Paulus W, Baur I, Oberer D M, Breakefield X O, Reeves S A
Department of Neurology, Massachusetts General Hospital and Harvard Medical School, Boston, USA.
J Neurosurg. 1997 Jul;87(1):89-95. doi: 10.3171/jns.1997.87.1.0089.
Because accurate regulation of toxin gene expression is critical for safe and effective gene therapy applications, the authors have examined the regulation of diphtheria toxin A (DTA) fragment expression in human glioma cell lines using two transcriptional control systems derived from Escherichia coli: the tetracycline (Tet) system and the lactose (Lac) system. The Tet system includes a tetracycline-controlled transactivator (tTA), a tTA-responsive minimum human cytomegalovirus (hCMV) promoter controlling the expression of the DTA gene, and tetracycline as an allosteric inhibitor. The Lac system includes the lac repressor (lacR), a lacR-regulated Rous sarcoma virus-long terminal repeat (RSV-LTR) promoter controlling the expression of the DTA gene, and isopropyl-thio-beta-D-galactoside (IPTG) as an allosteric inducer. Expression plasmids encoding either tTA or lacR were transfected into U-87MG and U-343MG glioma cells along with the responsive DTA plasmid. Cell killing was monitored by the ability of the toxin to abolish protein synthesis and was quantitated using a luciferase reporter gene. In the Tet system, tumor cell killing could be regulated by tetracycline up to 120-fold. In contrast, only a twofold IPTG-dependent regulation was obtained using the Lac system because of an incomplete repression of DTA expression in the uninduced state. Replacement of the RSV-LTR promoter with the heavy metal-inducible mouse metallothionein-1 promoter in the lacR-responsive unit, as well as the generation of a clonal glioma cell line expressing lacR, did not significantly enhance regulation of DTA in the Lac system. In conclusion, this study demonstrates that the Tet system is of potential use in gene therapy applications in which regulated expression of a therapeutic gene is an important issue.
由于毒素基因表达的精确调控对于安全有效的基因治疗应用至关重要，作者使用源自大肠杆菌的两种转录控制系统，研究了人胶质瘤细胞系中白喉毒素A（DTA）片段表达的调控：四环素（Tet）系统和乳糖（Lac）系统。Tet系统包括四环素调控的反式激活因子（tTA）、控制DTA基因表达的tTA反应性最小人巨细胞病毒（hCMV）启动子，以及作为变构抑制剂的四环素。Lac系统包括乳糖阻遏物（lacR）、控制DTA基因表达的lacR调控的劳氏肉瘤病毒长末端重复序列（RSV-LTR）启动子，以及作为变构诱导剂的异丙基硫代-β-D-半乳糖苷（IPTG）。将编码tTA或lacR的表达质粒与反应性DTA质粒一起转染到U-87MG和U-343MG胶质瘤细胞中。通过毒素消除蛋白质合成的能力监测细胞杀伤情况，并使用荧光素酶报告基因进行定量。在Tet系统中，四环素可将肿瘤细胞杀伤调控高达120倍。相比之下，由于在未诱导状态下DTA表达的不完全抑制，使用Lac系统仅获得了两倍的IPTG依赖性调控。在lacR反应单元中用重金属诱导型小鼠金属硫蛋白-1启动子替换RSV-LTR启动子，以及生成表达lacR的克隆胶质瘤细胞系，均未显著增强Lac系统中DTA的调控。总之，本研究表明Tet系统在治疗基因的调控表达是一个重要问题的基因治疗应用中具有潜在用途。
