Harper P A, Prokipcak R D, Bush L E, Golas C L, Okey A B
Division of Clinical Pharmacology and Toxicology, Hospital for Sick Children, Toronto, Ontario, Canada.
Arch Biochem Biophys. 1991 Oct;290(1):27-36. doi: 10.1016/0003-9861(91)90587-9.
The Ah (aromatic hydrocarbon) receptor mediates induction of aryl hydrocarbon hydroxylase (AHH; an enzyme activity associated with cytochrome P450IA1) by polycyclic aromatic hydrocarbon carcinogens such as 3-methylcholanthrene (MC) and benzo[a]pyrene (BP) and the halogenated toxin 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Until recently the AhR seemed to be present only at very low levels in human cells and tissue. With a modified assay (the presence of sodium molybdate and a reduction in the amount of charcoal used to adsorb "excess" ligand) we found that cytosol from LS180 cells contains a high concentration of AhR (400-500 fmol/mg cytosolic protein) when detected by [3H]TCDD or [3H]MC. Cytosolic receptor also was detected with [3H]BP but at a level that was 35% of that detected with [3H]TCDD or [3H]MC. These levels are similar to those found in mouse Hepa-1 hepatoma cells in which AhR has been extensively characterized. The apparent binding affinity (Kd) of the cytosolic receptor for [3H]TCDD and for [3H]MC was about 5 nM. As with Hepa-1, the human LS180 cytosolic AhR sedimented at about 9 S on sucrose gradients when detected with [3H]TCDD, [3H]BP or [3H]MC. The nuclear-associated ligand.receptor complex recovered from cells incubated in culture with [3H]TCDD sedimented at about 6.2 S. The 9.8 S cytosolic form corresponds to a multimeric protein of a relative molecular mass (Mr) of about 285,000 whereas the 6.2 S nuclear receptor corresponds to a multimeric protein of Mr 175,000. The smallest specific ligand-binding subunit (detected by sodium dodecyl sulfate-polyacrylamide electrophoresis under denaturing conditions of receptor photoaffinity labeled with [3H]TCDD) was about Mr 110,000. AHH activity was induced in cells exposed in culture to TCDD or benz[a]anthracene (BA). The EC50 was 4 x 10(-10) M for TCDD and 1.5 x 10(-5) M for BA. For both inducers the EC50 in LS180 cells was shifted about one log unit to the right as compared to the EC50 for AHH induction in mouse Hepa-1 cells. The lower sensitivity of the LS180 cells to induction of AHH activity by TCDD or BA is consistent with the lower affinity of TCDD and MC for binding to human AhR. The ligand-binding properties, physicochemical properties, and mode of action of the AhR in this human cell line are therefore very similar to those of the extensively characterized AhR in rodent cells and tissues.
芳烃（Ah）受体介导多环芳烃致癌物如3-甲基胆蒽（MC）和苯并[a]芘（BP）以及卤化毒素2,3,7,8-四氯二苯并对二恶英（TCDD）诱导芳烃羟化酶（AHH；一种与细胞色素P450IA1相关的酶活性）。直到最近，AhR似乎仅以非常低的水平存在于人类细胞和组织中。通过一种改良的检测方法（加入钼酸钠并减少用于吸附“过量”配体的活性炭量），我们发现当用[3H]TCDD或[3H]MC检测时，LS180细胞的胞质溶胶中含有高浓度的AhR（400 - 500 fmol/mg胞质蛋白）。用[3H]BP也检测到了胞质受体，但其水平是用[3H]TCDD或[3H]MC检测到的水平的35%。这些水平与在已对AhR进行广泛表征的小鼠Hepa - 1肝癌细胞中发现的水平相似。胞质受体对[3H]TCDD和[3H]MC的表观结合亲和力（Kd）约为5 nM。与Hepa - 1一样，当用[3H]TCDD、[3H]BP或[3H]MC检测时，人LS180胞质AhR在蔗糖梯度上约9 S处沉降。从用[3H]TCDD培养的细胞中回收的核相关配体 - 受体复合物在约6.2 S处沉降。9.8 S的胞质形式对应于相对分子质量（Mr）约为285,000的多聚体蛋白，而6.2 S的核受体对应于Mr为175,000的多聚体蛋白。最小的特异性配体结合亚基（在用[3H]TCDD进行受体光亲和标记的变性条件下，通过十二烷基硫酸钠 - 聚丙烯酰胺电泳检测）约为Mr 110,000。在培养中暴露于TCDD或苯并[a]蒽（BA）的细胞中诱导了AHH活性。TCDD的EC50为4×10^(-10) M，BA的EC50为1.5×10^(-5) M。与小鼠Hepa - 1细胞中AHH诱导的EC50相比，两种诱导剂在LS180细胞中的EC50均向右移动了约一个对数单位。LS180细胞对TCDD或BA诱导AHH活性的较低敏感性与TCDD和MC与人AhR结合的较低亲和力一致。因此，这种人类细胞系中AhR的配体结合特性、物理化学特性和作用方式与啮齿动物细胞和组织中已广泛表征的AhR非常相似。
