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

Combined activity of the redox-modulating compound setanaxib (GKT137831) with cytotoxic agents in the killing of acute myeloid leukemia cells

Acute myeloid leukemia (AML) cells harbor elevated levels of reactive oxygen species (ROS), which promote cell proliferation and cause oxidative stress. Therefore, the inhibition of ROS formation or elevation beyond a toxic level have been considered as therapeutic strategies. ROS elevation has recently been linked to enhanced NADPH oxidase 4 (NOX4) activity. Therefore, the compound Setanaxib (GKT137831), a clinically advanced ROS-modulating substance, which has initially been identified as a NOX1/4 inhibitor, was tested for its inhibitory activity on AML cells. Setanaxib showed antiproliferative activity as single compound, and strongly enhanced the cytotoxic action of anthracyclines such as daunorubicin in vitro. Setanaxib attenuated disease in a mouse model of FLT3-ITD driven myeloproliferation in vivo. Setanaxib did not significantly inhibit FLT3-ITD signaling, including FLT3 autophosphorylation, activation of STAT5, AKT, or extracellular signal regulated kinase 1 and 2 (ERK1/2). Surprisingly, the effects of Setanaxib on cell proliferation appeared to be independent of the presence of NOX4 and were not associated with ROS quenching. Instead, Setanaxib caused elevation of ROS levels in the AML cells and importantly, enhanced anthracycline-induced ROS formation, which may contribute to the combined effects. Further assessment of Setanaxib as potential enhancer of cytotoxic AML therapy appears warranted

The structure of the 5′-end of the protein-tyrosine phosphatase PTPRJ mRNA reveals a novel mechanism for translation attenuation

Analysis of the human protein-tyrosine phosphatase (PTP) PTPRJ mRNA detected three in-frame AUGs at the 5′-end (starting at nt +14, +191 and +356) with no intervening stop codons. This tandem AUG arrangement is conserved between humans and the mouse and is unique among the genes of the classical PTPs. Until now it was assumed that the principal open reading frame (ORF) starts at AUG356. Our experiments showed that: (i) translation of the mRNA synthesized under the PTPRJ promoter starts predominantly at AUG191, leading to the generation of a 55 amino acid sequence preceding the signal peptide; (ii) the longer form is being likewise correctly processed into mature PTPRJ; (iii) the translation of the region between AUG191 and AUG356 inhibits the overall expression, a feature which depends on the sequence of the encoded peptide. Specifically, a sequence of 13 amino acids containing multiple arginine residues (RRTGWRRRRRRRR) confers the inhibition. In the absence of uORF these previously unrecognized characteristics of the 5′-end of the mRNA present a novel mechanism to suppress, and potentially to regulate translation

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

Slow dynamics near glass transitions in thin polymer films

The $\alpha$-process (segmental motion) of thin polystyrene films supported on glass substrate has been investigated in a wider frequency range from 10$^{-3}$ Hz to 10$^4$ Hz using dielectric relaxation spectroscopy and thermal expansion spectroscopy. The relaxation rate of the $\alpha$-process increases with decreasing film thickness at a given temperature above the glass transition. This increase in the relaxation rate with decreasing film thickness is much more enhanced near the glass transition temperature. The glass transition temperature determined as the temperature at which the relaxation time of the $\alpha$-process becomes a macroscopic time scale shows a distinct molecular weight dependence. It is also found that the Vogel temperature has the thickness dependence, i.e., the Vogel temperature decreases with decreasing film thickness. The expansion coefficient of the free volume $\alpha_f$ is extracted from the temperature dependence of the relaxation time within the free volume theory. The fragility index $m$ is also evaluated as a function of thickness. Both $\alpha_f$ and $m$ are found to decrease with decreasing film thickness.Comment: 9 pages, 7 figures, and 2 table

Role of cAMP in the promotion of colorectal cancer cell growth by Prostaglandin E2

<p>Abstract</p> <p>Background</p> <p>Prostaglandin E2 (PGE2), a product of the cyclooxygenase (COX) reaction, stimulates the growth of colonic epithelial cells. It is inferred that the abrogation of prostaglandins' growth-promoting effects as a result of COX inhibition underlies the advantageous effects of non-steroidal anti-inflammatory drugs in colorectal carcinoma (CRC). Despite this appreciation, the underlying molecular mechanisms remain obscure since cell culture studies have yielded discrepant results regarding PGE2's mitogenicity.</p> <p>Methods</p> <p>We have employed several alternative approaches to score cell proliferation and apoptosis of 4 CRC cell lines exposed to PGE2 under various conditions. To investigate the role of cAMP in PGE2's functions, activation of the cAMP pathway was assessed at different levels (changes in cAMP levels and PKA activity) in cells subjected to specific manipulations including the use of specific inhibitors or prostanoid receptor-selective agonists/antagonists.</p> <p>Results</p> <p>Our data document that the dose-response curve to PGE2 is 'bell-shaped', with nano molar concentrations of PGE2 being more mitogenic than micro molar doses. Remarkably, mitogenicity inversely correlates with the ability of PGE2 doses to raise cAMP levels. Consistent with a major role for cAMP, cAMP raising agents and pertussis toxin revert the mitogenic response to PGE2. Accordingly, use of prostanoid receptor-selective agonists argues for the involvement of the EP3 receptor and serum deprivation of HT29 CRC cells specifically raises the levels of Gi-coupled EP3 splice variants.</p> <p>Conclusion</p> <p>The present data indicate that the mitogenic action of low PGE2 doses in CRC cells is mediated via Gi-proteins, most likely through the EP3 receptor subtype, and is superimposed by a second, cAMP-dependent anti-proliferative effect at higher PGE2 doses. We discuss how these findings contribute to rationalize conflictive literature data on the proliferative action of PGE2.</p

Phlebologie an deutschen Hautkliniken: eine Bestandsaufnahme im Auftrag der Deutschen Gesellschaft für Phlebologie

Reich-Schupke S, Alm J, Altmeyer P, et al. Phlebologie an deutschen Hautkliniken: Eine Bestandsaufnahme im Auftrag der Deutschen Gesellschaft für Phlebologie. Hautarzt. 2013;64(9):685-694.Phlebologic diseases have become extremely common and have major socio-economic impact. However, the percentage of dermatologists working in phlebology appears to be decreasing according to the data of the German Society of Phlebology (DGP). To investigate the reasons for this development, we-on behalf of the DGP-sent a questionnaire to 120 German Departments of Dermatology in autumn 2012. In 76 returned questionnaires, the number of physicians with additional fellowship training in phlebology averaged 1.5; the average number of those who fulfill the criteria for training fellows in phlebology was 0.9. In 71.1 % of the departments there was a phlebologist. A special phlebologic outpatient clinic existed in 73.7 % of the departments. Sonography with Doppler (89.5 %) and duplex (86.8 %) was used as the most frequent diagnostic tool. For therapy, compression (94.7 %), sclerotherapy (liquid 78.9 %, foam 63.2 %, catheter 18.4 %), endoluminal thermic procedures (radio wave 28.9 %, laser 17.1 %) and surgery (especially crossectomy and stripping 67.1 %, phlebectomy of tributaries 75 %) were used. The average number of treatments was very heterogenous in the different departments. Phlebology definitely plays an important role in dermatology. Most departments fulfill the formal criteria for the license to conduct advanced training in phlebology. A wide spectrum of phlebological diagnostic and therapeutic procedures is available.Hintergrund Venenleiden sind eine Volkskrankheit und haben eine enorme sozioökonomische Bedeutung. Dennoch scheint der Anteil der phlebologisch tätigen Dermatologen entsprechend den Zahlen der Deutschen Gesellschaft für Phlebologie (DGP) abzunehmen. Methoden Um die Ursachen dieser Entwicklung zu untersuchen, führten wir im Herbst 2012 im Auftrag der DGP eine Befragungsstudie zur Phlebologie an 120 deutschen Hautkliniken durch. Ergebnisse In 76 antwortenden Klinien gab es im Mittel 1,5 Phlebologen und 0,9 Ärzte mit Voraussetzungen zur Weiterbildungsermächtigung Phlebologie. In 71,1 % der Kliniken gab es einen Phlebologen, in 73,7 % eine phlebologische Sprechstunde. Zur Diagnostik wurden besonders die Doppler- (89,5 %) und Duplexsonographie (86,8 %) eingesetzt. In der Therapie führte die Kompressionstherapie (94,7 %), gefolgt von Sklerosierungstherapien (flüssig 78,9 %, Schaum 63,2 %, Katheter 18,4 %), den endoluminalen thermischen Verfahren (Radiowelle 28,9 %, Laser 17,1 %) und den operativen Eingriffen (vor allem Cross- und Saphenektomie 67,1 %, Seitenastexhairese 75 %). Die durchschnittlichen Behandlungszahlen waren sehr heterogen. Schlussfolgerung Die Phlebologie spielt in der Dermatologie weiterhin eine wichtige Rolle. Mehrheitlich sind in den Kliniken die Möglichkeiten zur Weiterbildung Phlebologie gegeben. Es wird ein breites Spektrum an phlebologischen Techniken zur Diagnostik und Therapie angeboten

The translation attenuating arginine-rich sequence in the extended signal peptide of the protein-tyrosine phosphatase PTPRJ/DEP1 is conserved in mammals.

The signal peptides, present at the N-terminus of many proteins, guide the proteins into cell membranes. In some proteins, the signal peptide is with an extended N-terminal region. Previously, it was demonstrated that the N-terminally extended signal peptide of the human PTPRJ contains a cluster of arginine residues, which attenuates translation. The analysis of the mammalian orthologous sequences revealed that this sequence is highly conserved. The PTPRJ transcripts in placentals, marsupials, and monotremes encode a stretch of 10-14 arginine residues, positioned 11-12 codons downstream of the initiating AUG. The remarkable conservation of the repeated arginine residues in the PTPRJ signal peptides points to their key role. Further, the presence of an arginine cluster in the extended signal peptides of other proteins (E3 ubiquitin-protein ligase, NOTCH3) is noted and indicates a more general importance of this cis-acting mechanism of translational suppression