Exposure to exogenous DNA can modify the sensitivity of the Fas apoptotic pathway.
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.
de Carvalho Bittencourt
Marcelo
M
Etablissement Français du Sang de Bourgogne Franche Comté, INSERM E0119 UPRES EA2284, Université de Franche-Comté, F-25020 Besançon Cedex, France.
Saas
Philippe
P
Fresnay
Stéphanie
S
Yerly-Motta
Véronique
V
Ferrand
Christophe
C
Perruche
Sylvain
S
Duperrier
Anne
A
Hervé
Patrick
P
Tiberghien
Pierre
P
Chalmers
David E
DE
D016475:3T3 Cells; D000818:Animals; D017209:Apoptosis / Q000235:genetics*; D018122:B7-1 Antigen / Q000235:genetics*; D004247:DNA / Q000235:genetics*; D019175:DNA Methylation; D053222:Fas Ligand Protein; D005354:Fibrosarcoma; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors; D006801:Humans; D008562:Membrane Glycoproteins / Q000235:genetics*; D051379:Mice; D010957:Plasmids; D012367:RNA, Viral; D012190:Retroviridae; D015398:Signal Transduction / Q000235:genetics; D014162:Transfection; D015854:Up-Regulation; D019014:fas Receptor / Q000235:genetics*
D018122:B7-1 Antigen; C505661:FASLG protein, human; D053222:Fas Ligand Protein; C505658:Fasl protein, mouse; D008562:Membrane Glycoproteins; D012367:RNA, Viral; D019014:fas Receptor; D004247:DNA