Effects of CD/5-FC Suicide Gene Therapy System
on Human Malignant Glioma Cells in Vitro

LV Sheng-Qing1,2*, YANG Hui2, HE Jia-Quan2,
WANG Bin2, YOSHIMURA Ichiro3, LIU Yun-Sheng1*

( 1 Department of Neurosurgery, Xiangya Hospital, Central South University, Changsha 410008, China;
2 Department of Neurosurgery, Xinqiao Hospital, Third Military Medical University, Chongqing 400037, China;
3 Department of Urology, National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama 359-8513, Japan )

Abstract The purpose of this paper is to investigate the antitumor effects of cytosine deaminase/5-fluorocytosine (CD/5-FC) suicide gene therapy system on human malignant glioma cells in vitro. The pCMVCD plasmid was constructed through the CD gene insertion in the multicloning site of eukaryotic expression vector pcDNA3.0, and confirmed by restriction endonuclease digestion/gene sequencing. The construct was subsequently transfected into the U251 human malignant glioma cells by using LipofectAMINE2000-mediated method. Resistant clones (named U251/CD cells) were isolated by screening with G418 presence. U251/CD cells were incubated with 5-FC in different concentrations to determine viability ratios (or cytotoxicity assay), measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The concentrations of 5-fluorouracil (5-FU) in the media were measured by high-performance liquid chromatography (HPLC) detector. Our results suggested that the untreated U251 cells were insensitive to 5-FC, with the IC50 about 6500 mmol/L. After transfection, the IC50 was dramatically reduced to about 10 mmol/L. Therefore, gene transfection made G418-resistant clones (U251/CD cells) be highly sensitive to 5-FC. HPLC analysis showed that 5-FU was detected in U251/CD cell medium. Study on U251 cells genetically modified by CD gene in vitro will play an essential role in glioma gene therapy in vivo. In conclusion, our results indicated that the CD/5-FC system was feasible to treat glioma.

Key words cytosine deaminase; 5-fluorocytosine; gene therapy; malignant glioma

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