Development of a transactivator in hepatoma cells that allows expression of phase I, phase II, and chemical defense genes
Development of a transactivator in hepatoma cells that allows expression of phase I, phase II, and chemical defense genes
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DOI：
10.1152/ajpcell.00133.2005
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发表时间：
2006-01-01
影响因子：
5.5
通讯作者：
Park, BK
中科院分区：
文献类型：
--
作者：
Goldring, CEP;Kitteringham, NR;Park, BK
精确控制细胞中蛋白质的表达水平，可以获得关于给定基因在正常细胞生理学中的作用以及暴露于化学品和药物的定量和时间信息。这是特别相关的肝细胞，其中许多蛋白质的表达，如第一阶段和第二阶段药物代谢酶，物种之间，人类个体之间，并在暴露于外源性物质的差异很大。最广泛使用的基因调控系统是tet-on/off方法。虽然最近描述了第二代tet-on反式激活因子，但尚未对其作为细胞中基因调节工具的潜力进行广泛研究，特别是在先前与第一代tet-on方法无关的细胞中，例如肝细胞衍生细胞。在这里，我们展示了两个人（HepG 2和HuH 7）和一个小鼠（Hepa 1c 1c 7）肝癌衍生细胞系的发展，将第二代强力霉素诱导的基因表达系统和应用的人线控制不同的转基因的表达。检测了两种人细胞系对与肝脏生物学相关的5种转基因的瞬时或稳定诱导，即I相（细胞色素P-450 2 E1; CYP 2 E1）和II相（谷胱甘肽S-转移酶P1; GST P1）药物代谢，以及对化学应激有反应的3种转录因子[核因子红细胞2 p45相关因子（NRF）1和2以及NF-κ B的NF KB 1亚基]。以时间和剂量依赖性方式获得高水平的功能表达。重要的是，强力霉素没有引起细胞蛋白质组的明显变化。总之，我们已经产生了肝细胞衍生的细胞系，其中基因的表达是完全可控的。
Precise control of the level of protein expression in cells can yield quantitative and temporal information on the role of a given gene in normal cellular physiology and on exposure to chemicals and drugs. This is particularly relevant to liver cells, in which the expression of many proteins, such as phase I and phase II drug-metabolizing enzymes, vary widely between species, among individual humans, and on exposure to xenobiotics. The most widely used gene regulatory system has been the tet-on/off approach. Although a second-generation tet-on transactivator was recently described, it has not been widely investigated for its potential as a tool for regulating genes in cells and particularly in cells previously recalcitrant to the first-generation tet-on approach, such as hepatocyte-derived cells. Here we demonstrate the development of two human (HepG2 and HuH7) and one mouse (Hepa1c1c7) hepatoma-derived cell lines incorporating a second-generation doxycycline-inducible gene expression system and the application of the human lines to control the expression of different transgenes. The two human cell lines were tested for transient or stable inducibility of five transgenes relevant to liver biology, namely phase I (cytochrome P-450 2E1; CYP2E1) and phase II (glutathione S-transferase P1; GSTP1) drug metabolism, and three transcription factors that respond to chemical stress [nuclear factor erythroid 2 p45-related factors (NRF)1 and 2 and NFKB1 subunit of NF-kappa B]. High levels of functional expression were obtained in a time- and dose-dependent manner. Importantly, doxycycline did not cause obvious changes in the cellular proteome. In conclusion, we have generated hepatocyte-derived cell lines in which expression of genes is fully controllable.