FasL induces Fas/Apo1-mediated apoptosis in human embryonic kidney 293 cells routinely used to generate E1-deleted adenoviral vectors.
Human embryonic kidney 293 cells contain the E1 region of adenovirus type 5, and thus sustain, through transcomplementation, the production of recombinant E1-deleted adenovirus vectors. During attempts to produce recombinant adenovirus expressing the apoptosis-inducing molecule Fas ligand (FasL) under the control of a very strong truncated major immediate-early human cytomegalovirus (MIEhCMV) promoter, we discovered that 293 cells were not surviving the initial cotransfection with a shuttle plasmid encoding the mouse FasL; and pJM17, a plasmid containing the genome of adenovirus type 5 with deletions in the E1-E3 regions, in an unpackagable form. Investigation of the reason for massive cell death after cotransfection led us to determine that 293 cells express the FasL receptor. Fas-Apo1 (CD95), and respond with apoptosis to the cross-linking of Fas-Apo1 with either IgM monoclonal antibodies or FasL. Therefore, we decided to generate adenoviral vectors expressing FasL, under the control of tissue-specific and/or-inducible promoter elements. Our findings can explain difficulties several groups have had in generating recombinant adenoviral vectors expressing FasL using 293 cells, as well as the lower titres reported.
Larregina
A T
AT
Department of Medicine, University of Manchester, UK.
Morelli
A E
AE
Dewey
R A
RA
Castro
M G
MG
Fontana
A
A
Lowenstein
P R
PR
D000256:Adenoviridae; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D002461:Cell Line, Transformed; D053222:Fas Ligand Protein; D005434:Flow Cytometry; D005822:Genetic Vectors; D006801:Humans; D007150:Immunohistochemistry; D007668:Kidney / Q000196:embryology* / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000378:metabolism / Q000494:pharmacology*; D015202:Protein Engineering; D014162:Transfection; D019014:fas Receptor / Q000378:metabolism*
C505661:FASLG protein, human; D053222:Fas Ligand Protein; C505658:Fasl protein, mouse; D008562:Membrane Glycoproteins; D019014:fas Receptor