title: Advanced two-step transcriptional amplification as a novel method for cancer-specific gene expression and imaging.
abstract: The two-step transcriptional amplification (TSTA) system was previously reported to enhance the tissue-specific gene expression driven by weak promoters, but the enhancement of the gene expression is limited to use in in vitro and in vivo experimental situations. To achieve robust tissue-specific gene expression using the TSTA system, we developed an advanced TSTA system which includes polyglutamines and rat glucocorticoid receptor sequences between the GAL4 and VP16 sequences in the region of the first step of transcription. We evaluated the advanced TSTA system as a method to enhance the human telomerase reverse transcriptase (hTERT) promoter-driving cancer-specific transcription in various cancer cell lines. As a result, the advanced TSTA enhanced cancer-specific luciferase gene expression in all of the examined cancer cell lines, when compared with both the one-step and conventional TSTA systems (an ~6- and ~17-fold enhancement, respectively). Notably, the enhancement of the hTERT driven expression by the conventional TSTA system was modest and even inferior to the one-step system in several cancer cell lines. We then constructed a luciferase gene encoding the adeno-associated virus vector in which the hTERT promoter-mediated expression was driven by the advanced TSTA or control systems. In an orthotopic liver tumor model, mice were treated with the vector via tail vein injection. An optical imaging device was used to visualize the in vivo luciferase expression in the orthotopic tumor. The advanced TSTA system significantly enhanced the luciferase expression compared with the one-step and conventional TSTA systems (18.0±1.0- and 15.9±0.85-fold gain, respectively). Therefore, the advanced TSTA system significantly improves hTERT-dependent cancer-specific gene expression both in vitro and in vivo when compared with the previous systems. Since the advanced TSTA method can also be applied to other site-specific gene expression systems using tissue-specific promoters, this approach is expected to become a valuable tool enabling in vivo site-specific targeting in the field of gene therapy and molecular imaging.
authors: Center for Gene and Cell Therapy, Department of Urology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, 2-5-1 Shikata-cho, Okayama 700-8558, Japan. mwcorrespondence@gmail.com
authors: Masami
authors: M
authors: Watanabe
authors: Hideo
authors: H
authors: Ueki
authors: Kazuhiko
authors: K
authors: Ochiai
authors: Peng
authors: P
authors: Huang
authors: Yasuyuki
authors: Y
authors: Kobayashi
authors: Yasutomo
authors: Y
authors: Nasu
authors: Katsumi
authors: K
authors: Sasaki
authors: Haruki
authors: H
authors: Kaku
authors: Yuji
authors: Y
authors: Kashiwakura
authors: Hiromi
authors: H
authors: Kumon
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D045744:Cell Line, Tumor; D005784:Gene Amplification; D015870:Gene Expression; D015316:Genetic Therapy / Q000379:methods; D006367:HeLa Cells; D006801:Humans; D008114:Liver Neoplasms, Experimental / Q000235:genetics; D008156:Luciferases / Q000235:genetics; D008297:Male; D051379:Mice; D008819:Mice, Nude; D008969:Molecular Sequence Data; D009369:Neoplasms / Q000235:genetics* / Q000628:therapy; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D019098:Telomerase / Q000235:genetics; D014158:Transcription, Genetic; D014162:Transfection; D014183:Transplantation, Heterologous
chemical_list: D008156:Luciferases; C509186:TERT protein, human; D019098:Telomerase