Antitumor efficiency of the cytosine deaminase/5-fluorocytosine suicide gene therapy system on malignant gliomas: an in vivo study.
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.


METHODS
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.
Lv
Sheng-Qing
SQ
Department of Neurosurgery, Xinqiao Hospital, The Third Military Medical University, Chongqing, P.R. China.
Zhang
Ke-Bin
KB
Zhang
Eric E
EE
Gao
Fang-You
FY
Yin
Chang-Lin
CL
Huang
Chun-Ji
CJ
He
Jia-Quan
JQ
Yang
Hui
H
D000818:Animals; D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid; D043525:Cytosine Deaminase / Q000235:genetics* / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli; D005437:Flucytosine / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D005472:Fluorouracil / Q000378:metabolism / Q000627:therapeutic use*; D015316:Genetic Therapy / Q000379:methods*; D005910:Glioma / Q000188:drug therapy / Q000235:genetics* / Q000628:therapy*; D015151:Immunoblotting; D051379:Mice; D008819:Mice, Nude; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013778:Tetrazolium Salts; D013844:Thiazoles
D017931:DNA Primers; D013778:Tetrazolium Salts; D013844:Thiazoles; D005437:Flucytosine; D043525:Cytosine Deaminase; C022616:thiazolyl blue; D005472:Fluorouracil