Identification of NOX2 regions for normal biosynthesis of cytochrome b558 in phagocytes highlighting essential residues for p22phox binding

X−-CGD mutant analysis provides the first indications of NOX2 regions essential for the synthesis of normal cytochrome b558 in phagocytes; the FAD-binding pocket, the first transmembrane passage and a cytosolic region are located between the membrane and the dehydrogenase domain of NOX2.

Influence of NADPH Oxidase Activity On the Reactive Oxygen Species Level in Human Leukemic Cells

Abstract 4801 Redox metabolism plays an important role in self-renewal and differentiation of hematopoietic and leukemic cells. Reactive oxygen species (ROS) level is highly regulated. This regulation involves antioxydative enzymes and it has been recently described that leukemic stem cells (LSC) overexpress glutathione peroxydase 3 (Herault O et al, J. Exp. Med, 2012). This overexpression is associated with a decrease in ROS level and p38MAPK inactivation. ROS level in leukemic cells could be also regulated by the activity of ROS producers, such as NADPH oxidase, known to catalyze an electron transfer from NADPH to oxygen producing superoxides which could generate other downstream ROS. The expression of this enzymatic complex (NOX family, 6 isoforms) has been established in the plasma cell membrane of normal CD34+ hematopoietic progenitors (Piccoli C et al, Biochem. Biophys. Res. Commun., 2007). The aim of this study was to decipher the expression of NADPH oxydase components in various human acute myeloid leukemia (AML) Different leukemic cell lines were used according FAB classification: KG1a (MO/M1), KG1 (M1), HL60 (M2), Kasumi 1 (M2), NB4 (M3), ML2 (M4), THP1 (M5), U937 (M5), MV4–11 (M5), K562 (M6). The cells were cultured (2.105 cells/mL, 37°C in 95% humidified air and 5% CO2) in RPMI 1640 with 20mmoL/L L-glutamine supplemented with 10% FCS, 100 units/mL penicillin G, and 100mg/mL streptomycin. The expression of NOX1, NOX2, NOX3, NOX4, NOX5, DUOX1, DUOX2, P22phox and P40phox, P47phox, P67phox, NOXO1, NOXA1 was quantified by RT-qPCR (Universal Probe Library, Roche). NOX2 and its regulatory subunits expression was quantified by SDS-PAGE and western-blot experiments. The effects of diphenylene iodonium (DPI), a specific NOX inhibitor, were evaluated by ROS quantification using dichlorodihydrofluorescein diacetate (DCF-DA) staining followed by fluorimetry and flow cytometry analyses. The cells were washed twice in the physiological saline buffer (PBS) without calcium and magnesium, then incubated in PBS complemented with 0.5M MgCl2, 0.9M CaCl2, 20mM glucose (Picciocchi A et al, J. Biol. Chem., 2011) with or without 20μM DPI for 1 hour. The cells were distributed at 106cells per 200μL well in 96 wells plates. DCF-DA (10μM) was added to quantify the ROS level (flow cytometry) and to monitor ROS production kinetic (fluorimetry). NOX family genes expression showed that phagocyte oxidase NOX2 is expressed in all leukemic cell lines. Conversely the NOX2 isoforms were not expressed, or very weakly expressed in leukemic cell lines (NOX3 in KG1a; NOX4 in K562; DUOX1 in KG1a, KG1; DUOX2 in KG1a, KG1, HL60). P22phox, the second cytochrome b558 component was also expressed in all cell lines, this expression being higher than NOX2. The cytochrome b558 components were more expressed in differentiated leukemic cells (granulocytic and monocytic) than in undifferentiated cells (KG1a, KG1). NOX2 regulatory subunits were expressed in all leukemic cell lines, the lower level (especially P40phox, P47phox) being observed in KG1a. Proteins quantification confirmed RNA results. Cytochrome b558 components and regulatory subunits were expressed in all cell lines with a higher level in differentiated leukemias. Interestingly, the regulatory subunits were not observed in KG1a cells. Functional flow cytometry and fluorimetry studies revealed a decrease in ROS production in DPI exposed leukemic cell lines. This effect was higher in monocytic cell lines than in granulocytic and undifferentiated leukemias. In conclusion, NADPH oxidases are present in the AML cell membrane, and NOX contribution to the ROS level is higher in differentiated cells than in immature leukemias. Altogether these results suggest that NADPH oxidase is constitutively active in leukemic cells and influences the ROS level, suggesting a role in the pathophysiology of AML. Disclosures: No relevant conflicts of interest to declare.