Intracellular Fas ligand expression causes Fas-mediated apoptosis in humanprostate cancer cells resistant to monoclonal antibody-induced apoptosis

Several laboratories have attempted with little success to induce Fas-media ted apoptosis in prostate cancer (PCa) cells, using different external Fas agonists, i.e., anti-fas antibodies and membrane-bound FasL. The present st udy confirms these earlier results using the anti-fas antibody CH-11 in fiv e human PCa cell lines (PPC-1, LNCaP, PC-3, TSU-Pr1, and DU145). However, i ntracellular murine FasL expression induced Fas-mediated apoptosis in all C H-11-resistant cell lines. Adenovirus (AdCFPFasL(TET)) was used to deliver a Murine FasL-GFP fusion gene into human PCa cells resulting in 70-98% apop tosis at 48 h as determined by the MTS assay. DU145 and PPC-1 cells treated with AdGFPFasL(TET) stained positive for the TUNEL assay, indicating that cell death was via apoptosis. Using immunofluorescent microscopy, Fas and G FPFasL colocalized to the same intracellular compartment. The anti-fas neut ralizing antibody ZB-4 was unable to block AdGFPFasL(TET)-mediated cell dea th, suggesting that intracellular FasL may ligate Fas within the Golgi and/ or endoplasmic reticulum. This is the first evidence suggesting that these two molecules interact prior to cell surface presentation. Collectively, th ese findings indicate that intracellular GFPFasL expression is superior to CH-11 at inducing Fas-mediated apoptosis in human PCa cells and may allow u se of AdGFPFasL(TET) for PCa gene therapy.