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Exposure to exogenous DNA can modify the sensitivity of the Fas apoptotic pathway
Marcelo de Carvalho Bittencourt1, Philippe Saas, Stéphanie Fresnay
1Etablissement Français du Sang de Bourgogne Franche Comté, INSERM E0119 UPRES EA2284, Université de Franche-Comté, F-25020 Besançon Cedex, France.
Background:
Gene-transfer techniques are commonly employed for both in vitro and in vivo studies. However, modifications of the target cell following the introduction of the gene of interest are not often examined. These modifications can alter the immunogenicity and/or the susceptibility of the target cell to apoptosis and may produce unwanted consequences in vivo.
Methods:
Gene transfer into the murine fibroblastic Psi-CRIP packaging cell line was performed using calcium phosphate precipitation, cationic liposome-DNA complexes or a retroviral RNA-mediated method. After gene transfer, Fas expression, cytokine production, and sensitivity to Fas ligand (FasL)-mediated death were assessed.
Results:
Following transfection of a FasL expression vector by calcium phosphate precipitation, an unexpected increase was observed in apoptotic cell death in previously Fas-resistant Psi-CRIP cells. This apoptosis was due to Fas upregulation and an increase of sensitivity to FasL-mediated death. Other plasmids coding non-cytotoxic factors also modulated this apoptotic pathway. The co-stimulatory molecule CD80 was also upregulated. Exposure to naked DNA alone elicited the same response. The effect was not dependent on the methylation status of exogenous DNA, but was found to be dependent on the target cell type and might be avoided by the use of an RNA-mediated retroviral system.
Conclusions:
Plasmid transfection or simple exposure to naked DNA can increase sensitivity to apoptosis. The generation of FasL packaging cell lines is therefore limited by an increase in FasL/Fas-mediated apoptosis. These findings should be considered when using genetically modified transplantable cells in order to prevent elimination by host cytotoxic cells and in particular when cells are engineered using FasL.
Insights
Gene transfer methods like plasmid transfection can unexpectedly increase cell sensitivity to apoptosis by upregulating Fas. This finding is crucial for genetically modified cell therapies to prevent immune rejection and ensure therapeutic success.
Area of Science:
- Molecular Biology
- Immunology
- Cell Biology
Background:
- Gene-transfer techniques are vital for in vitro and in vivo research.
- Cellular modifications post-gene introduction are often overlooked.
- These changes can impact immunogenicity and apoptosis susceptibility.
Purpose of the Study:
- To investigate cellular modifications following gene transfer into murine fibroblastic Psi-CRIP cells.
- To assess the impact of gene transfer methods on Fas expression and apoptosis sensitivity.
Main Methods:
- Gene transfer using calcium phosphate precipitation, cationic liposome-DNA complexes, or retroviral RNA-mediated methods.
- Assessment of Fas expression, cytokine production, and Fas ligand (FasL)-mediated cell death.
- Analysis of co-stimulatory molecule CD80 and the effect of naked DNA exposure.
Main Results:
- Plasmid transfection with a FasL vector unexpectedly increased apoptosis in Fas-resistant cells.
- Apoptosis resulted from Fas upregulation and increased sensitivity to FasL-mediated death.
- Naked DNA exposure and other plasmids also modulated apoptosis; retroviral systems may avoid this.
Conclusions:
- Plasmid transfection and naked DNA exposure can enhance apoptosis sensitivity.
- Increased FasL/Fas-mediated apoptosis limits the generation of FasL packaging cell lines.
- Consideration of these apoptotic effects is essential for transplantable cell therapies to prevent host immune elimination.