Effects of CD/5-FC suicide gene therapy system on human malignant glioma cells in vitro.
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 IC(50) about 6500 micromol/L. After transfection, the IC(50) was dramatically reduced to about 10 micromol/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.
Lv
Sheng-Qing
SQ
Department of Neurosurgery, Xiangya Hospital, Central South University, Changsha 410008, China. lvsq0518@yahoo.com.cn
Yang
Hui
H
He
Jia-Quan
JQ
Wang
Bin
B
Yoshimura
Ichiro
I
Liu
Yun-Sheng
YS
D016923:Cell Death; D002455:Cell Division / Q000187:drug effects; D003470:Culture Media; D043525:Cytosine Deaminase; D005437:Flucytosine / Q000378:metabolism*; D005472:Fluorouracil / Q000494:pharmacology*; D015316:Genetic Therapy; D005822:Genetic Vectors; D005910:Glioma / Q000473:pathology / Q000628:therapy; D006801:Humans; D009700:Nucleoside Deaminases / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014407:Tumor Cells, Cultured
D003470:Culture Media; D005437:Flucytosine; D009700:Nucleoside Deaminases; D043525:Cytosine Deaminase; D005472:Fluorouracil