Department of Cell Biology, Okayama University, Okayama 700-8558, Japan.
Department of Urology, Okayama University, Okayama 700-8558, Japan.
Oncol Rep. 2017 Aug;38(2):1108-1114. doi: 10.3892/or.2017.5710. Epub 2017 Jun 12.
We developed and validated a novel hTERT/CMV promoter element-driven gene expression cassette that can robustly enhance cancer-specific gene expression. The following gene expressional elements were located in tandem within the plasmid construct: [hTERT core promoter, cytomegalovirus (CMV) minimized promoter, RU5' sequence, an inserted gene, BGH polyA, hTERT enhancer]; this is hereafter referred to as the hT/Cm-R-hT construct. Using various human cancer cell lines and normal cells, the cancer-specific transcription of the green fluorescent protein (GFP) gene was examined by western blotting and fluorescence microscopy. Cancer-specific gene expression was robustly achieved in the hT/Cm-R-hT plasmid in comparison to the other control hT/Cm-driven construct. Notably, the expression level of GFP observed in the hT/Cm-R-hT-driven construct was superior to that of the control plasmid with the conventional CMV promoter in HEK293 cells, which are known to possess higher hTERT activity than normal cells. We next examined the availability of hT/Cm-R-hT in detecting the target GFP expressing cancer cells from human peripheral blood mononuclear cells (PBMCs). The hT/Cm-R-hT plasmid successfully induced cancer-specific gene expression; the robust expression of GFP was observed in target HeLa cancer cells, whereas GFP was not visibly expressed in normal PBMCs. The plasmid allowed for the selective visualization of viable HeLa cancer cells in mixed cell cultures containing up to 10000-fold more PBMCs. These findings indicate that the hT/Cm-R-hT expressional system is a valuable tool for detecting viable cancer cells mixed with normal cells. The current system can therefore be applied to the in vitro detection of cancer cells that are disseminated in the blood and other types of body fluid in vivo. Since the current system can also be applied to other types of vectors, including virus vectors, this approach using the hTERT promoter-based construct is expected to become a valuable tool for enhancing cancer-specific gene expression.
我们开发并验证了一种新型的 hTERT/CMV 启动子元件驱动的基因表达盒，可显著增强癌症特异性基因表达。该质粒构建物中串联排列了以下基因表达元件：[hTERT 核心启动子、巨细胞病毒（CMV）最小化启动子、RU5' 序列、插入基因、BGH 多聚 A、hTERT 增强子]；以下简称 hT/Cm-R-hT 构建体。使用各种人类癌细胞系和正常细胞，通过 Western blot 和荧光显微镜检查 GFP 基因的癌症特异性转录。与其他对照 hT/Cm 驱动的构建体相比，hT/Cm-R-hT 质粒中 GFP 的癌症特异性基因表达得到了显著增强。值得注意的是，与在具有更高 hTERT 活性的正常细胞中已知的常规 CMV 启动子的对照质粒相比，在 hT/Cm-R-hT 驱动的构建体中观察到的 GFP 表达水平更高。接下来，我们检查了 hT/Cm-R-hT 在从人外周血单核细胞（PBMCs）中检测表达靶 GFP 的癌症细胞中的可用性。hT/Cm-R-hT 质粒成功诱导了癌症特异性基因表达；在靶 HeLa 癌细胞中观察到 GFP 的强烈表达，而在正常 PBMCs 中 GFP 则不明显表达。该质粒允许在含有多达 10000 倍以上 PBMCs 的混合细胞培养物中选择性地可视化存活的 HeLa 癌细胞。这些发现表明，hT/Cm-R-hT 表达系统是一种用于检测与正常细胞混合的存活癌细胞的有价值的工具。因此，该系统可应用于检测在体内血液和其他类型体液中扩散的癌细胞。由于当前系统也可应用于包括病毒载体在内的其他类型载体，因此使用 hTERT 启动子构建体的这种方法有望成为增强癌症特异性基因表达的有价值的工具。
