Subcellular localization of the FLT3-ITD oncogene plays a significant role in the production of NOX- and p22phox-derived reactive oxygen species in acute myeloid leukemia

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 p22phox, 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 p22phox protein degradation

NOX-driven ROS formation in cell transformation of FLT3-ITD positive AML

In different types of myeloid leukemia, increased formation of reactive oxygen species (ROS) has been noted and associated with aspects of cell transformation including the promotion of leukemic cell proliferation and migration, as well as DNA-damage and accumulation of mutations. Work reviewed in this article has shown the involvement of NADPH oxidase (NOX)-derived ROS downstream of oncogenic protein-tyrosine kinases in both processes, and the related pathways have been partially identified. FLT3-ITD, an important oncoprotein in a subset of AML, causes activation of AKT and subsequently stabilization of p22phox, a regulatory subunit for NOX1-4. This process is linked to ROS formation and DNA damage. Moreover, FLT3-ITD signaling through STAT5 enhances expression of NOX4, ROS formation and inactivation of the protein-tyrosine phosphatase DEP-1/PTPRJ, a negative regulator of FLT3 signaling, by reversible oxidation of its catalytic cysteine residue. Genetic inactivation of NOX4 restored DEP-1 activity and attenuated cell transformation by FLT3-ITD in vitro and in vivo. Future work is required to further explore these mechanisms and their causal involvement in leukemic cell transformation, which may result in the identification of novel candidate targets for therapy

Pro-survival redox signalling in progesterone-mediated retinal neuroprotection

Retinitis pigmentosa (RP) is a group of hereditary retinal diseases, characterised by photoreceptor cell loss. Despite a substantial understanding of the mechanisms leading to cell death, an effective therapeutic strategy is sought. Our laboratory has previously demonstrated the neuroprotective properties of Norgestrel, a progesterone analogue, in the degenerating retina, mediated in part by the neurotrophic factor basic fibroblast growth factor (bFGF). In other retinal studies, we have also presented a pro-survival role for reactive oxygen species (ROS), downstream of bFGF. Thus, we hypothesized that Norgestrel utilises bFGF-driven ROS production to promote photoreceptor survival. Using the 661W photoreceptor-like cell line, we now show that Norgestrel, working through progesterone receptor membrane complex 1 (PGRMC1); generates an early burst of pro-survival bFGF-induced ROS. Using the rd10 mouse model of RP, we confirm that Norgestrel induces a similar early pro-survival increase in retinal ROS. Norgestrel-driven protection in the rd10 retina was attenuated in the presence of antioxidants. This study therefore presents an essential role for ROS signalling in Norgestrel-mediated neuroprotection in vitro and demonstrates that Norgestrel employs a similar pro-survival mechanism in the degenerating retina

Progesterone analogue protects stressed photoreceptors via bFGF-mediated calcium influx.

Retinitis pigmentosa (RP) is a degenerative retinal disease leading to photoreceptor cell loss. In 2011, our group identified the synthetic progesterone ‘Norgestrel’ as a potential treatment for RP. Subsequent research showed Norgestrel to work through progesterone receptor membrane component 1 (PGRMC1) activation and upregulation of neuroprotective basic fibroblast growth factor (bFGF). Using trophic factor deprivation of 661W photoreceptor-like cells, we aimed to further elucidate the mechanism leading to Norgestrel-induced neuroprotection. In the present manuscript, we show by flow cytometry and live-cell immunofluorescence that Norgestrel induces an increase in cytosolic calcium in both healthy and stressed 661Ws over 24h. Specific PGRMC1 inhibition by AG205 (1 μM) showed this rise to be PGRMC1-dependent, primarily utilising calcium from extracellular sources, for blockade of L-type calcium channels by verapamil (50 μM) prevented a Norgestrel-induced calcium influx in stressed cells. Calcium influx was also shown to be bFGF-dependent, for siRNA knock down of bFGF prevented Norgestrel-PGRMC1 induced changes in cytosolic calcium. Notably, we demonstrate PGRMC1-activation is necessary for Norgestrel-induced bFGF upregulation. We propose that Norgestrel protects through the following pathway: binding to and activating PGRMC1 expressed on the surface of photoreceptor cells, PGRMC1 activation drives bFGF upregulation and subsequent calcium influx. Importantly, raised intracellular calcium is critical to Norgestrel's protective efficacy, for extracellular calcium chelation by EGTA abrogates the protective effects of Norgestrel on stressed 661W cells in vitro

1000 Norms Project: Protocol of a cross-sectional study cataloging human variation

Background Clinical decision-making regarding diagnosis and management largely depends on comparison with healthy or ‘normal’ values. Physiotherapists and researchers therefore need access to robust patient-centred outcome measures and appropriate reference values. However there is a lack of high-quality reference data for many clinical measures. The aim of the 1000 Norms Project is to generate a freely accessible database of musculoskeletal and neurological reference values representative of the healthy population across the lifespan. Methods/design In 2012 the 1000 Norms Project Consortium defined the concept of ‘normal’, established a sampling strategy and selected measures based on clinical significance, psychometric properties and the need for reference data. Musculoskeletal and neurological items tapping the constructs of dexterity, balance, ambulation, joint range of motion, strength and power, endurance and motor planning will be collected in this cross-sectional study. Standardised questionnaires will evaluate quality of life, physical activity, and musculoskeletal health. Saliva DNA will be analysed for the ACTN3 genotype (‘gene for speed’). A volunteer cohort of 1000 participants aged 3 to 100 years will be recruited according to a set of self-reported health criteria. Descriptive statistics will be generated, creating tables of mean values and standard deviations stratified for age and gender. Quantile regression equations will be used to generate age charts and age-specific centile values. Discussion This project will be a powerful resource to assist physiotherapists and clinicians across all areas of healthcare to diagnose pathology, track disease progression and evaluate treatment response. This reference dataset will also contribute to the development of robust patient-centred clinical trial outcome measures

Nuclear membrane-localised NOX4D generates pro-survival ROS in FLT3-ITD-expressing AML

Internal tandem duplication of the juxtamembrane domain of FMS-like tyrosine kinase 3 (FLT3-ITD) is the most prevalent genetic aberration present in 20-30% of acute myeloid leukaemia (AML) cases and is associated with a poor prognosis. FLT3-ITD expressing cells express elevated levels of NADPH oxidase 4 (NOX4)generated pro-survival hydrogen peroxide (H2O2) contributing to increased levels of DNA oxidation and double strand breaks. NOX4 is constitutively active and has been found to have various isoforms expressed at multiple locations within a cell. The purpose of this study was to investigate the expression, localisation and regulation of NOX4 28 kDa splice variant, NOX4D. NOX4D has previously been shown to localise to the nucleus and nucleolus in various cell types and is implicated in the generation of reactive oxygen species (ROS) and DNA damage. Here, we demonstrate that FLT3ITD expressing-AML patient samples as well as -cell lines express the NOX4D isoform resulting in elevated H2O2 levels compared to FLT3-WT expressing cells, as quantified by flow cytometry. Cell fractionation indicated that NOX4D is nuclear membrane-localised in FLT3-ITD expressing cells. Treatment of MV4-11 cells with receptor trafficking inhibitors, tunicamycin and brefeldin A, resulted in deglycosylation of NOX4 and NOX4D. Inhibition of the FLT3 receptor revealed that the FLT3-ITD oncogene is responsible for the production of NOX4D-generated H2O2 in AML. We found that inhibition of the PI3K/AKT and STAT5 pathways resulted in down-regulation of NOX4D-generated pro-survival ROS. Taken together these findings indicate that nuclear membrane-localised NOX4D-generated pro-survival H2O2 may be contributing to genetic instability in FLT3-ITD expressing AML

1000 Norms Project : health-related quality of life across the lifespan

Aims: Patient-reported outcomes (PROs) provide valuable information regarding health-related quality of life (HRQOL) in healthcare, clinical trials and economic evaluation of health services. The aims of this study were to generate HRQOL normative reference data and investigate the relationship between HRQOL, socio-demographic factors and physical function in a sample of healthy children and adults. Methods: This study was conducted within the 1000 Norms Project, an observational study investigating objective and subjective measures of health and function in 1000 healthy Australians aged 3–101 years. Data for adult participants were analysed. Age, weight, height, waist circumference, highest educational level and marital status were collected. Physical activity level, workability and measures of physical function were also collected (Table 1). HRQOL was investigated using the AQoL-8D utility instrument assessing eight dimensions: independent living, pain, physical senses, self-worth, relationships, coping, happiness and mental health. Scores were converted to two super-dimensions (physical and mental) and a global 'utility' total score. Results: To date data from 516 participants aged 18–91 years have been analysed. Adults aged 80+ years reported significantly lower scores for the independent living and senses dimensions and the physical super-dimension (p.05) (Fig. 1). No significant associations were found between AQoL-8D utility scores and gender, marital status, education or physical activity (p>.05). Workability was associated with AQoL-8D utility and both super-dimension scores (rho = .266, .354 and .159, p<.001). Physical super-dimension scores were negatively correlated with BMI, waist circumference and weight (rho = -.290, -.293 and -.155, p<.001), and positively with 6-min walking distance (Fig. 2), timed up and down stairs, chair stand, and BOT balance scores (rho = .404, .410, .332, .342, p<.001). Conclusions: Physical super-dimension scores decreased in later life from 80 years. Mental super-dimension scores remained unchanged yet were surprisingly low across the sample. Low perceived workability, higher body mass, and poorer lower limb function were associated with lower physical super-dimension scores. By September 2015 data from all 1000 participants, including children and adolescents, will be available.1 page(s

Defining health and disease: setting the boundaries for physiotherapy. Are we undertreating or overtreating? How can we tell?

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The synthetic progestin norgestrel modulates Nrf2 signaling and acts as an antioxidant in a model of retinal degeneration

Retinitis pigmentosa (RP) is one of the most common retinal degenerative conditions affecting people worldwide, and is currently incurable. It is characterized by the progressive loss of photoreceptors, in which the death of rod cells leads to the secondary death of cone cells; the cause of eventual blindness. As rod cells die, retinal-oxygen metabolism becomes perturbed, leading to increased levels of reactive oxygen species (ROS) and thus oxidative stress; a key factor in the secondary death of cones. In this study, norgestrel, an FDA-approved synthetic analog of progesterone, was found to be a powerful neuroprotective antioxidant, preventing light-induced ROS in photoreceptor cells, and subsequent cell death. Norgestrel also prevented light-induced photoreceptor morphological changes that were associated with ROS production, and that are characteristic of RP. Further investigation showed that norgestrel acts via post-translational modulation of the major antioxidant transcription factor Nrf2; bringing about its phosphorylation, subsequent nuclear translocation, and increased levels of its effector protein superoxide dismutase 2 (SOD2). In summary, these results demonstrate significant protection of photoreceptor cells from oxidative stress, and underscore the potential of norgestrel as a therapeutic option for RP