Background
Suicide gene therapy, particularly that utilizing the cytosine deaminase/5-fluorocytosine (CD/5-FC) system, represents a novel and attractive methodology of cancer research. Mechanistically, the CD enzyme can convert the antifungal agent 5-FC into the antimetabolite agent 5-fluorouracil (5-FU), thereby killing tumor cells. The purpose of this study was to investigate the antitumor efficiency of the CD/5-FC system in malignant gliomas using a nude mouse model.
Material and Method
The eukaryotic expression plasmid pCMV-CD was transfected into U251 malignant glioma cells. Resistant clones (labeled U251/CD cells) were subsequently isolated and further confirmed by reverse transcription polymerase chain reaction (RT-PCR), immunofluoroscence, and immunoblot. Then U251/CD cells were incubated with 5-FC at various concentrations to measure viability ratios as determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. 5-FU concentrations in the media were measured by high-performance liquid chromatography (HPLC). Finally, the volumes and weights of tumors from glioma-bearing nude mice after 5-FC intervention were evaluated.
Results
The results revealed that the untreated U251 cells were insensitive to 5-FC whereas the U251/CD cells were highly sensitive. Apoptosis and cell death were observed on the U251/CD cells after 5-FC administration. HPLC analysis showed that 5-FU was detected in the U251/CD cell media. These in vivo animal data showed that the volumes and weights of the implanted tumors were dramatically decreased due to cell apoptosis and tumor necrosis.
Conclusions
The in vivo results encourage a further investigation in a controlled trial on the treatment of malignant gliomas via the CD/5-FC gene therapy system.

Keywords: Reverse Transcriptase Polymerase Chain Reaction, Thiazoles, Tetrazolium Salts, Immunoblotting, Glioma - therapy, Genetic Therapy - methods, Fluorouracil - therapeutic use, Flucytosine - metabolism, Fluorescent Antibody Technique, Escherichia coli, DNA Primers - genetics, Cytosine Deaminase - metabolism, Chromatography, High Pressure Liquid