Abstracts
1998 Digestive Disease Week

#1042

EFFECTIVE TOXIN GENE EXPRESSION VIA ADENOVIRAL INFECTION OF HUMAN CHOLANGIOCARCINOMA CELLS IN THE PRESENCE OF HUMAN BILE. L.C. Pederson, D.J. Buchsbaum, M.A. Stackhouse, D.T. Curiel and S.M. Vickers, Departments of Surgery, Radiation Oncology, and Gene Therapy, University of Alabama at Birmingham. Birmingham AL.

Perihilar cholangiocarcinoma is a virtually incurable tumor resistant to current clinical interventions. We have demonstrated cytotoxicity to human cholangiocarcinoma in vitro and in vivo via adenoviral delivery of E. coli cytosine deaminase gene (AdCMVCD) with 5-fluorocytosine (5-FC) and radiation. The biliary milieu may present obstacles to clinical gene therapy applications due to ineffective gene transfer in situ. Therefore, the purpose of this study was to determine whether toxin gene (CD or herpes simplex virus thymidine kinase (HSV-tk)), transfer to cholangiocarcinoma cells occurs in the presence of human bile.

Methods: To determine the transduction efficiency of carcinoma cells in the presence of human bile, the recombinant adenoviral vector AdCMVLacZ, encoding the E. coli LacZ reporter gene was used. Human cholangiocarcinoma cell lines (SK-ChA-1 and Oz) and HeLa, cervical carcinoma cells were infected at 100 plaque forming units (pfu)/cell in serum free media and in media with 10% human bile (filtered sterilized from a patient after Whipple resection). Cells were analyzed for FDG staining by fluorescence activated cell sorting analysis. Susceptibility of cholangiocarcinoma cells to adenoviral delivered CD or HSV-tk, in the presence of bile was also analyzed. Cells were infected with AdCMVCD, or AdCMVHSV-tk, either in serum free media or 10% bile, and treated with 5-FC or ganciclovir (GCV). Cell proliferation assays were performed. Results: SK-ChA-1 and Oz cells were efficiently transduced by AdCMVLacZ in the presence of human bile (98 and 78%), compared to serum free medium (98 and 85%), respectively. Cholangiocarcinoma cytotoxicity was induced with the addition of 5-FC and GCV following adenoviral infection, both in the control medium and bile. For SK-ChA-1 cells infected with AdCMVCD and exposed to 5-FC, 84% were killed in bile compared to 62% in control medium. For Oz cells infected with AdCMVHSV-tk and exposed to GCV, 40% were killed in bile compared to 64% in control medium. Conclusions: These data demonstrate effective adenoviral mediated toxin gene function within cholangiocarcinoma cells in a biliary physiologic context. Verification of therapeutic gene transfer to the biliary epithelium in the presence of bile is critical to the clinical application of these gene therapy strategies.

Copyright 1996 - 1998, SSAT, Inc. Revised 29 June 1998.