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Generation of Recombinant Influenza Virus from Plasmid DNA
Published on: August 3, 2010
Tumor cell death after electrotransfer of plasmid DNA is associated with cytosolic DNA sensor upregulation
Katarina Znidar1, Masa Bosnjak2, Nina Semenova3
1Faculty of Health Sciences, University of Primorska, Izola, Slovenia.
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.
Insights
Tumor cells possess cytosolic DNA sensors that activate inflammatory responses and cell death pathways upon plasmid DNA electrotransfer. The specific cell death outcome (necrosis or apoptosis) depends on the electrotransfer protocol, impacting gene therapy strategies.
Area of Science:
- Immunology
- Molecular Biology
- Cancer Research
Background:
- Cytosolic DNA sensors, a type of pattern recognition receptor (PRR), detect aberrant DNA in the cell's cytosol.
- Sensor activation triggers pro-inflammatory cytokine and chemokine release, potentially leading to cell death.
- Tumor cells, specifically TS/A mammary adenocarcinoma and WEHI 164 fibrosarcoma, express these sensors and inflammatory mediators.
Purpose of the Study:
- To investigate the presence and activation of cytosolic DNA sensors in tumor cells.
- To analyze the effects of plasmid DNA (pDNA) electrotransfer on sensor expression and cell viability.
- To determine the types of cell death induced by different electrotransfer protocols.
Main Methods:
- Real-time reverse transcription polymerase chain reaction (RT-PCR) for mRNA analysis.
- Western blotting and enzyme-linked immunosorbent assay (ELISA) for protein and cytokine detection.
- Annexin V and 7-AAD assays to differentiate apoptosis and necrosis.
Main Results:
- Upregulation of DAI/ZBP1, DDX60, and p204 mRNAs in tumor cells post-pDNA electrotransfer, dependent on pulse protocol.
- Reduced cell survival observed, influenced by cell type, DNA concentration, and electrotransfer parameters.
- Distinct cell death modalities (necrosis and apoptosis) were induced by varying electrotransfer protocols.
Conclusions:
- Tumor cells harbor functional cytosolic DNA sensors that can be activated by delivered pDNA.
- Electrotransfer protocols significantly influence the type of cell death (apoptosis vs. necrosis) induced.
- Findings offer crucial insights for optimizing gene therapy and DNA vaccination protocols involving electrotransfer.
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