Regulation of CMV promoter‐driven exogenous gene expression with doxorubicin in genetically modified cells |
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| Authors: | Atsushi Kinoshita Daisuke Kobayashi Yasuhide Hibino Tomokazu Isago Katsuyoshi Uchino Keiko Yagi Midori Hirai Yukiya Saitoh Fusao Komada |
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| Institution: | 1. Division of Drug Informatics, Faculty of Pharmaceutical Sciences, Himeji Dokkyo University, Kamiono 7‐2‐1, Himeji, Hyogo 670‐8524, Japan;2. Department of Drug Informatics, Faculty of Pharmaceutical Sciences, Josai University, 1‐1, Keyakidai, Sakado, Saitama 350‐0295, Japan;3. Laboratory of Immunobiochemistry, Department of Clinical Dietetics and Human Nutrition, Faculty of Pharmaceutical Sciences, Josai University, 1‐1 Keyakidai Sakado, Saitama 350‐0295, Japan;4. Department of Pharmaceutical Health Care and Sciences, Tokyo University of Pharmacy and Life Sciences, 1432‐1 Horinouchi, Hachioji, Tokyo 192‐0392, Japan;5. Department of Clinical Pharmacy, Kobe Pharmaceutical University, 4‐19‐1 Motoyamakita‐machi, Higashinada‐ku, Kobe, Hyogo 658‐8558, Japan;6. Department of Hospital Pharmacy, School of Medicine, Kobe University, 7‐5‐2 Kusunoki‐cho, Chuo‐ku, Kobe, Hyogo 650‐0017, Japan |
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| Abstract: | The regulation of gene expression after the introduction of an exogenous gene is a problematic aspect of gene therapy. The purpose of this study was to use doxorubicin to regulate exogenous gene expression in a vector containing the cytomegalovirus (CMV) promoter. The pQBI25 vector, which encodes the CMV promoter and the cDNA for red‐shifted green fluorescent protein (rsGFP), was transfected into a rat skin fibroblast cell line (FR cells). The pEGFP vector, encoding the CMV promoter and enhanced green fluorescent protein (EGFP) cDNA, was transfected into human hepatoma HepG2 cells. FR‐pQBI25 cells were then continuously exposed to doxorubicin and methotrexate for 96 and 48 h, respectively; HepG2‐pEGFP cells were continuously exposed to doxorubicin for 48 h. The levels of c‐fos, c‐jun and rsGFP mRNA, as well as the levels of rsGFP protein, in the FR‐pQBI25 cells were found to be significantly higher following exposure to doxorubicin. However, the level of rsGFP protein was not changed by exposure to methotrexate. The level of EGFP protein in the HepG2‐pEGFP cells was also significantly higher following exposure to doxorubicin. To examine the effect of cessation of doxorubicin exposure, FR‐pQBI25 cells that had been exposed to doxorubicin for 48 h were re‐plated in fresh medium without doxorubicin for a further 48 h. The increased levels of c‐fos, c‐jun and rsGFP mRNA and rsGFP protein seen after treatment with doxorubicin had reduced by 48 h after the cessation of exposure to doxorubicin. These findings suggest that CMV‐driven exogenous gene expression may be regulated by doxorubicin. |
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