Effects of CD/5-FC Suicide Gene Therapy System

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

LV
Sheng-Qing^1,2*, YANG Hui², HE Jia-Quan²,
WANG Bin², YOSHIMURA Ichiro³, LIU Yun-Sheng^1*

( 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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