Activating RAS mutations are one of the most frequent molecular abnormalities associated with acute myeloid leukemia (AML) and have also been linked to induction of reactive oxygen species (ROS). ROS play an important role in immune defense but also regulate intracellular signaling and are contributing factors in several cancer models especially those associated with oncogenic Ras activity. Here we report that constitutively active Ras expression strongly promotes production of ROS in human CD34+ cells and that this is linked to growth-factor independent survival and hyper-phosphorylation of kinases in these cells. Expression of H-RasG12V in human CD34+ cells was achieved by retroviral infection using a vector co-expressing green fluorescent protein (GFP). Using luminol-based chemiluminescence, we found that mutant Ras induced constitutive production of superoxide anions (O2) in CD34+ cells (6.3±2.1 fold greater than controls; p<0.01). Electron paramagnetic resonance spectroscopy confirmed the presence of O2. Hydrogen peroxide (H2O2) (which forms via dismutation of O2minus;) was also elevated (2.7±0.1μM H2O2 vs <0.5μM in controls, measured using Amplex Red). Superoxide (and H2O2) can be produced via the NOX enzyme family of oxidases. NOX inhibitor treatment dramatically suppressed O2 production induced by mutant Ras (100±0.1% inhibition with diphenyleneiodonium, p<0.001) whereas Rotenone, a mitochondrial O2 inhibitor had no effect, suggesting NOX proteins are the predominant source of ROS in these cells. Consistent with this, Ras expressing cells showed greater accumulation of the NOX2 regulatory proteins Rac, p47phox and p67phox in the plasma membrane. Ras also strongly promoted the survival of human CD34+ cells following incubation for 48 hours in medium without growth factors or serum (Ras 59±3.3% viable vs control 26±3.9%; p<0.001, as determined by Annexin V and 7-AAD staining). Growth factor-independent survival of Ras-expressing (but not control) cells decreased significantly (p<0.001; ANOVA) in a cell density-dependent manner, suggesting the presence of autocrine/paracrine pro-survival factors. In order to determine whether these factors could influence the survival of control cells in a paracrine fashion, control CD34+ progenitors (expressing GFP) were co-cultured with an increasing proportion of cells expressing mutant Ras (co-expressing DsRed), enabling analysis of individual cell populations in mixed culture by flow cytometry. Ras expressing cells significantly promoted survival of co-cultured control cells in a ‘dose’-dependent manner; control cells 33±1.5% viable in 1:1 co-culture vs 21±5.1% when cultured alone (p<0.01, ANOVA). In addition, initial data shows treatment with catalase suppressed control cell survival under co-culture conditions. This suggests that the presence of H2O2 is necessary in mediating this pro-survival effect. Ras-expressing cells also demonstrated constitutive phosphorylation of a wide variety of proteins associated with growth and survival including Akt, PDK-1 and PKC which have previously shown to be a critical effectors of Ras in CD34+cells (Darley et al, Blood, 2007). H2O2 promotes protein phosphorylation via phosphatase inhibition (Rhee, Science, 2007), suggesting that phosphorylation of these molecules could be dependent on the high levels of H2O2 produced by Ras CD34+ cells. In support of this, we found that the phosphorylation of PKC in Ras (but not control) cells increased in a density-dependent manner. Treatment with catalase reduced pPKC levels in mutant Ras expressing cells in a dose-dependent manner, supporting a role for H2O2 in promoting protein phosphorylation. In summary, these data show for the first time that Ras strongly promotes ROS production via NOX family proteins in normal human progenitor cells and that ROS are likely to play a key role in promoting cell survival and phosphorylation of intracellular proteins.

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