Advanced two-step transcriptional amplification as a novel method for cancer-specific gene expression and imaging
Advanced two-step transcriptional amplification as a novel method for cancer-specific gene expression and imaging
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DOI：
10.3892/or.2011.1371
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发表时间：
2011-10-01
期刊：
影响因子：
4.2
通讯作者：
Kumon, Hiromi
中科院分区：
文献类型：
--
作者：
Watanabe, Masami;Ueki, Hideo;Kumon, Hiromi
两步转录扩增（TSTA）系统以前被报道可以增强由弱启动子驱动的组织特异性基因表达，但是基因表达的增强仅限于在体外和体内实验情况下使用。为了使用TSTA系统实现稳健的组织特异性基因表达，我们开发了一种先进的TSTA系统，其包括在转录的第一步区域中的GAL 4和VP 16序列之间的多聚谷氨酰胺和大鼠糖皮质激素受体序列。我们评估了先进的TSTA系统作为一种方法，以提高人类端粒酶逆转录酶（hTERT）启动子驱动的癌症特异性转录在各种癌细胞系。结果，与一步和常规TSTA系统相比，高级TSTA增强了所有检查的癌细胞系中的癌症特异性荧光素酶基因表达（分别增强了6倍和17倍）。值得注意的是，在几种癌细胞系中，传统TSTA系统对hTERT驱动的表达的增强是适度的，甚至不如一步系统。然后，我们构建了一个荧光素酶基因编码的腺相关病毒载体，其中的hTERT启动子介导的表达是由先进的TSTA或控制系统驱动。在原位肝肿瘤模型中，通过尾静脉注射用载体处理小鼠。使用光学成像装置来可视化原位肿瘤中的体内荧光素酶表达。与一步和常规TSTA系统相比，先进的TSTA系统显著增强了荧光素酶表达（分别增加18.0+/-1.0和15.9+/-0.85倍）。因此，先进的TSTA系统显着提高hTERT依赖性癌症特异性基因表达在体外和体内相比，以前的系统。由于先进的TSTA方法也可以应用于其他位点特异性基因表达系统使用组织特异性启动子，这种方法有望成为一个有价值的工具，使在基因治疗和分子成像领域的体内位点特异性靶向。
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 similar to 6- and similar to 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.