Co-expression of functional human Heme Oxygenase 1, Ecto-5′-Nucleotidase and ecto-nucleoside triphosphate diphosphohydrolase-1 by “self-cleaving” 2A peptide system

Giorgi, M. De; Cinti, Alessandro; Pelikant‐Małecka, Iwona; Chisci, Elisa; Lavitrano, Marialuisa; Giovannoni, Roberto; Smolenski, Ryszard T.

doi:10.1016/j.plasmid.2015.03.004

Cited by 9 publications

(12 citation statements)

References 37 publications

(49 reference statements)

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“…Our studies highlighted that expression of CD73 could be protective from several aspects of endothelium damage that occur during xenograft rejection [63]. Development of strategies that involve overexpression of CD73 alone or in combination with other genes could be fundamental for success of xenotransplantation [64,65]. All these examples confirm that the development of therapies involving the CD73 activation could be a new effective treatment strategy in vascular pathologies.…”

Section: Therapeutic Strategy That Involves Activation Of Cd73supporting

confidence: 56%

The role of ecto-5′-nucleotidase in endothelial dysfunction and vascular pathologies

Zukowska

Kutryb–Zajac

Toczek

et al. 2015

Pharmacological Reports

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Ecto-5'-nucleotidase (e5NT, CD73) is an enzyme that is highly expressed in endothelium and is involved in the extracellular nucleotide catabolism. CD73 converts AMP to adenosine that via specific subtypes of P1 receptor mediates cytoprotection involving diverse mechanisms such as vasodilatation, suppression of inflammation, inhibition of thrombosis and anti-adrenergic effect. Physiological intravascular concentration of adenosine is in nanomolar range, but could become micromolar in response to various forms of stress. Endothelium is a major site for both CD73 mediated production of adenosine and its cytoprotective effect. Nucleotides (predominantly ATP or ADP) that could be released from different cells via controlled specific of unspecific mechanisms constitute a major source of substrate for adenosine production via CD73. Direct effects of extracellular nucleotides (mediated by P2 receptors) are typically opposite to adenosine P1 mediated activities. Retention of nucleotides and decreased adenosine production due to loss of CD73 function may have negative implications and could be important cause of various pathologies. Protective role of CD73 was indicated in ectopic calcification, atherosclerosis, rejection after xenotransplantation and thrombosis. Reduced activity of CD73 due to lymphocyte contact with endothelium increases its permeability that leads to enhanced leukocyte transmigration. Upregulation of endothelial CD73 may therefore be protective in a number of cardiovascular pathologies. Such effect has been confirmed for some common drugs such as statins and it could be part of its pleiotropic portfolio. Activation of CD73 could be a new target for specific treatment strategy that in particular will enhance endothelial protection.

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Section: Therapeutic Strategy That Involves Activation Of Cd73supporting

confidence: 56%

The role of ecto-5′-nucleotidase in endothelial dysfunction and vascular pathologies

Zukowska

Kutryb–Zajac

Toczek

et al. 2015

Pharmacological Reports

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“…The 2A peptide links two coding sequences in one ORF, which is transcribed into one mRNA molecule, whereas it is translated into two different, function-independent proteins (24). The 'self-cleavage' characteristic of 2A peptide qualifies it to co-express two separate molecules by same vector efficiently (35,36). The present study was the first to constructed an adenovirus, Ad-XAF-1&TNF-α, which co-expressed XAF-1 and TNF-α efficiently.…”

Section: Construction Of Adenovirus Co-expressing Xaf-1 and Tnf-αmentioning

confidence: 79%

Adenovirus encoding XAF-1 and TNF-α in the same open reading frame efficiently inhibits hepatocellular cancer cells

et al. 2016

Molecular Medicine Reports

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X‑linked inhibitor of apoptosis (XIAP)‑associated factor 1 (XAF‑1), a tumor suppressor, is downregulated in most human malignant tumors. However, the tumor suppressive role of XAF‑1 in hepatocellular carcinoma (HCC) and its therapeutic value require further elucidation. The present study examined the expression of XAF‑1 at the mRNA and protein level in the HCC and paired peritumor tissue specimens, as well as in HCC cell lines and a normal liver cell line. A recombinant adenovirus which co‑expressed XAF‑1 and TNF‑α was then constructed, and its effects on the proliferation and colony formation ability of the MHCC97H HCC cell line were assessed using apoptosis induction, flow cytometry, trypan blue staining assay and a clonogenic assay. The results demonstrated that the expression of XAF‑1 was significantly reduced in HCC tissues compared with that in their matched peritumor specimens, and a significant correlation with the tumor size, stage and tumor ‑ nodes ‑ metastasis stage was identified. The reduced levels of XAF‑1 were further confirmed the HCC cell lines MHCC97L, HepG2 and MHCC97H compared with those in the L02 normal liver cell line. The recombinant adenovirus Ad‑XAF‑1&TNF‑α, which co‑expressed XAF‑1 and TNF‑α, was shown to efficiently express the two proteins at the mRNA and protein level. Furthermore, infection with Ad‑XAF‑1&TNF‑α synergistically induced apoptosis, reduced the proliferation and colony formation ability of MHCC97L cells to a significantly greater extent than overexpression of XAF‑1 or TNF‑α individually. To the best of our knowledge, the present study was the first to construct an adenovirus which co‑expressed XAF‑1 and TNF‑α in the same open reading frame and expressed them proportionally. As Ad‑XAF‑1&TNF‑α inhibited HCC cells with enhanced efficiency, it may be applicable for the treatment of HCC.

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“…The triple cistronic vector was prepared following a strategy similar to those previously reported by Ryan et al . [ 31 ] and by our group [ 32 ]. Briefly, the coding sequence of the plasmid pcDNA3.1-hHO1-F2A1-hE5NT-F2A2-hENPTD1 [ 32 ] was excised and ligated into pCX-C1 plasmid (a pCX-EGFP plasmid [ 33 ] to which a neomycin resistance cassette has been added) to form the final pCX-hHO1-F2A1-hE5NT-F2A2-hENTPD1-C1, which was called pCX-TRI-2A.…”

Section: Methodsmentioning

confidence: 99%

“…[ 31 ] and by our group [ 32 ]. Briefly, the coding sequence of the plasmid pcDNA3.1-hHO1-F2A1-hE5NT-F2A2-hENPTD1 [ 32 ] was excised and ligated into pCX-C1 plasmid (a pCX-EGFP plasmid [ 33 ] to which a neomycin resistance cassette has been added) to form the final pCX-hHO1-F2A1-hE5NT-F2A2-hENTPD1-C1, which was called pCX-TRI-2A. Control plasmids, expressing only one of the three human proteins at once, were prepared by ligation of the PCR-amplified coding sequence into pCX-C1 plasmid, to obtain pCX-hHO1, pCX-hE5NT and pCX-hENTPD1 respectively.…”

Section: Methodsmentioning

confidence: 99%

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Simultaneous Overexpression of Functional Human HO-1, E5NT and ENTPD1 Protects Murine Fibroblasts against TNF-α-Induced Injury In Vitro

et al. 2015

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Several biomedical applications, such as xenotransplantation, require multiple genes simultaneously expressed in eukaryotic cells. Advances in genetic engineering technologies have led to the development of efficient polycistronic vectors based on the use of the 2A self-processing oligopeptide. The aim of this work was to evaluate the protective effects of the simultaneous expression of a novel combination of anti-inflammatory human genes, ENTPD1, E5NT and HO-1, in eukaryotic cells. We produced an F2A system-based multicistronic construct to express three human proteins in NIH3T3 cells exposed to an inflammatory stimulus represented by tumor necrosis factor alpha (TNF-α), a pro-inflammatory cytokine which plays an important role during inflammation, cell proliferation, differentiation and apoptosis and in the inflammatory response during ischemia/reperfusion injury in several organ transplantation settings. The protective effects against TNF-α-induced cytotoxicity and cell death, mediated by HO-1, ENTPD1 and E5NT genes were better observed in cells expressing the combination of genes as compared to cells expressing each single gene and the effect was further improved by administrating enzymatic substrates of the human genes to the cells. Moreover, a gene expression analyses demonstrated that the expression of the three genes has a role in modulating key regulators of TNF-α signalling pathway, namely Nemo and Tnfaip3, that promoted pro-survival phenotype in TNF-α injured cells. These results could provide new insights in the research of protective mechanisms in transplantation settings.

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Co-expression of functional human Heme Oxygenase 1, Ecto-5′-Nucleotidase and ecto-nucleoside triphosphate diphosphohydrolase-1 by “self-cleaving” 2A peptide system

Cited by 9 publications

References 37 publications

The role of ecto-5′-nucleotidase in endothelial dysfunction and vascular pathologies

The role of ecto-5′-nucleotidase in endothelial dysfunction and vascular pathologies

Adenovirus encoding XAF-1 and TNF-α in the same open reading frame efficiently inhibits hepatocellular cancer cells

Simultaneous Overexpression of Functional Human HO-1, E5NT and ENTPD1 Protects Murine Fibroblasts against TNF-α-Induced Injury In Vitro

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