Radioiodide Uptake And Retention Mediated By Adenovirus Transfer Of The Sodium Iodide Symporter And Thyroperoxidase Gene Into Hepatocellular Carcinoma Cell

Objective:¹³¹I therapy is a widely accepted treatment for differentiated thyroid cancer. Transfer of the NIS into nonthyroid tumors by coupling radioactive iodide administration has been proposed as a new principle of cancer gene therapy. Although efficient iodide uptake is achieved in the tumors, no obvious therapeutic effect could be observed with ¹³¹I, most probably because the iodide retention time in the target cells is short. We propose to accumulation and organify the iodide by coexpression NIS and TPO in hepatocellular cancer cells to circumvent this problem. Mehtods:1. hNIS and hTPO coding region were amplified from Graves' patient's thyroid tissue by RT-PCR, subcloned into plasmid vector, then constructed recombinant plasmid pGEM-Teasy-NIS and PBK-CMV-TPO , and verify them by restriction enzyme analysis and DNA sequencing.2. hNIS and hTPO coding region were subcloned into shuttle plasmid pAdTrack-CMV which contained a green fluorescent protein (GFP) gene respectively, and further homologous recombined in the bacteria BJ5183 that already contained AdEasy-1 plasmid . Positive recombinant adenovirus vectors were selected, packaged, and amplified in the 293 cells. After purification, viral titers were calculated. Using Western-Blotting to test the protein expressing of AdNIS and AdTPO.3. HepG2 cells were transfected with AdNIS or cotransfected with AdNIS and AdTPO. Iodide uptake and efflux were evaluated, cell killing with 131I and colongenic assay were also assessed between two groups.4. Intratumoral injection AdNIS in tumor-bearing nude mice, expression of the NIS protein in the tumor was confirmed by immunohistological analysis. Organs' radioactivity were counted after injected ' 5I, then calculated the percentage of injected dose per gram (%ID/g).' I was administered intravenously through the tail vein for the imaging of tumor-bearing nude mice.Results:1. Recombinant plasmids pGEM-Teasy-NIS and PBK-CMV-TPO were correctly constructed and confirmed by restriction enzyme analysis and DNA Sequencing2. Recombinant AdNIS and AdTPO were correctly constructed and confirmed by restriction enzyme analysis and PCR. The viral titer was 1.5 X 109 efu/ml, 2.0 X 109efu/ml respectively. Special 90kD and HOkD protein were found by SDS-PAGE electrophoresis, and could immunoreact with mouse monoclonal hNIS and hTPO-specific antibody, respectively.3. The cells transfected with AdNIS showed 125I uptake capability, 24 times higher than that of noninfected cells. Iodide uptake could be specifically inhibited by perchlorate anions.Radioiodide efflux was rapid ,with a T1/2 efflux of 16min, co-transfection with AdNIS and AdTPO genes could not increase iodide uptake (p > 0.05) , but could organify partial radioiodide, which rentention in the cell was mildly increase,Ti/2 efflUx ²5min, 40 %of AdNIS-transfected tumor cells were selectively killed by 131I, while greater cytotoxicity was observed in cotransfected tumor cells.4. Immunohistological analysis confirmed the expression of the NIS protein in the tumor and localization in the surface of cells. AdNIS-treated tumor uptake of 125I was shown (10.10 ± 1.81%ID/g) at 2 h after injection, the biological half-life was 2.35h. Imaging study of tumor-bearing nude mice model showed tumor could be clearly observed with 13II at 2h after injection.Conclusion:hNIS and hTPO coding region were successfully cloned and adenovirus AdNIS and AdTPO were constructed by using convenient Adeasier system. The cells transfected with AdNIS showed 125I uptake capability, cotransfection with AdNIS and AdTPO could organify partial radioiodide, but could not significantly improve radioiodide rentenion in the targeted cells. Tumor could uptake radioiodide and clear tumor imaging was performed. However, the retention time was short, which suggests should choose other proper shorter physical half-life radionuclide to obtain efficient treatment.