Abstracts
1997 Digestive Disease Week

Molecular chemotherapy combined with radiation therapy enhances killing of cholangiocarcinoma.

LC Pederson, M Stackhouse, SM Vickers, DJ Buchsbaum, D Raben, DT Curiel. Departments of Surgery, Radiation Oncology, Medicine, and Gene Therapy Program. University of Alabama at Birmingham, Birmingham, AL.

Cholangiocarcinoma is a virtually incurable tumor resistant to current surgical, medical and radiotherapeutic interventions. We applied the novel gene therapy strategy of local toxin gene conversion of prodrug to active antineoplastic agent in combination with radiation therapy to the treatment of cholangiocarcinoma. Five-fluorouracil (5-FU) is an accepted radiosensitizing chemotherapeutic agent currently utilized in cancer therapy. The Escherichia coli (E. coli) enzyme cytosine deaminase (CD) converts the non-toxic prodrug 5-fluorocytosine (5-FC) to 5-FU. Methods: The transduction efficiency of SK-ChA-1 human cholangiocarcinoma cell line (gift of Dr. A. Knuth, Ludwig Institute for Cancer Research, London UK) to infection with adenovirus was determined by fluorescent activated cell sorting analysis, following infection. The recombinant adenoviral vector AdCMVlacZ, which contains the E. coli ß-galactosidase reporter gene, was used to infect SK-ChA-1 cells at 100 plaque forming units (pfu)/cell. To evaluate CD mediated conversion of 5-FC to 5-FU and subsequent cytotoxicity, SK-ChA-1 cells were infected with recombinant adenovirus, AdCMVCD (at 100 pfu/cell), which encodes CD. Cells were exposed to 5-FC (with conversion to 5-FU) for 3 days, prior to plating for proliferation assay. Cell proliferation assays (measuring tetrazolium salt (MTS) conversion to formazan by colorimetric assay) were performed daily beginning 6 days after plating. Synergistic cytotoxicity by addition of external beam radiation was evaluated by ⁶⁰Cobalt exposure (8 Gy) following AdCMVCD infection, and 3 day exposure to 5-FC (with conversion to 5-FU). Results: SK-ChA-1 cells were efficiently transduced (99%) by 100 pfu of AdCMVlacZ. Nine to 23% of SK-ChA-1 cells were killed following infection with 10 pfu of AdCMVCD and 3 day exposure to 5-FC (10-20 µg/ml). Higher pfu/cell infection with higher 5-FC concentrations resulted in greater cell killing. Radiation treatment enhanced cell killing (70-81.5%) of infected cells treated with a sub-lethal dose of 5-FC (10-20 µg/ml), and the effect was more pronounced as a function of time. Conclusions: Human cholangiocarcinoma cells were transduced with a recombinant adenovirus in vitro at high efficiency, and were susceptible to AdCMVCD mediated conversion of 5-FC to 5-FU. Enhanced cytotoxicity was seen with addition of external beam radiation. These results provide further foundation for multimodality therapy to human cholangiocarcinoma. In addition, molecular chemotherapy offers unique treatment specificity, with potentially significant disease control.