Abstract
Cytosolic DNA sensors are a subgroup of pattern recognition receptors (PRRs) and are activated by the abnormal presence of the DNA in the cytosol. Their activation leads to the upregulation of pro-inflammatory cytokines and chemokines and can also induce cell death. The presence of cytosolic DNA sensors and inflammatory cytokines in TS/A murine mammary adenocarcinoma and WEHI 164 fibrosarcoma cells was demonstrated using real time reverse transcription polymerase chain reaction (RT-PCR), western blotting and enzyme-linked immunosorbent assay (ELISA). After electrotransfer of plasmid DNA (pDNA) using two pulse protocols, the upregulation of DNA-depended activator of interferon regulatory factor or Z-DNA binding protein 1 (DAI/ZBP1), DEAD (Asp-Glu-Ala-Asp) box polypeptide 60 (DDX60) and interferon-inducible protein 204 (p204) mRNAs was observed in both tumor cell lines, but their expression was pulse protocol dependent. A decrease in cell survival was also observed; it was cell type, DNA concentration and pulse protocol dependent. Furthermore, the different protocols of electrotransfer led to different cell death outcomes, necrosis and apoptosis, as indicated by an annexin V and 7AAD assays. The obtained data provide new insights on the presence of cytosolic DNA sensors in tumor cells and the activation of different types of cells death after electrotransfer of pDNA. These observations have important implications on the planning of gene therapy or DNA vaccination protocols.
Highlights
Electroporation (EP) is a delivery method in which cells are exposed to electric pulses with specific intensities and durations in order to increase the permeability of the cell membrane, enabling transition of polar molecules into the cells
Cytosolic DNA sensors are a subgroup of pattern recognition receptors (PRRs) and are activated by the abnormal presence of the DNA in the cytosol
Transfection efficiency, cell survival, and Adenosine triphosphate (ATP) levels were quantified after electrotransfer into TS/A and WEHI 164 cells using three different pulse protocols
Summary
Electroporation (EP) is a delivery method in which cells are exposed to electric pulses with specific intensities and durations in order to increase the permeability of the cell membrane, enabling transition of polar molecules into the cells In biomedicine, this technique is widely used to transform bacteria and transfect eukaryotic cells and tissues [1]. ECT is performed in more than 140 cancer centers in Europe and is included in the national guidelines for treatment in UK, Germany, Slovenia and other EU countries Another application of electroporation that has reached clinical trials is gene electrotransfer, where electroporation is used for transfection of cells within tissues with plasmid DNA (pDNA), generally for the www.oncotarget.com purpose of cancer therapy, cancer vaccines, or infectious disease vaccines. Several clinical trials are ongoing for several types of cancer using pDNA encoding different therapeutic molecules, either immunomodulatory molecules such as interleukin-12 [7] or tumor associated antigens [8,9,10,11]
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