title: Prodrug-activating Gene Therapy with Rabbit Cytochrome P450 4B1/4-Ipomeanol or 2-Aminoanthracene System in Glioma Cells.
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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Molecular Imaging Research Center, Korea Institute of Radiological and Medical Sciences (KIRAMS), 75 Nowon-gil, Gongneung-Dong, Nowon-Gu, Seoul, 139-706 Republic of Korea.
authors.forename: Su Jin
authors.initials: SJ
authors.lastname: Jang
authors.affiliation: Molecular Imaging Research Center, Korea Institute of Radiological and Medical Sciences (KIRAMS), 75 Nowon-gil, Gongneung-Dong, Nowon-Gu, Seoul, 139-706 Republic of Korea.
authors.forename: Joo Hyun
authors.initials: JH
authors.lastname: Kang
authors.affiliation: Molecular Imaging Research Center, Korea Institute of Radiological and Medical Sciences (KIRAMS), 75 Nowon-gil, Gongneung-Dong, Nowon-Gu, Seoul, 139-706 Republic of Korea.
authors.forename: Tae Sup
authors.initials: TS
authors.lastname: Lee
authors.affiliation: Molecular Imaging Research Center, Korea Institute of Radiological and Medical Sciences (KIRAMS), 75 Nowon-gil, Gongneung-Dong, Nowon-Gu, Seoul, 139-706 Republic of Korea.
authors.forename: Sung Joo
authors.initials: SJ
authors.lastname: Kim
authors.affiliation: Molecular Imaging Research Center, Korea Institute of Radiological and Medical Sciences (KIRAMS), 75 Nowon-gil, Gongneung-Dong, Nowon-Gu, Seoul, 139-706 Republic of Korea.
authors.forename: Kwang Il
authors.initials: KI
authors.lastname: Kim
authors.affiliation: Molecular Imaging Research Center, Korea Institute of Radiological and Medical Sciences (KIRAMS), 75 Nowon-gil, Gongneung-Dong, Nowon-Gu, Seoul, 139-706 Republic of Korea.
authors.forename: Yong Jin
authors.initials: YJ
authors.lastname: Lee
authors.affiliation: Molecular Imaging Research Center, Korea Institute of Radiological and Medical Sciences (KIRAMS), 75 Nowon-gil, Gongneung-Dong, Nowon-Gu, Seoul, 139-706 Republic of Korea ; Department of Nuclear Medicine, Korea Institute of Radiological and Medical Sciences (KIRAMS), 75 Nowon-gil, Gongneung-Dong, Nowon-Gu, Seoul, 139-706 Republic of Korea.
authors.forename: Gi Jeong
authors.initials: GJ
authors.lastname: Cheon
authors.affiliation: Department of Nuclear Medicine, Korea Institute of Radiological and Medical Sciences (KIRAMS), 75 Nowon-gil, Gongneung-Dong, Nowon-Gu, Seoul, 139-706 Republic of Korea.
authors.forename: Chang Woon
authors.initials: CW
authors.lastname: Choi
authors.affiliation: Department of Nuclear Medicine, Korea Institute of Radiological and Medical Sciences (KIRAMS), 75 Nowon-gil, Gongneung-Dong, Nowon-Gu, Seoul, 139-706 Republic of Korea.
authors.forename: Sang Moo
authors.initials: SM
authors.lastname: Lim
keywords: Cytochrome P450 4B1; Firefly luciferase; Glioma; Prodrug-activating gene therapy