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58P Medical Research Society for AW-464 + L I P vs 22.0f0.7ngIml for IL-18 alone, p<O.OI, n=3). AW-464 (30pM) had no effect on A549 cell viability as assessed by the M l T assay. NF-KB-DNA binding activity of A549 cell lysates was assessed using Trans-AMT" assay kits. Preincubation with 30pM AW-464 reduced NF-KB activity in response to 0.3ngIml L I P (OD4,dng protein 42325.0 for AW-464 + IL-Ip vs 121.5f10.3 for IL-Ip alone, p<O.OOl, n=3). Discussion. Our data show that AW-464, an inhibitor of intracellular Trx activity, reduces inflammatory activation of A549 cells in response to certain inflammatory mediators. These effects do not reflect general inhibition of protein synthesis (as AW-464 had no effect on ICAM-I induced by IFNy) or a reduction in cell viability. Instead this antiinflammatory action may be mediated through inhibition of NF-KB activity induced by inflammatory mediators. Trx has been shown to upregulate NF-KB DNA binding by reducing a cysteine residue on the p50 subunit of NF-KB, and AW-464 may interfere with this process. Our data suggest that Trx is a key intracellular mediator in the inflammatory activation of A549 cells, and may represent a novel therapeutic target in lung diseases characterised by a dysregulated immune response. M158 ACCELERATION OF HUMAN NEUTROPHIL APOPTOSIS BY TUMOR NECROSIS FACTOR-RELATED APOPTOSISINDUCING LIGAND (TRAIL). Stephen A. Renshaw*, Jasvir S Parmart, Vanessa Singleton*, Sarah J. Rowe*, Steven K. Dower*, David 11. Dockrell*, Colin D. Bingle*, Edwin R Chilverst and Moira K.B. Whyte*. *Respiratory Medicine Unit, Section of Functional Genomics, Division of Genomic Medicine, University of Sheffeld, Royal Hallamhire Hospital, Glossop Road, Shefield, S10 U F , United Kingdom t Respiratory Medicine Division, Department of Medicine, University of Cambridge School of Medicine, Addenbrooke's and Papworth Hospitals, Hills Road, Cambridge CB2 2QQ, United Kingdom. Neutrophil granulocytes have a short lifespan, with their survival limited by a constitutive programme of apoptosis. Acceleration of neutrophil apoptosis following ligation of the Fas death receptor is well documented and TNFa also has a transient pro-apoptotic effect. TRAIL is a related ligand, which transduces its signal through binding to two cell surface death receptors, TRAIL-RI and -R2, and two non-signalling "decoy receptors", TRAIL-R3 and -R4. We have studied the role of TRAIL in human neutrophils. We identified the presence of mRNAs for TRAIL, TRAIL-R2 and TRAIL-R3, and cell surface expression of TRAIL-R2 and -R3 in neutrophil populations. Neutrophil apoptosis is specifically accelerated by exposure to a leucine-zipper tagged form of TRAIL, which mimics cell surface TRAIL (e.g. at 6h 15.6f2.98 control vs. 32.7f5.38 LZ-TRAIL treated, n=6, p=0.003). Using blocking antibodies to TRAIL receptors and TRAIL-RI:Fc receptor fusion proteins, we have excluded a role for TRAIL in regulating constitutive neutrophil apoptosis. The effects of TRAIL upon human neutrophils differed from both FasL and TNFa. Unlike TNFa, TRAIL does not activate NF-KB and unlike FasL does not induce a chemotactic response in human neutrophils. The susceptibility of neutrophils to TRAILmediated apoptosis suggests a role for TRAIL in the regulation of inflammation and may provide a mechanism for clearance of neutrophils from sites of inflammation. M159 The avP5 integrin induces anoikis in Squamous Cell carcinoma (SCC) cells by activating the intrinsic and extrinsic death pathways and inhibiting an AkVPKB survival signal. S M Janes and F M Watt. Cancer Research UK, 44 Lincoln's IM Fields, London, WC2A 3PX, UK. Introduction. Focal or extensive loss of a v p 5 is a feature of the most poorly differentiated SCCs, while an increase in avp6 is associated with invasiveness and metastatic spread (Watt Dev Suppl 1993:185-92). Epithelial cells normally undergo apoptosis on detachment from their extracellular matrix (anoikis), but transformed cells do not. We hypothesised that expression of a v on H357 cells (which completely lack a v ) and SCC4 cells (which have low expression) would restore their ability to undergo anoikis. Methods. a v or a 4 integrin subunits were transfected into H357 and SCC4 cells using the pBabepuro retroviral vector giving high expressing polyclonal populations. Anoikis was assessed by FACS analysis for sub-GI cells and by TUNEL staining. Signalling and apoptotic pathways were investigated with small molecule inhibitors, a constituitively active Akt, and a dominant negative FADD construct. Anoikis activation was examined using a chimeric molecule with the cytoplasmic domain of p5 attached to the extracellular domain of 86. Results. The QV subunit formed a functional heterodimer with 85 as measured by FACS and adhesion to vitronectin. Anoikis was dramatically increased in the a v infected cells (both H357s and SCC4s) compared to the parental populations, empty vector controls and a 4 infected cells at 48 and 72 hours (p< 0.01). Anoikis was not increased in avp6 expressing cells. The pathways involved in avo5 induced anoikis include a suppression of activation of the survival factor AkVPKB, possibly via the cytoplasmic domain of 8 5 and both the intrinsic (mitochondrial) and extrinsic (death receptor mediated) cell death pathways. Conclusion. Re-introduction of the a v integrin subunit increased SCCs ability to undergo anoikis via the a v p 5 heterodimer. Both the intrinsic and extrinsic apoptotic pathways are required, and a cell survival mechanism of activating AkVPKB is suppressed. M160 Transforming Growth Factor-p Activation is Diminished in Fibrosis-Resistant Neutrophil Elastase-Deficient Mice Felix Chua', Sarah E. Dunsmore', Peter Clingen', Anthony W. Segal', Jurgen Roes' & Geoffrey J. Laurent' 'Centre For Respiratory Research, *Department of Oncology and 3Department of Medicine, University College London, LONDON WClE 655. Rationale. Enhanced activation of transforming growth factor-beta (TGF-o), a key fibrogenic mediator, is widely implicated in the development of major fibrotic disorders. We have previously shown that neutrophil elastase-deficient (NE.'.) mice are protected from bleomycin-induced pulmonary fibrosis. Crucially, levels of active TGF-8 are significantly diminished in bronchoalveolar lavage fluid from these animals. In the present studies, we sought to assess whether defective pulmonary TGF-P activation might account for the fibrosis resistance phenotype of NB'. animals. Approach. Wild type (WT) and NE.'. mice were injected intratracheally with 0.05U bleomycin or saline and lung tissue collected seven days later. Matrix-bound TGF-B was extracted,