ENHANCEMENT OF BYSTANDER EFFECTS WITH THE COMBINATION OF IMMUNO-GENE THERAPY AND PRO-DRUG GENE THERAPY

本研究分析了免疫基因治疗与前药物基因治疗相结合对增强旁观者效应的作用。作为本项目的主要部分，分析了尿嘧啶磷酸核糖基转移酶(UPRT)基因治疗膀胱癌的效果。目的：观察腺病毒介导的UPRT基因对5-FU对实验性膀胱癌的增敏抗肿瘤作用。材料与方法：以人膀胱癌T24细胞为研究对象，进行体内外实验研究。从血清型5型野生型腺病毒(Ad5)出发，将CAG启动子与E.coliLacZ基因和E.coliUprt基因分别插入基因组EIA区，缺失E1B和E3区，构建了两种复制缺陷型腺病毒载体ADCA-LacZ和ADCA-UPRT。体外药敏试验：T24细胞接种于96孔板中，密度为1000cell/孔，感染…多用ADCA-LacZ或ADCA-UPRT(MOI：0~100)，然后用5-FU治疗。6天后，用四甲基偶氮唑盐比色法测定药物敏感性。对于体内肿瘤的治疗，采用皮下注射T24细胞(10^7细胞)。肿瘤细胞注射后12天(第12天)，肿瘤显影，平均体重147 mg(108-199 mg)，随机分为两组，分别直接注射ADCA-LacZ或ADC A-UPRT(1×10~8pfu/10ul)，随后(第2天：第14天)腹腔注射生理盐水或5-FU(20 mg/kg)，连续10天(第14~23天)。结果：ADCA-UPRT感染可增加T24细胞对5-FU的敏感性，且呈MOI依赖性。在MOI为50的ADCA-Uprt感染细胞中，5-FU的IC_(50)由对照细胞的5，М移至0.05М，表明ADCA-Uprt感染致敏100倍。与体外实验结果一致，联合应用ADCA-UPRT和全身应用5-FU可在第28天抑制肿瘤生长，与对照组相比，对照组之间有无差异。结论：UPRT基因治疗联合5-FU可增敏5-FU的抗肿瘤作用。因此，该方法为肿瘤基因治疗提供了一种新的化疗增敏策略，为膀胱癌的治疗提供了一种更可行的方法。较少

In this research, enhancement of bystander effects with the combination of immuno-gene therapy and pro-drug gene therapy was analyzed. As a main portion of this project, effects of UPRT (uracil phosphoribosyltransferase) gene therapy (new type of pro-drug gene therapy) for bladder cancer was analyzed. Here we show the summary of them.Objective : We evaluated whether adenovirus-mediated UPRT genes could generate sensitized antitumor effect of 5-FU on experimental bladder cancer.Material and Method : Human bladder cancer cell T24 was used for in vitro and in vivo study. Two recombinant replication-deficient adeno virus vectors, AdCA-LacZ and AdCA-UPRT, were constructed from a serotype 5 wild-type adenovirus (Ad5) by inserting CAG promoter with an E. coli LacZ gene and an E. coli UPRT gene, respectively, into the EIA region of the genome, deleting the E1B and E3 region. For in vitro chemosensitivity assay, T24 cells were seeded in 96-well plates at a density of 1000 cells/well, infected w … More ith AdCA-LacZ or AdCA-UPRT (MOI :0-100), and then exposed to 5-FU. Six days later, the sensitivity was assessed with MTT assay. For in vivo tumor treatment, subcutaneous tumors were employed by injection of T24 cells (10^7 cells). Twelve days after tumor cell injection (day 12) when tumor becomes palpable with mean estimated weight 147mg (108 -199mg), mouse were randomized to be directly injected with AdCA-LacZ or AdC A-UPRT (1X10^8 pfu/10ul) subsequently (two days later : day14) with either saline or 5-FU(2Omg/kg) intraperitoneally for 10days (from day14 to 23).Results : AdCA-UPRT infection increased the sensitivity of T24 cells to 5-FU in a MOI-dependent manner. IC50 to 5-FU shifted from 5,М in, control cells to 0.05М in AdCA-UPRT-infected cells at MOI of 50 indicating 100 fold sensitization by AdCA-UPRT infection. Consistent with in vitro studies, administration of AdCA-UPRT into the tumor of T24 cells, together with systemic 5-FU administration, resulted in sup pression of tumor growth at day28, compared with the control tumor groups : Among control groups, difference was not observed in comparis on with the presence or absence of 5-FU administrationConclusion : These results suggest that UPRT gene therapy with 5-FU can sensitize the antitumor effect of 5-FU. Consequently, this approach is a new chemosensitizing strategy for cancer gene therapy and a more feasible modality for the treatment of bladder cancer. Less