We are interested in how Fas and Fas ligand interact with one another. Three different constructs were studied. Full length Fas was used as a positive control. A chimera composed of extracellular Fas and intracellular= TNFR was made to compare the two signalling pathways. Lastly, it had been previously shown that deleting the last fifteen amino acids of Fas removed a= regulatory domain that rendered the cell more sensitive to Fas mediated death. These constructs were transfected into EL4 cells (murine T lymphoma). This cell line was chosen because it has low murine Fas expression and is resistant to PMA induced (Fas-dependent) killing. Using = a 51)Cr release assay it was shown that B6aBALB stimulated cells could kill E6 (Jurkatt - human T lymphoma) as well as EL4 cells transfected with human Fas. This shows that human Fas and murine ligand interact with one another and that intracellular elements from murine cells= can interact with the intracelhilar domain of hFas. Surprisingly, the chimera gave enhanced killing in this assay but the deletion showed no change in the degree of killing as compared to full length Fas. On the other hand, when antibody mediated killing was observed with these cell lines, full length Fas was killed with antibody alone and enhanced killing= was observed with antibody and cycloheximide. As before the chimera was more sensitive to antibody mediated death than full length Fas but the deletion also showed increased sensitivity compared to full length Fas. This demonstrated the limited usefulness of using antibodies in an attempt to mimic physiological conditions.
|State||Published - Dec 1 1996|