stress and damage that leads to decline of tissue and organ systems in aging and disease. However, emerging data show that ROS produced in certain situations can also contribute to physiology and increased fitness. This Perspective provides a focused discussion on what factors lead ROS molecules to become signal and/or stress agents, highlighting how increasing knowledge of the underlying chemistry of ROS can lead to advances in understanding their disparate contributions to biology. An important facet of this emerging area at the chemistry-biology interface is the development of new tools to study these small molecules and their reactivity in complex biological systems.