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In vitro efficacy of Fas ligand gene therapy for the treatment of bladder cancer

Cancer Gene Therapy volume 12, pages 12–18 (2005)Cite this article

Abstract

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.

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Acknowledgements

We thank Rick Peppler for acquiring all the flow cytometry data with the use of the MUSC Flow Cytometry Facility equipment. We also thank Dr Jeffrey Bergelson for kindly providing the RcmB anti-CAR antibody. This study was supported by NIH/NIC PO1 CA97132.

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Authors and Affiliations

  1. Department of Microbiology and Immunology, Medical University of South Carolina, Charleston, South Carolina, USA

    Sunil Sudarshan, David H Holman, Marc L Hyer, Christina Voelkel-Johnson, Jian-Yun Dong & James S Norris

  2. Department of Urology, Medical University of South Carolina, Charleston, South Carolina, USA

    Sunil Sudarshan

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  1. Sunil Sudarshan
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  2. David H Holman
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  3. Marc L Hyer
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Correspondence to James S Norris.

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Sudarshan, S., Holman, D., Hyer, M. et al. In vitro efficacy of Fas ligand gene therapy for the treatment of bladder cancer. Cancer Gene Ther 12, 12–18 (2005). https://doi.org/10.1038/sj.cgt.7700746

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