Li W, Harper P A, Tang B K, Okey A B
Department of Pharmacology, University of Toronto, Ontario, Canada.
Biochem Pharmacol. 1998 Sep 1;56(5):599-612. doi: 10.1016/s0006-2952(98)00208-1.
It has been difficult to study the regulation of cytochrome P4501A2 (CYP1A2) because expression of this enzyme is reported to be limited or absent in cell culture. We found that CYP1A2 can be induced significantly by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 3-methylcholanthrene (MC), or benz[a]anthracene in the human colon carcinoma cell line LS180. TCDD and MC each caused a dramatic elevation of CYP1A2 mRNA, as assessed by reverse transcription-polymerase chain reaction or by northern blot analysis. TCDD also increased immunoreactive CYP1A2 protein and the activity of phenacetin-O-deethylase, a diagnostic catalytic marker for CYP1A2. The induction of CYP1A2 at all levels (mRNA, protein, catalytic activity) was concentration- and time-dependent: the EC50 for mRNA induction by TCDD = 0.5 nM, and by MC = 1.4 microM. Inducible CYP1A2 mRNA also was detected at lower levels in two other human cell lines, the hepatoma cell line HepG2 and the breast carcinoma cell line MCF-7. CYP1A1 and CYP1B1, additional CYP1 enzymes regulated by the aryl hydrocarbon receptor (AHR), also were inducible by TCDD and MC in LS180 cells; their concentration-dependent induction was highly correlated with induction of CYP1A2 at mRNA, protein, and catalytic levels. CYP1B1 was constitutively expressed and inducible in the LS180, MCF-7, and HepG2 cell lines as well as in the human choriocarcinoma cell line JEG-3 and the squamous cell carcinoma line A431. CYP1A2 was neither constitutively expressed nor inducible in A431 or JEG-3 cells. The expression of mRNAs encoding the regulators of CYP1 enzymes-the AHR and its heterodimerization partner, the ARNT (AH receptor nuclear translocator) protein-was not altered by treatment with TCDD or MC. However, the cytosolic content of AHR protein and ARNT protein was depleted substantially following treatment with TCDD. The LS180 cell line should constitute a good model for further mechanistic studies on AHR-regulated CYP1A2 expression.
由于据报道这种酶在细胞培养中的表达有限或不存在，因此研究细胞色素P4501A2（CYP1A2）的调控一直很困难。我们发现，在人结肠癌细胞系LS180中，2,3,7,8-四氯二苯并对二恶英（TCDD）、3-甲基胆蒽（MC）或苯并[a]蒽可显著诱导CYP1A2。通过逆转录-聚合酶链反应或Northern印迹分析评估，TCDD和MC均导致CYP1A2 mRNA显著升高。TCDD还增加了免疫反应性CYP1A2蛋白以及非那西丁-O-脱乙基酶的活性，后者是CYP1A2的一种诊断性催化标志物。CYP1A2在所有水平（mRNA、蛋白、催化活性）的诱导均呈浓度和时间依赖性：TCDD诱导mRNA的EC50 = 0.5 nM，MC诱导mRNA的EC50 = 1.4 μM。在另外两个人类细胞系，即肝癌细胞系HepG2和乳腺癌细胞系MCF-7中也检测到较低水平的可诱导性CYP1A2 mRNA。CYP1A1和CYP1B1是另外两种受芳烃受体（AHR）调控的CYP1酶，在LS180细胞中也可被TCDD和MC诱导；它们的浓度依赖性诱导与CYP1A2在mRNA、蛋白和催化水平的诱导高度相关。CYP1B1在LS180、MCF-7和HepG2细胞系以及人绒毛膜癌细胞系JEG-3和鳞状细胞癌细胞系A431中组成性表达且可被诱导。CYP1A2在A431或JEG-3细胞中既不组成性表达也不可被诱导。编码CYP1酶调节剂——AHR及其异源二聚体伴侣ARNT（芳烃受体核转运蛋白）蛋白——的mRNA表达不受TCDD或MC处理的影响。然而，用TCDD处理后，AHR蛋白和ARNT蛋白的胞质含量大幅减少。LS180细胞系应构成一个用于进一步研究AHR调控CYP1A2表达机制的良好模型。
