Abstract:

Abstract:Objective
To investigate the enhancement of hypoxia responsive element (HRE) on  killing effect of retrovirus mediated HSV-tk/gancyclovir (GCV) suicide gene system controlled by human telomerase reverse transcriptase (hTERT) promoter on   tumor  cells under hypoxia condition.Methods The recombinant retroviral vectors including the HRE hypoxia responsive element,the hTERT promoter and suicide gene tk based on retroviral vector pLNCL were constructed．The recombinant retroviral vector was transfected into PA317 cells for package by liposome transfection， 2 weeks after G418 selection，G418-resistant PA317 colonies were obtained and amplified．The supernatant of cell culture was harvested and used to infect NIH3T3 cells to measure the viral titer of recombinant retrovirus.The viral supernatant was used to infect lung cancer cells SPC-A1 and liver cancer cells Bel-7402.The positive clones were obtained by G418 selection.The experiment was divided into control group,SPC-A1/tk group and Bel-7402/tk group.Under the hypoxia (1%O2 ，94%N2) condition,GCV-mediated cell growth inhibition was determined with method of tetrazolium( MTT).FCM combined with PI and AnnexinV-FITC double pigmention methods were used to observe the apoptosis and cell cycle of two tumor  cell lines infected by the recombinant retrovirus.
Results As compared with control group, SPC-A1/tk and Bel-7402/tk showed high sensitivity to GCV in vitro under hypoxia condition,the apoptotic rate was  increased significantly (P<0.001).As compared with group of recombinant retrovirus with tk driven by the hTERT promoter,the survival rate in group of recombinant retrovirus with tk driven by HRE-modified hTERT promoter was decreased significantly (P<0.01),the apoptotic rate was  increased significantly (P<0.01),and both the cell populations at G₁ and G₀ phases were increased significantly (P<0.05),and the cell population at S phases was decreased significantly (P<0.05).Conclusion  HRE can enhance the  killing effect of HSV-tk/GCV suicide gene system controlled by the hTERT promoter on tumor  cells line under hypoxia condition.