Putranto Endy Widya, Kinoshita Rie, Watanabe Masami, Sadahira Takuya, Murata Hitoshi, Yamamoto Ken-Ichi, Futami Junichiro, Kataoka Ken, Inoue Yusuke, Winarsa Ruma I Made, Sumardika I Wayan, Youyi Chen, Kubo Miyoko, Sakaguchi Yoshihiko, Saito Kenji, Nasu Yasutomo, Kumon Hiromi, Huh Nam-Ho, Sakaguchi Masakiyo
Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama 700-8558, Japan.
Department of Pediatrics, Dr Sardjito Hospital/Faculty of Medicine, Gadjah Mada University, Yogyakarta 55281, Indonesia.
Oncol Lett. 2017 Jul;14(1):1041-1048. doi: 10.3892/ol.2017.6201. Epub 2017 May 17.
Reduced expression in immortalized cells (REIC)/Dickkopf-3 (Dkk-3) overexpression, induced using an adenovirus (Ad)-REIC, has been revealed to have a dramatic therapeutic effect on multiple types of cancer. To achieve an improved therapeutic effect from Ad-REIC on cancer, our group previously developed an enhanced gene expression system, the C-TSC cassette [cytomegalovirus (CMV)-RU5' located upstream (C); another promoter unit composed of triple tandem promoters, human telomerase reverse transcriptase (hTERT), simian virus 40 and CMV, located downstream of the cDNA (TSC); plus a polyadenylation (polyA) signal]. When applied to the conventional Ad-REIC, this novel system induced the development of an enhanced product, Ad-C-TSC-REIC, which exhibited a noticeable anticancer effect. However, there were difficulties in terms of Ad-C-TSC-REIC productivity in HEK293 cells, which are a widely used donor cell line for viral production. Productivity of Ad-C-TSC-REIC was significantly reduced compared with the conventional Ad-REIC, as the Ad-C-TSC-REIC had a significantly higher ability to induce apoptotic cell death of not only various types of cancer cell, but also HEK293 cells. The present study aimed to overcome this problem by modifying the C-TSC structure, resulting in an improved candidate: A C-T cassette (C: CMV-RU5' located upstream; T: another promoter unit composed of a single hTERT promoter, located downstream of the cDNA plus a polyA signal), which demonstrated gene expression comparable to that of the C-TSC system. The improved adenovirus REIC/Dkk-3 product with the C-T cassette, named Ad-C-T-REIC, exhibited a higher expression level of REIC/Dkk3, similar to that of Ad-C-TSC-REIC. Notably, the vector mitigated the cell death of donor HEK293 cells, resulting in a higher rate of production of its adenovirus. These results indicated that Ad-C-T-REIC has the potential to be a useful tool for application in cancer gene therapy.
使用腺病毒（Ad）-REIC诱导永生化细胞中REIC/Dickkopf-3（Dkk-3）表达降低，已显示对多种类型癌症具有显著治疗效果。为了提高Ad-REIC对癌症的治疗效果，我们团队先前开发了一种增强基因表达系统，即C-TSC盒式结构[巨细胞病毒（CMV）-RU5'位于上游（C）；另一个由三重串联启动子、人端粒酶逆转录酶（hTERT）、猿猴病毒40和CMV组成的启动子单元，位于cDNA下游（TSC）；加上一个聚腺苷酸化（polyA）信号]。当应用于传统的Ad-REIC时，这个新系统诱导产生了一种增强型产物Ad-C-TSC-REIC，其表现出显著的抗癌效果。然而，在用于病毒生产的广泛使用的供体细胞系HEK293细胞中，Ad-C-TSC-REIC的生产存在困难。与传统的Ad-REIC相比，Ad-C-TSC-REIC的产量显著降低，因为Ad-C-TSC-REIC不仅对多种类型癌细胞，而且对HEK293细胞都具有显著更高的诱导凋亡细胞死亡的能力。本研究旨在通过修改C-TSC结构来克服这个问题，从而得到一个改进的候选物：C-T盒式结构（C：CMV-RU5'位于上游；T：另一个由单个hTERT启动子组成的启动子单元，位于cDNA下游加上一个polyA信号），其显示出与C-TSC系统相当的基因表达。带有C-T盒式结构的改进型腺病毒REIC/Dkk-3产物，命名为Ad-C-T-REIC，表现出与Ad-C-TSC-REIC相似的更高水平的REIC/Dkk3表达。值得注意的是，该载体减轻了供体HEK293细胞的死亡，导致其腺病毒的生产率更高。这些结果表明，Ad-C-T-REIC有潜力成为癌症基因治疗中一种有用的工具。
