Jakob Troppmair

THE INS AND OUTS OF RAF KINASES

Raf kinases are signal-integrating enzymes that have the ability to switch tyrosine kinase signalling to serine/threonine phosphorylation and connect growth factor receptors with transcription factors. The connection involves a cascade of protein kinases that is essential for cellular proliferation and differentiation of species ranging from worms to humans. This cascade also mediates transformation by most oncogenes.

Apoptosis suppression by Raf-1 and MEK1 requires MEK- and phosphatidylinositol 3-kinase-dependent signals.

ABSTRACT Two Ras effector pathways leading to the activation of Raf-1 and phosphatidylinositol 3-kinase (PI3K) have been implicated in the survival signaling by the interleukin 3 (IL-3) receptor. Analysis of apoptosis suppression by Raf-1 demonstrated the requirement for mitochondrial translocation of the kinase in this process. This could be achieved either by overexpression of the antiapoptotic protein Bcl-2 or by targeting Raf-1 to the mitochondria via fusion to the mitochondrial protein Mas p70. Mitochondrially active Raf-1 is unable to activate extracellular signal-related kinase 1 (ERK1) and ERK2 but suppresses cell death by inactivating the proapoptotic Bcl-2 family member BAD. However, genetic and biochemical data also have suggested a role for the Raf-1 effector module MEK-ERK in apoptosis suppression. We thus tested for MEK requirement in cell survival signaling using the interleukin 3 (IL-3)-dependent cell line 32D. MEK is essential for survival and growth in the presence of IL-3. Upon growth factor withdrawal the expression of constitutively active MEK1 mutants significantly delays the onset of apoptosis, whereas the presence of a dominant negative mutant accelerates cell death. Survival signaling by MEK most likely results from the activation of ERKs since expression of a constitutively active form of ERK2 was as effective in protecting NIH 3T3 fibroblasts against doxorubicin-induced cell death as oncogenic MEK. The survival effect of activated MEK in 32D cells is achieved by both MEK- and PI3K-dependent mechanisms and results in the activation of PI3K and in the phosphorylation of AKT. MEK and PI3K dependence is also observed in 32D cells protected from apoptosis by oncogenic Raf-1. Additionally, we also could extend these findings to the IL-3-dependent pro-B-cell line BaF3, suggesting that recruitment of MEK is a common mechanism for survival signaling by activated Raf. Requirement for the PI3K effector AKT in this process is further demonstrated by the inhibitory effect of a dominant negative AKT mutant on Raf-1-induced cell survival. Moreover, a constitutively active form of AKT synergizes with Raf-1 in apoptosis suppression. In summary these data strongly suggest a Raf effector pathway for cell survival that is mediated by MEK and AKT.

TD-GC-MS Analysis of Volatile Metabolites of Human Lung Cancer and Normal Cells In vitro

The aim of this study was to confirm the existence of volatile organic compounds (VOC) specifically released or consumed by the lung cancer cell line A549, which could be used in future screens as biomarkers for the early detection of lung cancer. For comparison, primary human bronchial epithelial cells (HBEpC) and human fibroblasts (hFB) were included. VOCs were detected in the headspace of cell cultures or medium controls following adsorption on solid sorbents, thermodesorption, and analysis by gas chromatography mass spectrometry. Using this approach, we identified VOCs that behaved similarly in normal and transformed cells. Thus, concentrations of 2-pentanone and 2,4-dimethyl-1-heptene were found to increase in the headspace of A549, HBEpC, and hFB cell cultures. In addition, the ethers methyl tert-butyl ether and ethyl tert-butyl ether could be detected at elevated levels in the case of A549 cells and one of the untransformed cell lines. However, especially branched hydrocarbons and alcohols were seen increased more frequently in untransformed than A549 cells. A big variety of predominantly aldehydes and the ester n-butyl acetate were found at decreased concentrations in the headspace of all cell lines tested compared with medium controls. Again, more different aldehydes were found to be decreased in hFB and HBEpC cells compared with A549 cells and 2-butenal was metabolized exclusively by both control cell lines. These data suggest that certain groups of VOCs may be preferentially associated with the transformed phenotype. Cancer Epidemiol Biomarkers Prev; 19(1); 182–95

Release of volatile organic compounds (VOCs) from the lung cancer cell line CALU-1 in vitro

BackgroundThe aim of this work was to confirm the existence of volatile organic compounds (VOCs) specifically released or consumed by lung cancer cells.Methods50 million cells of the human non-small cell lung cancer (NSCLC) cell line CALU-1 were incubated in a sealed fermenter for 4 h or over night (18 hours). Then air samples from the headspace of the culture vessel were collected and preconcentrated by adsorption on solid sorbents with subsequent thermodesorption and analysis by means of gas chromatography mass spectrometry (GC-MS). Identification of altogether 60 compounds in GCMS measurement was done not only by spectral library match, but also by determination of retention times established with calibration mixtures of the respective pure compounds.ResultsThe results showed a significant increase in the concentrations of 2,3,3-trimethylpentane, 2,3,5-trimethylhexane, 2,4-dimethylheptane and 4-methyloctane in the headspace of CALU-1 cell culture as compared to medium controls after 18 h. Decreased concentrations after 18 h of incubation were found for acetaldehyde, 3-methylbutanal, butyl acetate, acetonitrile, acrolein, methacrolein, 2-methylpropanal, 2-butanone, 2-methoxy-2-methylpropane, 2-ethoxy-2-methylpropane, and hexanal.ConclusionOur findings demonstrate that certain volatile compounds can be cancer-cell derived and thus indicative of the presence of a tumor, whereas other compounds are not released but seem to be consumed by CALU-1 cells.

Bag1 is essential for differentiation and survival of hematopoietic and neuronal cells

Bag1 is a cochaperone for the heat-shock protein Hsp70 that interacts with C-Raf, B-Raf, Akt, Bcl-2, steroid hormone receptors and other proteins. Here we use targeted gene disruption in mice to show that Bag1 has an essential role in the survival of differentiating neurons and hematopoietic cells. Cells of the fetal liver and developing nervous system in Bag1−/− mice underwent massive apoptosis. Lack of Bag1 did not disturb the primary function of Akt or Raf, as phosphorylation of the forkhead transcription factor FKHR and activation of extracellular signal–regulated kinase (Erk)-1/2 were not affected. However, the defect was associated with the disturbance of a tripartite complex formed by Akt, B-Raf and Bag1, in addition to the absence of Bad phosphorylation at Ser136. We also observed reduced expression of members of the inhibitor of apoptosis (IAP) family. Our data show that Bag1 is a physiological mediator of extracellular survival signals linked to the cellular mechanisms that prevent apoptosis in hematopoietic and neuronal progenitor cells.

Molecular analysis of volatile metabolites released specifically by staphylococcus aureus and pseudomonas aeruginosa

BackgroundThe routinely used microbiological diagnosis of ventilator associated pneumonia (VAP) is time consuming and often requires invasive methods for collection of human specimens (e.g. bronchoscopy). Therefore, it is of utmost interest to develop a non-invasive method for the early detection of bacterial infection in ventilated patients, preferably allowing the identification of the specific pathogens. The present work is an attempt to identify pathogen-derived volatile biomarkers in breath that can be used for early and non- invasive diagnosis of ventilator associated pneumonia (VAP). For this purpose, in vitro experiments with bacteria most frequently found in VAP patients, i.e. Staphylococcus aureus and Pseudomonas aeruginosa, were performed to investigate the release or consumption of volatile organic compounds (VOCs).ResultsHeadspace samples were collected and preconcentrated on multibed sorption tubes at different time points and subsequently analyzed with gas chromatography mass spectrometry (GC-MS). As many as 32 and 37 volatile metabolites were released by S. aureus and P. aeruginosa, respectively. Distinct differences in the bacteria-specific VOC profiles were found, especially with regard to aldehydes (e.g. acetaldehyde, 3-methylbutanal), which were taken up only by P. aeruginosa but released by S. aureus. Differences in concentration profiles were also found for acids (e.g. isovaleric acid), ketones (e.g. acetoin, 2-nonanone), hydrocarbons (e.g. 2-butene, 1,10-undecadiene), alcohols (e.g. 2-methyl-1-propanol, 2-butanol), esters (e.g. ethyl formate, methyl 2-methylbutyrate), volatile sulfur compounds (VSCs, e.g. dimethylsulfide) and volatile nitrogen compounds (VNCs, e.g. 3-methylpyrrole).Importantly, a significant VOC release was found already 1.5 hours after culture start, corresponding to cell numbers of ~8*106 [CFUs/ml].ConclusionsThe results obtained provide strong evidence that the detection and perhaps even identification of bacteria could be achieved by determination of characteristic volatile metabolites, supporting the clinical use of breath-gas analysis as non-invasive method for early detection of bacterial lung infections.

Regulation of c-myc expression by Ras/Raf signalling

The c-myc gene is induced upon growth factor stimulation of arrested cells. The interaction of a mitogen with a transmembrane receptor triggers a variety of parallel signal transduction cascades. In order to analyse the role of the Ras/Raf cascade in the regulation of c-myc expression we have established fibroblast cell lines harboring conditional systems activating or inhibiting this pathway. Fusion of the c-Raf-1 kinase domain with the hormone binding domain of the estrogen receptor (c-Raf-1-BxB-ERTM) provides a 4-hydroxytamoxifen regulated form of the oncogenic c-Raf-1 kinase. We have generated NIH3T3 cells stably expressing the chimeric Raf protein (N-BxB-ERTM). 4-hydroxytamoxifen mediated activation of the fusion protein in serum starved N-BxB-ERTM induces the expression of the c-myc gene within 2 – 6 h. Deletion of the c-Raf-1 kinase domain generates a mutant c-Raf-1 protein (c-Raf-1-C4B), which can directly interact with the effector domain of the Ras protein and thereby block Ras mediated signalling. We have established a NIH3T3 based cell line expressing the c-Raf-1-C4B protein under the control of a tetracycline responsive promoter (N-C4B-tet). Serum starved cells expressing the c-Raf-1-C4B protein exhibit a significantly reduced induction of c-myc expression following serum stimulation compared to the same cells not expressing the Ras inhibitor. The induction of c-myc mRNA following the activation of the isolated Raf/Mek/Erk cascade in addition to the partial inhibition of serum mediated induction of c-myc expression in the presence of the Ras inactivating c-Raf-1-C4B mutant strongly indicates an involvement of the Ras/Raf pathway in the regulation of c-myc expression.

Mitochondrial ROS production under cellular stress : comparison of different detection methods

AbstractReactive oxygen species (ROS) are involved in the regulation of many physiological processes. However, overproduction of ROS under various cellular stresses results in cell death and organ injury and thus contributes to a broad spectrum of diseases and pathological conditions. The existence of different cellular sources for ROS and the distinct properties of individual ROS (their reactivity, lifetime, etc.) require adequate detection methods. We therefore compared different models of cellular stress and various ROS-sensitive dyes—2′,7′-dichlorodihydrofluorescein diacetate (DCF-DA), MitoSOX™, and MitoTracker® red CM-H2XRos—using a confocal fluorescent imaging approach, which has the advantage of not only detecting but also of localizing intracellular sources for ROS. Confocal acquisition of DCF-DA fluorescence can be combined with ROS detection by the mitochondria-specific probes MitoSOX™ and MitoTracker® red CM-H2XRos. Specificity was controlled using various antioxidants such as Trolox and N-acetylcysteine. Using different fluorescent ROS-sensitive probes, we detected higher ROS production equally under cell starvation (IL-3 or serum depletion), hypoxia–reoxygenation, or treatment of cells with prooxidants. The detected increase in ROS was approximately threefold in IL-3-depleted 32D cells, approximately 3.5-fold in serum-deprived NIH cells, and 2.5-fold to threefold in hypoxic HL-1 cells, and these findings agree well with previously published spectrofluorometric measurements. In some cases, electron spin resonance (ESR) spectroscopy was used for the validation of results from confocal fluorescent imaging. Our data show that confocal fluorescent imaging and ESR data are in good agreement. Under cellular stress, mitochondrial ROS are released into the cytoplasm and may participate in many processes, but they do not escape from the cell. Online abstractMitochondrial ROS production under cellular stress

Specific function of B-Raf in mediating survival of embryonic motoneurons and sensory neurons

Embryonic sensory and motoneurons depend on neurotrophic factors for survival. Here we show that their survival requires B-Raf, which, in this function, cannot be substituted by C-Raf. Sensory and motoneurons from b-raf-deficient mice do not respond to neurotrophic factors for their survival. However, these primary neurons can be rescued by transfection of a b-raf expression plasmid. In contrast, c-raf-deficient neurons survive in response to neurotrophic factors, similarly to neurons from wild-type mice. This points to an essential and specific function of B-Raf in mediating survival of sensory and motoneurons during development.

Signaling Through RAS-RAF-MEK-ERK: from Basics to Bedside

Aberrant signaling caused by mutations in the RAS-RAF-MEK-ERK pathway and its upstream activators critically contributes to human tumor development. Strategies, which aim at inhibiting hyperactive signaling molecules, appear conceptually straight forward, but their translation into clinical practice has been hampered by many setbacks. Understanding structure, function and regulation of this intracellular pathway as well as its crosstalk with other signaling activities in the cell will be essential to ensure reasonable usage of new therapeutic possibilities. This review provides an understanding of this signaling cascade as revealed by genetic and biochemical approaches and discusses the existing or arising possibilities to interfere with unphysiological activation in cancer. Signaling aberrations and signal transduction therapies will be discussed exemplary for two types of hematological neoplasia, acute myeloid leukemia (AML) and the myelodysplastic syndromes (MDS). In the future understanding the role of tumor stem cells, both as a source of tumor recurrence and tumor heterogeneity, the signals controlling their fate as well as epigenetic changes in cancer will be the next critical steps to further advance the applicability of these novel therapeutic strategies.