title: Experimental studies on PNP suicide gene therapy of hepatoma.
abstract: To investigate the killing effect of PNP/MeP-dR suicide gene system on hepatoma cells, pcDNA3.0/PNP, an eukaryotic expression vector harboring E. coli PNP gene, was transfected into human hepatoma HepG2 cells by liposome-mediated method. A HepG2 cell line with stable PNP gene expression, HepG2/PNP, was established with presence of G418 selection. The cell growth curves were determined with trypan blue staining. The sensitivity of HepG2/PNP to MeP-dR and bystander effects were assayed by MTT and FCM methods. The enzymatic activity of the product of PNP gene was determined by HPLC method. The cytotoxic effects of MeP-dR on HepG2/PNP cells were obvious (IC50 = 4.5 micromol/L) and all HepG2/PNP cells were killed 4 days after the treatment with 100 micromol/L MeP-dR. In mixed cultures containing increasing percentages of HepG2/PNP cells, total population killing was demonstrated when HepG2/PNP cells accounted for as few as 5% of all HepG2 cells 8 days after the treatment with 100 micromol MeP-dR. High-pressure liquid chromatography (HPLC) demonstrated that the PNP enzyme could convert MeP-dR into 6-MP. PNP/MeP-dR suicide gene system had an advantage over traditional suicide gene systems for hepatoma gene therapy. Our e results suggest that high-level bystander effects of this system result in significant anti-tumor responses to hepatoma gene therapy, especially in vivo.
authors.affiliation: Institute for Liver Disease of Tongji Hospital, Tongji School of Medicine, Huazhong University of Science and Technology, Wuhan 430030, China.
authors.forename: Xiaokun
authors.initials: X
authors.lastname: Cai
authors.forename: Junli
authors.initials: J
authors.lastname: Zhou
authors.forename: Jusheng
authors.initials: J
authors.lastname: Lin
authors.forename: Xuemei
authors.initials: X
authors.lastname: Sun
authors.forename: Xiulan
authors.initials: X
authors.lastname: Xue
authors.forename: Chao
authors.initials: C
authors.lastname: Li
mesh_terms: D024201:Bystander Effect / Q000502:physiology; D006528:Carcinoma, Hepatocellular / Q000628:therapy*; D045744:Cell Line, Tumor; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D015316:Genetic Therapy / Q000379:methods*; D006801:Humans; D008113:Liver Neoplasms / Q000628:therapy*; D011355:Prodrugs / Q000737:chemistry / Q000627:therapeutic use; D011683:Purine-Nucleoside Phosphorylase / Q000235:genetics* / Q000378:metabolism; D011687:Purines / Q000737:chemistry / Q000627:therapeutic use
chemical_list: D011355:Prodrugs; D011687:Purines; D011683:Purine-Nucleoside Phosphorylase