Study Of Radioiodine Therapy Mediated By Co-transfection Of HNIS And HTPO Genes Into Glioma Cells Controlled By HTERT Promoter

Background and objectivesGene therapy is currently one of the major focuses in cancer research. Yet gene therapy is faced with the problems of how to improve its efficacy and to enhance targeted gene product expressions. Radioiodine therapy on differentiated thyroid cancers (DTC) exhibits high efficiency and specificity. The molecular basis of radioiodine uptake and storage by DTC cells is the cellular membrane expression of human sodium iodide symporter (hNIS) and human thyroid peroxidase (hTPO) in DTC cells.Therefore, this study aimed to construct and utilize recombinant adenovirus (as a gene transfer vector), then to transfect hNIS and hTPO genes into glioma cells. And we also intended to assess the iodine uptake abilities of the transfected cells, to investigate the radioiodine metabolism dynamics in the cells, and to further evaluate the anti-tumor activities of radioiodine on the transfected cells. We then planned to introduce human telomerase reverse transcriptase (hTERT) in adenovirus vector, afterwards hNIS expression changes were analyzed in the transfected glioma cells.Methods1. Using RT-PCR and real-time fluorescent quantitative PCR (FQ-PCR, or qPCR) to detect the expression of hTERT of a variety of tumor cell lines and normal cells. By using PCR and pMD-18T vector to amplify and clone the hTERT core promoter. To construct recombinant plasmid containing the hTERT promoter and hNIS gene with pGL3-Basic vector, and to detect promoting efficiency of hTERT promoter with luciferase activity test.2. By using Adeasy System to build recombinant adenovirus vector containing the target genes, and the virus titer and the expression of targeted genes in transfected cells were analyzed.3. Using recombinant adenovirus Ad-hTERT-hNIS single transfected and Ad-hTERT-hNIS plus Ad-CMV-hTPO co-transfected U251 and U87 glioma cells to determine the optimal multiplicity of infection (MOI). Metabolism dynamic changes in the glioma cells were tested by 125I uptake,125I influx and efflux experiments. The effectiveness of hNIS and hTPO were tested by NaC104 inhibition experiment and organic measurement experiment. And then clonogenic assay was adopted to determine the anti-tumor activities of 131I on differently transfected cell groups.Results1. RT-PCR results showed that the expressions of hTERT mRNA existed in H460, U251, U87, ARO and FRO cells, yet their levels of activities were different. But in normal cells of MRC-5 no expression of hTERT mRNA was found. FQ-PCR results showed that in U251 and U87 glioma cells the expressions of hTERT were very high, their relative concentrations were 0.18% and 0.12% compared with the internal reference ofβ-actin.2. Successfully constructed and identified plasmids pMD-18T and the pGL3 with 3 different length hTERT promoter sequences (260 bp,456 bp and 1453 bp). And successfully constructed and identified plasmid pGL3-hNIS with 260 bp hTERT promoter and hNIS as well.3. Luciferase activity assay showed that, in the above tumor cells, all three kinds of hTERT promoter fragments could induce the expression of fluorescent proteins, but differences existed in their efficiencies. Promoter fragment (260 bp) of pGL3-204 in FRO, H460 and U251 cells possessed the strongest efficiencies, which could reach 80% of the SV40 promoter.4. Construction and purification of the recombinant adenovirus Ad-CMV-hTPO. Measured by plaque formation assay, its titer was shown as approximately 1.0×109 pfu/ml. After infection of 293 cells, Western blot was performed to demonstrate a positive band at 110 kD, which was the target gene expression of protein hTPO.5. Iodine uptake experiment demonstrated that U251 and U87 glioma cells in single transfection group (Ad-hTERT-hNIS) had enhanced iodine uptake abilities of 30.76 fold and 29.66 fold. And in co-transfection group (Ad-hTERT-hNIS/Ad-CMV-hTPO), the iodine uptake abilities were increased further to 35.55 fold and 32.88 fold, respectively.6.125I influx and efflux experiments showed that in single transfected glioma cells iodine uptake was rapid, the peak was about 40 minutes. But a rapid efflux of iodine was also observed with Te about 11-12 minutes. However, the co-transfection group could delay the efflux of 125I to a Te of about 18-20 minutes.7. Perchlorate inhibition test showed that different concentrations of NaC104 could inhibit transfected glioma cells to uptake 125I, the inhibition rate was about 93.50-94.48%. Organic measurement experiment showed that the co-transfected cells possessed higher 125I binding ability (about 3 times) by intracellular proteins than single transfection group.8. Clonogenic assay demonstrated that after 131I treatment the co-transfection group had the lowest rate of colony formation, with U251 and U87 reduced by 11.37 and 14.52 folds. And the co-transfection group was followed by single transfection group, the reductions were 5.52 and 6.05 folds (P<0.01).Conclusion1. In U251 and U87 glioma cells and other tumor cells different expressions and activities of hTERT existed, so the hTERT promoter could be used to guide therapeutic genes to specific expressions in glioma cells. HTERT core promoter had high promoting efficiency, and could be used as a replace of the full length promoter to guide hNIS gene expression.2. Recombinant plasmid and adenovirus vector containing HTERT promoter, hNIS gene and hTPO gene were constructed successfully. Single transfected glioma cells with Ad-hTERT-hNIS alone could enable the tumor cells to acquire a strong iodine uptake ability, which could mediate the radioiodine treatment on tumor cells.3. In single transfection glioma cells of hNIS gene, iodine efflux happened very quickly. And the co-transfection with Ad-CMV-hTPO could increase protein binding of iodine, prolong entrapment of iodine in the cells, and to improve radioiodine therapy on glioma cells.