ABSTRACT
Objective To explore the possibility of using 131I as a targeted therapy method for malignant glioma by infecting U87 and U251 cells with conditionally replicative adenovirus Ad-Tp-E1a-Gp-NIS.Methods Human telomerase reverse transcriptase (hTERT) promoter and glial fibrillary acidic protein (GFAP) promoter were cloned and their transcriptional activities were detected by luciferase assay.The conditionally replicative adenovirus Ad-Tp-E1 a-Gp-NIS was constructed,purified,and transfected into U87 and U251 glioma cells.For these transfected cells,the selective replication ability was evaluated by plaque forming assay,and protein expression was detected by Western blot assay.125I-iodide uptake and exflux,the clonoy formation of 131I-iodide treated cells were also measured.Results Transcriptions activity of the GFAP and hTERT promoters was 59.75％-62.10％ (F =11.89,P ＜ 0.01) in U87 cells and 37.31％-49.00％ (F =5.87,P ＜ 0.05) in U251 cells.The Ad-Tp-E1a-Gp-NIS could be selectively replicated and the hNIS gene was successfully expressed in the hTERT-positive and GFAP-positive glioma cells which showed two protein bands with relative molecular mass of 120 × 103 and 49 × 103 in Western blot assay.After infection with Ad-Tp-E1a-Gp-NIS,the cell ability of 125I uptake was increased by 78.80 (F =2 914.58,P ＜0.01) and 92.48 (F =2 275.91,P ＜0.01) times in U87 and U251 cells,respectively.The GFAP-negative MRC-5 cells could not take in 125I.The in vitro clonogenic assay indicated that,after 131I treatment,more than 90％ of the transfected cells were killed,while only about 65％ (t =11.73-78.33,P ＜ 0.01) of control cells were killed.Conclusions The Ad-Tp-E1a-Gp-NIS has a good ability in selective replication and the enhancement of antitumor therapy effect by increasing tumor-specific iodide uptake in malignant glioma cells.
ABSTRACT
Objective To construct the conditionally replicative adenovirus vector pAd-Egr1-TRAIL-hTERT-E1A-E1Bp-E1B55K carrying early growth response gene-1 (Egr1)promoter and tumor necrosis factor related apoptosis inducing ligand (TRAIL)gene, and to observe the effects of the vector combined with 2 Gy irradiation on the TRAIL expression in MDA-MB-231 cells.Methods Egr-1 promotor sequence was cloned from pMD18 T-Egr1, TRAIL was constructed the downstream of Egr1 promoter, pShuttle-Egr1-TRAIL-hTERT-E1A-E1Bp-E1B55K (CRAd.pEgr1-TRAIL)was constructed,after the adenovirus vector was packaged successfully,MDA-MB-231 cells were infected with them and irradiated with X-rays.Real time PCR method and ELISA were used to detect the expression levels of TRAIL mRNA and protein, respectively. Six groups in the experiment were set up:control, 2 Gy,CRAd.p,CRAd.pEgr1-TRAIL,CRAd.p + 2 Gy and CRAd.pEgr1-TRAIL + 2 Gy. Results The recombinant adenovirus vector pAd-Egr1-TRAIL-hTERT-E1A-E1Bp-E1B55K was constructed and packaged successfully.The expression level of TRAIL mRNA in MDA-MB-231 cells transfected with the vector of 5 MOI for 24 h following 2.0 Gy X-rays irradiation began to increase and arrived to the top 8 h later in various groups,then declined.The expression level of TRAIL protein in MDA-MB-231 cells began to increase 6 h after irradiation and reached to the peak 24 h later,then declined 48 h later.There were significant differences in the expression levels of TRAIL protein between CRAd.pEgr1-TRAIL + 2.0 Gy and other groups at the same time point (P<0.01). Conclusion The recombinant adenovirus vector is obtained successfully, and the TRAIL mRNA and protein expression levels in MDA-MB-231 cells can be increased significantly by the vector combined with 2.0 Gy X-rays irradiation.
ABSTRACT
Objective: To construct an E1 A-deleted 24-bp triple regulated replicative adenovirus vector SG600/interleukin24 (IL24), which was driven by both hTERT promoter and HRE promoter. The level of IL24 in liver cancer cells was determined and the replication capacity of SG600/ IL24 and its killing effects on liver cancer cells were observed. Methods: SG600-IL24 vector was constructed using DNA cloning and recombination techniques. The IL24 gene expression in liver cancer cell lines SMMC-7721 and BEL-7404 and normal cell line BJ was detected by ELISA assay. The replications of SG600/IL-24 in different cell lines were determined by evaluating TCID50 (50% tissue culture infectious dose) at 49 and 96 h. In vitro cell-killing effects of SG600/IL24 on the three liver cancer cell lines were analyzed by MTT assay and CPE (cytopathic effect) staining method at different MOI values. Results: IL24 was over-expressed in both SMMC-7721 and BEL-7404 cells but was weakly expressed in BJ cells. At 48 and 96 h post infection the replication of SG600/IL-24 were 794 and 7940 folds in SMMC-7721 cells; 622 and 7 810 folds in BEL-7404 cells; 20 and 200 folds in BJ cells. MTT assay showed that the MOI values of SG600/IL24 for killing 50% and 90% cells were 0.3 and 5 for SMMC-7721 cells; 3 and 20 for BEL-7404 cells; 50 and 150 for BJ cells. CPE staining demonstrated that SG600/IL24 had significant killing effects on both liver cancer cells SMMC-7721 and BEL-7404 but had no significant influence on BJ cells. The cell-killing capability of SG600/IL24 was superior than that of replicative adenovirus ZD55/IL24 and non replicative adenovirus Ad-IL24. Conclusion: After SMMC-7721 and BEL-7404 liver cancer cells are infected with SG600/1124 at high efficiency, the virus replication is active and the expression of IL24 increases greatly. SG600/IL24 has specific cell-killing effects on the two liver cancer cell lines but has no significant influence on normal cells. This study provides a basis for further investigating the effect of SG600/IL24 on liver cancer in vivo.