Roberts E A, Harper P A, Wong J M, Wang Y, Yang S
Metabolism Programme, The Hospital for Sick Children Research Institute, University of Toronto, Ontario, Canada.
Arch Biochem Biophys. 2000 Dec 1;384(1):190-8. doi: 10.1006/abbi.2000.2059.
The Ah receptor mediates the induction of cytochrome P450 1A1 (CYP1A1) and toxicities of 2,3,7,8tetrachlorodibanzo-p-dioxin (TCDD). It has been detected in tissues of many species and in murine and human hepatoma lines. We show that the human hepatoma line SK-Hep-1 has cytosolic Ah receptor detectable by specific binding of [3H]TCDD. Concentrations of Ah receptor were low (mean = 43 +/- 3 fmol/mg cytosol protein compared to 430 fmol/mg protein in Hepa-1); the estimated number of receptor sites per cell is approximately 9,000, compared to 35,000 in Hepa-1. Ah receptor in SK-Hep-1 cells was physicochemically similar to Ah receptor in C57BL/6 mouse liver and in other human hepatoma lines studied to date except that binding affinity for TCDD, the most avidly bound ligand, was lower (estimated Kd was 14 nM by Woolf plot analysis). Translocation of the Ah receptor-ligand complex to the nucleus was shown; binding of the activated Ah receptor-ligand complex to an XRE in the 5'-upstream region of the CYP1A1 gene was demonstrated by gel-shift analysis. However, after SK-Hep-1 cells were incubated with typical PAHs including 3-methylcholanthrene, benzanthracene, and dibenz(a,h)anthracene, each over a wide range of concentrations, no induction of aryl hydrocarbon hydroxylase activity was detectable. On Northern analysis, no message for human CYP1A1 was detected in mRNA prepared from noninduced SK-Hep-1 cells or from cells treated for 24 h with 13 microM dibenz(a,h)anthracene. Further analysis by RT-PCR did not detect the induction of CYP1A1, CYP1A2, or CYP1B1 message in response to 10(-7) M TCDD, 10(-5) M benzanthracene, or 10(-5) M 3-methylcholanthrene. Transient transfection of reporter constructs containing either a minimal promoter or the CYP1A1 promoter fused to a reporter gene (luciferase) did not show any expression in response to increasing concentrations of TCDD up to 10(-8) M. Estimation of the size of the transcripts for AhR and ARNT protein revealed normal sizes, 2.7 and 2.4 kb, respectively. Together, these data suggest that SK-Hep-1 cells express an Ah receptor defective at the level of trans-activation of gene expression. SK-Hep-1 is the first human hepatoma line described with a demonstrable defect in CYP1A1 or its regulation.
芳烃受体介导细胞色素P450 1A1（CYP1A1）的诱导以及2,3,7,8-四氯二苯并-对-二恶英（TCDD）的毒性作用。它已在许多物种的组织以及小鼠和人类肝癌细胞系中被检测到。我们发现人类肝癌细胞系SK-Hep-1具有可通过[3H]TCDD特异性结合检测到的胞质芳烃受体。芳烃受体的浓度较低（平均为43±3 fmol/mg胞质溶胶蛋白，而Hepa-1细胞中为430 fmol/mg蛋白）；每个细胞中受体位点的估计数量约为9000个，而Hepa-1细胞中为35000个。SK-Hep-1细胞中的芳烃受体在物理化学性质上与C57BL/6小鼠肝脏以及迄今为止研究的其他人类肝癌细胞系中的芳烃受体相似，只是对TCDD（结合最紧密的配体）的结合亲和力较低（通过伍尔夫图分析估计Kd为14 nM）。已证明芳烃受体-配体复合物易位至细胞核；通过凝胶迁移分析证明活化的芳烃受体-配体复合物与CYP1A1基因5'上游区域的XRE结合。然而，在SK-Hep-1细胞与包括3-甲基胆蒽、苯并蒽和二苯并(a,h)蒽在内的典型多环芳烃在广泛浓度范围内孵育后，未检测到芳烃羟化酶活性的诱导。在Northern分析中，从未诱导的SK-Hep-1细胞或用13 μM二苯并(a,h)蒽处理24小时的细胞制备的mRNA中未检测到人类CYP1A1的信息。通过RT-PCR进一步分析未检测到CYP1A1、CYP1A2或CYP1B1信息在响应10^(-7) M TCDD、10^(-5) M苯并蒽或10^(-5) M 3-甲基胆蒽时的诱导。将含有最小启动子或与报告基因（荧光素酶）融合的CYP1A1启动子的报告构建体进行瞬时转染，在TCDD浓度增加至10^(-8) M时，未显示出任何表达。对AhR和ARNT蛋白转录本大小的估计显示大小正常，分别为2.7 kb和2.4 kb。总之，这些数据表明SK-Hep-1细胞表达一种在基因表达反式激活水平上有缺陷的芳烃受体。SK-Hep-1是第一个被描述在CYP1A1或其调控方面存在明显缺陷的人类肝癌细胞系。
