Zhang Jane, Ramesh Nagarajan, Chen Yu, Li Yuanhao, Dilley Jeanette, Working Peter, Yu De-Chao
Cell Genesys, Inc., Foster City, California 94404, USA.
Cancer Res. 2002 Jul 1;62(13):3743-50.
Uroplakins (UPs) are a group of integral membrane proteins that are synthesized as the major differentiation products of mammalian urothelium. UPII gene expression is bladder specific and differentiation dependent, but very little is known about its transcription response elements. To identify the promoter elements, a DNA fragment of 2239 bp upstream of the UPII gene was amplified by PCR and linked to a promoterless firefly luciferase reporter gene. Transient transfection experiments showed that the DNA segment located between -1809 and +1 bp resulted in preferential expression in bladder carcinoma cells with negligible expression in nonurothelial cells. This promoter was engineered into adenovirus (Ad) type 5 to drive the expression of the E1A and E1B genes and to create an attenuated replication-competent Ad variant, termed CG8840. Viral replication and the cytopathic effect of CG8840 were evaluated by virus yield and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays in bladder transitional cell carcinoma (TCC) cell lines RT4 and SW780; nonbladder cancer cell lines G361 (melanoma), LNCaP (prostate cancer), PA-1 (ovarian cancer), and U118 (brain cancer); and human primary cells including lung fibroblasts, bladder smooth muscle cells, and mammary epithelial cells. CG8840 replicated in and eliminated bladder TCC efficiently with high specificity (10,000:1) in comparison with nonbladder cells. The antitumor activity of CG8840 was examined in BALB/c nu/nu mice carrying s.c. human TCC xenografts. Intratumoral and i.v. administration of CG8840 in RT4 human bladder cancer xenografts caused significant (P < 0.01) inhibition of tumor growth. Synergistic antitumor efficacy was observed when CG8840 was combined with docetaxel, resulting in significant regression of RT4 bladder cancer xenograft tumors within 6 weeks after i.v. administration of CG8840 (3.33 x 10(9) plaque-forming units/animal on day 1) and docetaxel (20 mg/kg on days 2, 6, and 9). These results demonstrate the utility of the UPII promoter in the generation of urothelium-specific adenoviral vectors and provide a potential foundation for the development of bladder tumor-specific oncolytic viral therapies.
尿血小板溶素（UPs）是一组整合膜蛋白，作为哺乳动物尿路上皮的主要分化产物而合成。UPII基因表达具有膀胱特异性且依赖于分化，但对其转录反应元件了解甚少。为了鉴定启动子元件，通过聚合酶链反应（PCR）扩增了UPII基因上游2239 bp的DNA片段，并将其连接到无启动子的萤火虫荧光素酶报告基因上。瞬时转染实验表明，位于-1809至+1 bp之间的DNA片段在膀胱癌细胞中优先表达，而在非尿路上皮细胞中的表达可忽略不计。将该启动子构建到5型腺病毒（Ad）中，以驱动E1A和E1B基因的表达，并创建一种减毒的具有复制能力的Ad变体，称为CG8840。通过病毒产量和3-（4,5-二甲基噻唑-2-基）-2,5-二苯基四氮唑溴盐（MTT）测定法，在膀胱移行细胞癌（TCC）细胞系RT4和SW780；非膀胱癌细胞系G361（黑色素瘤）、LNCaP（前列腺癌）、PA-1（卵巢癌）和U118（脑癌）；以及包括肺成纤维细胞、膀胱平滑肌细胞和乳腺上皮细胞在内的人原代细胞中评估了CG8840的病毒复制和细胞病变效应。与非膀胱细胞相比，CG8840在膀胱TCC中高效且高度特异性（10000:1）地复制并消除了膀胱TCC。在携带人TCC皮下异种移植物的BALB/c nu/nu小鼠中检测了CG8840的抗肿瘤活性。在RT4人膀胱癌异种移植物中瘤内和静脉内给予CG8840可显著（P < 0.01）抑制肿瘤生长。当CG8840与多西他赛联合使用时，观察到协同抗肿瘤效果，在静脉内给予CG8840（第1天3.33×10⁹ 噬斑形成单位/动物）和多西他赛（第2、6和9天20 mg/kg）后6周内，RT4膀胱癌异种移植瘤显著消退。这些结果证明了UPII启动子在生成尿路上皮特异性腺病毒载体中的实用性，并为开发膀胱肿瘤特异性溶瘤病毒疗法提供了潜在基础。
