Study on killing effects of purine nucleoside phosphorylase/fludarabine (PNP/fludarabine) suicide gene system on human hepatocellular carcinoma cell line BEL7402 in vitro

Background: Expression of suicide gene in tumor cells can convert nontoxic prodrugs into highly toxic metabolites to kill cancer cells; purine nucleoside phosphorylase (PNP)/fludarabine suicide gene system may have the strongest bystander effect of any enzyme/prodrug system described, and is the hot topic in present gene therapy area. Aims: To investigate the killing effects of PNP/fludarabine suicide gene system on human hepatocellular carcinoma cell line BEL7402 in vitro. Methods: The PNP gene was amplified from Escherichia coli K12 genomic DNA by polymerase chain reaction (PCR) and cloned into the eukaryotic expression vector pEGFP-N1. Between PNP gene and EGFP gene, a linker including 8 glycine was used. The BEL7402 cells were transfected with pEGFP-N-PNP mediated by Lipofectamine 2000, and screened by G418 to yield the positive clone, which was termed BEL7402/PNP. The expression of PNP gene was identified by reverse transcriptase (RT)-PCR and Northern blotting. Followed the prodrug fludarabine incubation, the cytotoxic effect, bystander effect and apoptosis induction of PNP/fludarabine suicide gene system on BEL7402 cells were detected by methyl thiazolyl tetrazolium (MTT) colorimetric method and Annexin V-FITC apoptosis detection kit, respectively. Results: RT-PCR and Northern blotting analysis confirmed the expression of PNP gene in BEL7402 and BEL7402/PNP cells. There was notable cytotoxic effect of low-dose fludarabine on BEL7402 cells transfected with PNP gene; When BEL7402/PNP cells were mixed with parental BEL7402 cells at a ratio of 10:90, significant bystander effect was observed after incubated with low-dose fludarabine, the killing rate was 90%; Apoptosis analysis indicated that the PNP/fludarabine suicide gene system had apoptotic effect on BEL7402 cells. Conclusions: The PNP/fludarabine suicide gene system has strong killing and by-stander effects on human hepatocellular carcinoma cell line BEL7402.