Expression of tumor suppressor REIC/Dkk-3 by a newly improved adenovirus vector with insertion of a hTERT promoter at the 3'-side of the transgene

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

doi:10.3892/ol.2017.6201

Expression of tumor suppressor REIC/Dkk-3 by a newly improved adenovirus vector with insertion of a hTERT promoter at the 3'-side of the transgene

Authors:
- Endy Widya Putranto
- Rie Kinoshita
- Masami Watanabe
- Takuya Sadahira
- Hitoshi Murata
- Ken‑Ichi Yamamoto
- Junichiro Futami
- Ken Kataoka
- Yusuke Inoue
- I. Made Winarsa Ruma
- I. Wayan Sumardika
- Chen Youyi
- Miyoko Kubo
- Yoshihiko Sakaguchi
- Kenji Saito
- Yasutomo Nasu
- Hiromi Kumon
- Nam‑Ho Huh
- Masakiyo Sakaguchi
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Affiliations: Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama 700‑8558, Japan, Department of Urology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama 700‑8558, Japan, Department of Medical and Bioengineering Science, Okayama University Graduate School of Natural Science and Technology, Okayama 700‑8530, Japan, Department of Life Science, Faculty of Science, Okayama University of Science, Okayama 700‑0005, Japan, Faculty of Science and Technology, Division of Molecular Science, Gunma University, Kiryu, Gunma 376‑8515, Japan, Department of Microbiology, Kitasato University School of Medicine, Sagamihara, Kanagawa 252‑0374, Japan, Niimi College, Niimi, Okayama 718‑8585, Japan
Published online on: May 17, 2017 https://doi.org/10.3892/ol.2017.6201
Pages: 1041-1048

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Abstract

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.

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