Radioiodide Uptake And Therapy Of Tumor Cells Mediated By Adenovirus Containing The Human Sodium Iodide Symporter Gene Under The Control Of The Human Telomerase Transcriptase Promoter

Objective: To construct a recombinant adenovirus containing the human sodium iodide symporter (hNIS) under the control of the human telomerase reverse transcriptase (hTERT) core promoter to express specially on tumor cells, and to investigate the feasibility of radioiodine therapy mediated by hNIS gene controlled with hTERT promoter.Methods: (1) The hTERT core promoter was amplified from genomic DNA of Hepatoma cells (HepG2) by PCR, subcloned into plasmid vector pcDNA3.1 (+), and then verified by restriction enzyme and DNA sequence analysis. (2)The hTERT promoter was subcloned into plasmid vector FL*-hNIS/pcDNA3 containing the full-length hNIS cDNA, and then the hTERT-hNIS sequence was subcloned into the shuttle plasmid pAdTrack. The Recombinant adenovirus Ad-hTERT-hNIS was constructed by AdEasy system. In addition, a positive control of adenovirus Ad-CMV-hNIS containing the CMV promoter followed with hNIS gene and a negative control of adenovirus Ad-CMV not containing hNIS gene were created by Adeasy system. (3) The cell-specific transcriptional activity of hTERT was examined by RT-PCR in transiently transfected A549 cell lines. Western blot, Iodide uptake assays were used to confirm hNIS expression and function. Toxic effects of 131I on tumor cells were studied by in vitro clonogenic assay.Results: (1) The hTERT core promoter was cloned successfully, and confirmed by restriction enzyme and DNA sequence analysis. (2)Recombinant Ad-hTERT-hNIS was correctly constructed and confirmed by restriction enzyme analysis and PCR. (3)RT-PCR showed that hNIS cDNA could be amplified from transfected cells. Western blot analysis revealed a band at approximately 55~72 kDa reacting with hNIS-specific antibodies. (4) A 23-fold and 31-fold increase in iodide uptake was observed in Ad-hTERT-hNIS and Ad-CMV-hNIS infected A549 cells respectively. No significant iodide uptake increase was detected in cells infected with the negative control virus. The iodide uptake in transfected cells was inhibited by sodium perchlorate. (5) In vitro clonogenic assay revealed approximately 70% of Ad-hTERT-hNIS and 80% of Ad-CMV-hNIS transfected A549 cells were killed by exposure to Iodide-131 respectively, while only about 10% of NIS-negative control cells were killed.Conclusion: The hTERT promoter was successfully cloned, and the recombinant adenovirus Ad-hTERT-hNIS was constructed. This study demonstrates that hNIS is a potential therapy gene which can allow radioiodine therapy on transfected extra-thyroid tumors. The radioiodine therapy following tumor cells transfected with recombinant adenovirus Ad-hTERT-hNIS can afford a novel gene targeting therapy strategy.