TITLE: Simultaneous over-expression of graft protective hHO-1, hCD73 and hCD39: effects against cell cytotoxicity and cell death induced by the inflammatory cytokine TNF- in an in vitro model AUTHORS: Alessandro Cinti1, Marco De Giorgi2, Claudia Arena1, Elisa Chisci1, Gloria Galimberti1, Cristina Bugarin3, Maria Grazia Cerrito1, Giuseppe Gaipa3, Ryszard Tom Smolenski1,2, Roberto Giovannoni1, and Marialuisa Lavitrano1 AFFILIATIONS: 1Department of Surgery and Interdisciplinary Medicine, University of Milano-Bicocca, Monza, Italy; 2Department of Biochemistry, Medical University of Gdansk, Gdansk, Poland. 3Pediatric Department, Laboratory of Cell Therapy Stefano Verri, University of Milano-Bicocca, San Gerardo Hospital, Monza, Italy AIM: In order to obtain a donor animal that is compatible for xenotransplantation several modifications of the genome are required. We sought to test the effects of simultaneous expression of a combination of three selected human genes, namely Heme Oxygenase 1 (hHO1), Ecto 5’ Nucleotidase (hCD73) and Ecto Nucleoside Triphosphate Diphosphohydrolase 1 (hCD39), involved in the down-regulation of the inflammatory response, in an in vitro model of TNF--induced injury. METHODS AND RESULTS: A triple cistronic vector, named pCX TRI-2A, harbouring hHO-1, hCD73 and hCD39 coding sequences, has been produced by cloning in frame the sequences of the three genes by using the F2A-based technology. This vector has been stably transfected in mouse NIH3T3 cell line. The simultaneous expression of the three genes has been demonstrated at RNA level by RT-PCR and at protein level by immunofluorescence, fluorescence activated cell sorting (FACS) and western blotting. Transgenic (TG) and wild type (WT) NIH3T3 were tested in a TNF- mediated cytotoxicity assay and caspase activation assay. Both test showed a significant reduction in cytotoxicity (-26% at 24h, -44% at 48h) and caspase activation (-27% at 16h) in TG cells in comparison with WT cells. Moreover, there is a significant reduction in number of dead cells in TG compared to WT cells, as assayed by trypan blue exclusion counting (-10% at 24h, -46% at 48h) and by FACS detection of Propidium Iodide (PI) staining (-35% PI positive at 24h, -37% PI positive at 48h). Incubation of TG and WT NIH3T3 with enzymatic substrates of HO-1 and CD39/CD73, hemin and ATP respectively, promotes resistance of murine cells to inflammatory stimuli (TNF-). This effect is more evident and further increased in TG cells over-expressing the three genes. In fact, 24 hours of TNF- treatment induced a cytotoxicity of about 27% and 20% in WT and TG cells respectively, while the addition of hemin and ATP decreased the cytotoxicity up to 16% in WT cells and markedly more in TG cell reaching only 5% of cytotoxicity. The differences in the activation of TNF-a signaling pathway were analysed at molecular level by using an RT2 Profiler TM PCR array system in which the expression of 84 TNF-related pathway genes were evaluated by real-time PCR. The activity of the hCD39/hCD73 genes in TNF--treated TG cells maintained up-regulated the anti-inflammatory Tnfaip3 gene and the activity of hHO1 in TNF--treated TG cells up-regulated the anti-apoptotic Traf3 gene as compared to WT cells. CONCLUSIONS: The simultaneous over-expression of hHO-1, hCD73 and hCD39 in murine cells is protective against human TNF-alpha mediated cytotoxicity and apoptosis. The protective effect given by the simultaneous expression of hHO1, hCD73 and hCD39 genes against TNF-alpha toxicity is characterized by the up-regulation of specific genes inhibiting the NfkB pathway.

Simultaneous over-expression of graft protective hHO-1, hCD73 and hCD39: effects against cell cytotoxicity and cell death induced by the inflammatory cytokine TNF-α in an in vitro model

Giovannoni R;
2013

Abstract

TITLE: Simultaneous over-expression of graft protective hHO-1, hCD73 and hCD39: effects against cell cytotoxicity and cell death induced by the inflammatory cytokine TNF- in an in vitro model AUTHORS: Alessandro Cinti1, Marco De Giorgi2, Claudia Arena1, Elisa Chisci1, Gloria Galimberti1, Cristina Bugarin3, Maria Grazia Cerrito1, Giuseppe Gaipa3, Ryszard Tom Smolenski1,2, Roberto Giovannoni1, and Marialuisa Lavitrano1 AFFILIATIONS: 1Department of Surgery and Interdisciplinary Medicine, University of Milano-Bicocca, Monza, Italy; 2Department of Biochemistry, Medical University of Gdansk, Gdansk, Poland. 3Pediatric Department, Laboratory of Cell Therapy Stefano Verri, University of Milano-Bicocca, San Gerardo Hospital, Monza, Italy AIM: In order to obtain a donor animal that is compatible for xenotransplantation several modifications of the genome are required. We sought to test the effects of simultaneous expression of a combination of three selected human genes, namely Heme Oxygenase 1 (hHO1), Ecto 5’ Nucleotidase (hCD73) and Ecto Nucleoside Triphosphate Diphosphohydrolase 1 (hCD39), involved in the down-regulation of the inflammatory response, in an in vitro model of TNF--induced injury. METHODS AND RESULTS: A triple cistronic vector, named pCX TRI-2A, harbouring hHO-1, hCD73 and hCD39 coding sequences, has been produced by cloning in frame the sequences of the three genes by using the F2A-based technology. This vector has been stably transfected in mouse NIH3T3 cell line. The simultaneous expression of the three genes has been demonstrated at RNA level by RT-PCR and at protein level by immunofluorescence, fluorescence activated cell sorting (FACS) and western blotting. Transgenic (TG) and wild type (WT) NIH3T3 were tested in a TNF- mediated cytotoxicity assay and caspase activation assay. Both test showed a significant reduction in cytotoxicity (-26% at 24h, -44% at 48h) and caspase activation (-27% at 16h) in TG cells in comparison with WT cells. Moreover, there is a significant reduction in number of dead cells in TG compared to WT cells, as assayed by trypan blue exclusion counting (-10% at 24h, -46% at 48h) and by FACS detection of Propidium Iodide (PI) staining (-35% PI positive at 24h, -37% PI positive at 48h). Incubation of TG and WT NIH3T3 with enzymatic substrates of HO-1 and CD39/CD73, hemin and ATP respectively, promotes resistance of murine cells to inflammatory stimuli (TNF-). This effect is more evident and further increased in TG cells over-expressing the three genes. In fact, 24 hours of TNF- treatment induced a cytotoxicity of about 27% and 20% in WT and TG cells respectively, while the addition of hemin and ATP decreased the cytotoxicity up to 16% in WT cells and markedly more in TG cell reaching only 5% of cytotoxicity. The differences in the activation of TNF-a signaling pathway were analysed at molecular level by using an RT2 Profiler TM PCR array system in which the expression of 84 TNF-related pathway genes were evaluated by real-time PCR. The activity of the hCD39/hCD73 genes in TNF--treated TG cells maintained up-regulated the anti-inflammatory Tnfaip3 gene and the activity of hHO1 in TNF--treated TG cells up-regulated the anti-apoptotic Traf3 gene as compared to WT cells. CONCLUSIONS: The simultaneous over-expression of hHO-1, hCD73 and hCD39 in murine cells is protective against human TNF-alpha mediated cytotoxicity and apoptosis. The protective effect given by the simultaneous expression of hHO1, hCD73 and hCD39 genes against TNF-alpha toxicity is characterized by the up-regulation of specific genes inhibiting the NfkB pathway.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11568/945492
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