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.