Glial Fibrillary Acidic Protein And Human Telomerase Reverse Transcriptase Dual-promoters Direct Sodium Iodide Symporter Expression For Malignant Glioma Radioiodine Therapy

Objective Radioiodine is a routine therapy for differentiated thyroid cancers. The non-thyroid cancers can be treated with radioiodine following transfection with the human sodium iodide symporter (hNIS) gene. The human telomerase reverse transcriptase (hTERT) promoter and the glial fibrillary acidic protein(GFAP) promoter are the effective tumor-specific promoter of gene expression and thus may be useful in targeted gene therapy of Malignant glioma.Methods The hTERT promoter, the adenoviral early region1A (E1A),the GFAP promoter and the NIS gene were amplified by PCR or DNA replication. Detected the transcriptional activity of the hTERT and GFAP gene promoter. The GFAP promoter and hNIS gene segments,the hTERT promoter and E1A gene were ligated, respectively,and package the recombinant adenovirus Ad-Tp-E1A-Gp-NIS. The U87and U251glioma cells was tranfected by Ad-Tp-ElA-Gp-NIS, then the selective replication ability of the conditionally replicative adenovirus was evaluated by plaque forming assay, and proceeded western blots,125I uptake and exflux, clonogenecity following131I treatment.At last, on the basis of the cells-level experimental data, we had established U87tumor xenografts in nude mice and proceeded radioiodine therapy in U87tumors nude mice.Results Genes had cloned into plasmids. The activity of GFAP promoter and hTERT promoter had reached to as much as about60%and43%of SV40promoter,respectively. The recombined adenovirus Ad-Tp-E1A-Gp-NIS was constructed successfully and tranfected the glioma cells. The western blots revealed bands of approximately43,70,120,49,110kDa, consistent with the β-acting, GFAP, hTERT,hNIS and hTPO proteins. The adenovirus selective-eplication experiment was proved the efficiency of Ad-Tp-E1A-Gp-NIS and the hTERT promoter could controled the eplication of Ad-Tp-E1A-Gp-NIS in the cell-lines. After transfection with the hNIS gene, the cells could intake125I-iodide. In clonogenic assay, transfected cells were killed, compared to control cells (transfected with Ad-CMV-EGFP) after incubated with131I. The U87tumor nude mice was evaluated. After treatment with131I and injection of Ad-Tp-E1A-Gp-NIS, the survival period of U87tumor loading nude mice were prolonged and the growth of tumor in mice was inhibited. Rude mice harboring xenografts after injection of Ad-Tp-E1A-Gp-NIS, could taken the99mTc.Conclusions The experiments demonstrated that after transfected replication-selective adeno viruses Ad-Tp-E1A-Gp-NIS, the therapy of131I was achieved effectively in malignant glioma cell lines following induction of tumor-specific iodide uptake activity by GFAP promoters directed hNIS gene expression in vitro and in vivo.