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Introduction: We have previously demonstrated decreased cell-surface but abundant cytoplasmic expression of the death receptor Fas and resistance to Fas-mediated apoptosis in esophageal adenocarcinoma (EA). While exploring the hypothesis that this phenotypic Fas expression results in alternative signaling, we observed that abundant Fas Ligand (FasL) expression through adenoviral delivery induced massive apoptosis in cells that are resistant to Fas-mediated apoptosis via extracellular treatments. Methods: Two EA cell lines (Seg-1 and Flo-1) that abundantly express cytoplasmic (but not membranous) Fas and the Fas-null breast cancer cell line MDA-468 were infected with either empty adenovirus (Ad-Ψ5) or human Fas ligand (FasL)-producing adenovirus (Ad-FasL) at a range of MOI (10-100). FasL expression and the phosphorylation status of Erk were determined by immunoblotting. Cell viability and apoptosis were assessed by FACScan analysis of Annexin V- and PI-labeled cells and by immunoblotting for caspase-3 and PARP cleavage following adenoviral infection as described above or treatment with activating anti-Fas antibody and recombinant protein G. All studies were performed in the presence or absence of the pan-caspase inhibitor zVad. Results: Ad-FasL infection resulted in cytoplasmic FasL expression and Erk phosphorylation in both EA cell lines and in MDA-468 cells. Dramatic morphologic changes of the cultured cells suggestive of apoptosis were apparent within 4 hours of infection in the EA cell lines but not in MDA-468 cells. By FACScan analysis, Ad-FasL infection, but not activating anti-Fas antibody treatment, induced cell death in a MOI-dependent fashion resulting in complete loss of the EA cultures by 24 hours. Apoptosis was confirmed by demonstration of caspase-3 and PARP cleavage. Laser-scanning confocal microscopy confirmed FasL expression predominantly localized to the cytoplasm. Interestingly, pre-treatment with zVad diminished Erk phosphorylation and augmented the apoptotic response at early time points (6 hours) in both Seg-1 (50% increase) and Flo-1 (400% increase) cells. Ad-FasL infection did not induce apoptosis in MDA-468 cells. Conclusions: Resistance to Fas-mediated apoptosis is mediated by decreased cell-surface Fas expression in EA cells and can be overcome by cytoplasmic expression of FasL. Erk is activated following intracellular stimulation of Fas, and this appears to be mediated in part by zVad-inhibited caspases.

[Proc Amer Assoc Cancer Res, Volume 47, 2006]