In vitro efficacy of Fas ligand gene therapy for the treatment of bladder cancer.
Previous investigations have revealed that bladder cancer cells are generally resistant to Fas-mediated apoptosis by conventional Fas agonists. However, the ability of these cell lines to undergo Fas-mediated apoptosis may have been underappreciated. As a result, we investigated the in vitro efficacy of Fas ligand gene therapy for bladder cancer. Three human bladder cancer lines (T24, J82, and 5637) were treated with the conventional Fas agonist CH-11, a monoclonal antibody to the Fas receptor. Cells were also treated with a replication-deficient adenovirus containing a modified murine Fas ligand gene fused to green fluorescent protein (GFP), AdGFPFasL. A virus containing the GFP gene alone was used to control for viral toxicity (AdGFP). Cell death was quantified using a tetrazolium-based (MTS) assay. Cells were also evaluated by Western blotting to evaluate poly (ADP-ribose) polymerase, caspase 8, and caspase 9 cleavage and by flow cytometry to determine the presence of coxsackie/adenovirus receptor (CAR). These studies confirmed bladder cancer resistance to cell death by the anti-Fas monoclonal antibody CH-11. This resistance was overcome with AdGFPFasL at a multiplicity of infection (MOI) of 1000 achieving over 80% cell death in all cell lines. Furthermore, greater than 80% cell death was evident in 5637 cells treated with low-dose AdGFPFasL (MOI=10). 5637 cells expressed significantly higher levels of surface CAR than J82 or T24 cells (P<.05). AdGFPFasL is cytotoxic to bladder cancer cells that would otherwise be considered Fas resistant, supporting its in vivo potential. Enhanced sensitivity to AdGFPFasL may be in part due to increased cell surface CAR levels.
Sudarshan
Sunil
S
Department of Microbiology and Immunology, Medical University of South Carolina, Charleston, South Carolina, USA.
Holman
David H
DH
Hyer
Marc L
ML
Voelkel-Johnson
Christina
C
Dong
Jian-Yun
JY
Norris
James S
JS
D000256:Adenoviridae; D000906:Antibodies / Q000494:pharmacology; D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D017209:Apoptosis / Q000235:genetics*; D002135:Calcium-Binding Proteins; D005136:Eye Proteins; D053222:Fas Ligand Protein; D015316:Genetic Therapy; D005822:Genetic Vectors; D006801:Humans; D008024:Ligands; D008074:Lipoproteins; D008562:Membrane Glycoproteins; D051603:Recoverin; D014407:Tumor Cells, Cultured; D001749:Urinary Bladder Neoplasms / Q000235:genetics* / Q000628:therapy*; D019014:fas Receptor
D000906:Antibodies; D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; C481004:CH-11 anti-fas antibody, human; D002135:Calcium-Binding Proteins; D005136:Eye Proteins; C505661:FASLG protein, human; D053222:Fas Ligand Protein; D008024:Ligands; D008074:Lipoproteins; D008562:Membrane Glycoproteins; C496731:RCVRN protein, human; D019014:fas Receptor; D051603:Recoverin