Nucl Med Mol Imaging.  2010 Sep;44(3):193-198.

Prodrug-activating Gene Therapy with Rabbit Cytochrome P450 4B1/4-Ipomeanol or 2-Aminoanthracene System in Glioma Cells

• Jang SJ
• Kang JH
• Lee TS
• Kim SJ
• Kim KI
• Lee YJ
• Cheon GJ
• Choi CW
• Lim SM

Abstract

OBJECTIVE
We determined the cytotoxic properties of cytochrome P450 4B1 (CYP4B1) activated 4-ipomeanol (4-ipo) and 2-aminoanthracene (2-AA) in rat glioma to verify the CYP4B1/4-ipo or 2-AA system for prodrug-activating gene therapy.
METHODS
The cyp4B1 cDNA was cloned into pcDNA3.1/Hygro from rabbit lung total RNA (pcDNA-cyp4B1). Lentiviral vector encoding firefly luciferase (fLuc) was infected into C6 (rat glioma), and the fLuc-expressing cell was selected (C6-L). After transfection with pcDNA-cyp4B1 vector into C6-L, the single clone expressing cyp4B1 gene was selected (C6-CL). Prodrug for various concentrations of 4-ipo or 2-AA was treated for 72 h and 96 h. The cell survival rate of C6-CL was determined using MTT assay and trypan-blue dye exclusion methods.
RESULTS
By RT-PCR analysis, fLuc and CYP4B1 expression was detected in C6-CL, but not in C6. MTT assay and trypan-blue dye exclusion showed that IC50 of C6-CL was 0.3 mM and <0.01 mM after 4-ipo or 2-AA treatment at 96 h or 72 h exposure, respectively. Cell survivals of C6-CL were more rapidly reduced after treatment with 4-ipo or 2-AA than those of C6-L cells. The cell survival rate with MTT and trypan-blue dye exclusion assay was well correlated with fLuc activity in C6-CL cells.
CONCLUSION
CYP4B1-based prodrug-activating gene therapy may have the potential to treat glioma and the cytotoxic effects of CYP4B1 enzyme activated 4-ipo or 2-AA in C6, and could be clearly determined by bioluminescent activity in C6-CL.

Keyword

Cytochrome P450 4B1; Firefly luciferase; Glioma; Prodrug-activating gene therapy

MeSH Terms

Animals
Anthracenes
Aryl Hydrocarbon Hydroxylases
Cell Survival
Clone Cells
Cytochrome P-450 Enzyme System
Cytochromes
DNA, Complementary
Fireflies
Genetic Therapy
Glioma
Inhibitory Concentration 50
Luciferases
Lung
Rats
RNA
Terpenes
Transfection
Anthracenes
Aryl Hydrocarbon Hydroxylases
Cytochrome P-450 Enzyme System
Cytochromes
DNA, Complementary
Luciferases
RNA
Terpenes