Anticancer Effects Of Fusion Suicide Gene Fcy:: Fur/5-Fluorocytosine Therapies To Human Ovarian Cancer Cell Line HO-8910 Mediated By Retroviral Vector

ObjectiveThe purpose is to study anticancer effects of fusion suicide gene Fcy::Fur/5-fluorocytosine therapies to human ovarian cancer cell line HO-8910.Methods1. Two pairs of PCR primers for Fcy: :Fur genes were designed according to the multiclone site of retroviral expression vector pLXSN, LZRSpBMN-Z and the sequence of Fcy::Fur , in which EcoR I and BamH I sites were introduced at the two 5' ends of the primers respectively. Fcy::Fur genes were amplified from the pORF5-Fcy::Fur using PCR . After DNA sequencing and analysis , Fcy::Fur genes were further inserted into pLXSN and LZRSpBMN-Z respectively with sense open reading frame. The recombinant expression plasmids, which had been confirmed by enzymes digestion, were transfected into packing cells PT67and Pheonix differently. Supernatant of the cloned packing cells were harvested and tested for the presence of the recombinant retrovivus.2. HO-8910 was infected with two kinds of recombinant retrovivus and selected by neomycin(G418) and puromycin separately ,and the HO-8910 cell lines expressing Fcy::Fur stably were then obtained (named HO-8910-Fcy::Fur ). PCR and RT-PCR were taken to demonstrate if Fcy::Fur gene had been intergrated into the chromosome of resistant HO-8910 and expressed correctly .3. After administration of 5-FC, the changes of these cells were observed through inverted and phase-contrast microscopy .The cytotoxicity efficacy was evaluated by MTT methods, then the growth inhibition rate(GIR) was counted. Immunohistochemical assay (SABC method) was used to detect the expression of Fas and FasL, and the apoptosis was analysed by flow cytometry.Results:1. HO-8910 cells were infected with the recombinant retrovirus acquired from the transfected packing cells, then, after selected by antibiotic we obtained HO-8910-Fcy ::Fur . PCR and RT-PCR demonstrated that Fcy::Fur gene had been intergrated correctly into the chromosome of resistant HO-8910 .2. .With the administration of 5-FC, morphologic variation and cells mortality were observed in HO-8910-Fcy::Fur compared with no obvious changes in control by inverted and phase-contrast microscopy.3. The cytotoxicity efficacy appeared remarkablely by MTT methods in HO-8910-Fcy::Fur when the final concentration of 5-FC >10 u mol/L. At 1000 u mol/L, 75 % of HO-8910-Fcy::Fur presented growth inhibition. Whereas the 5-FC had no evident cytotoxicity to normal HO-8910 at the same final concentration of 5-FC .4. A manifest apoptosis peak appeared prior to the normal Go/G] peak in HO-8910-Fcy::Fur .The percentage of apoptosis ofHO-8910-Fcy::Fur was 33.8% after the administration of 5-FC calculated by the flow cytometry, compared with 2.86% of the control.5.The expression of Fas and FasL showed positive in HO-8910-Fcy::Fur after the 96 hours administration of 5-FC compared with negative in normal HO-8910 by immunohistochemical assay.Conclusion:Fusion suicide gene Fcy::Fur combined with 5-fluorocytosine could significantly kill HO-8910, and the growth inhibition rate(GIR) increased remarkablely compared with the control. The percentage of apoptosis calculated by the flow cytometry was much higher than that of the control, so we could infer that the apoptosis might play a role in killing mechanism. Immunohistochemical assay demonstrated the upregulation of Fas and Fas-L after the administration of 5-FC, which suggested that the killing mechanism of Fcy::Fur/5-fluorocytosine therapies mightbe associated with apoptosis induced by Fas and Fas-L.