Sadek C M, Allen-Hoffmann B L
Department of Pathology, University of Wisconsin, Madison 53706.
J Biol Chem. 1994 Dec 16;269(50):31505-9.
We have recently demonstrated that release of normal human epithelial cells from cell-substratum and/or cell-cell adhesion generates cellular signals that induce the expression of CYP1A1 in the absence of xenobiotic polycyclic aromatic hydrocarbons (Sadek, C. M., and Allen-Hoffmann, B. L. (1994) J. Biol. Chem. 169, 16067-16074). To directly test the involvement of the Ah receptor signal transduction pathway in CYP1A1 induction following suspension of epithelial cells, we analyzed wild-type Hepa 1c1c7 cells, a subclone of the Hepa-1c1 mouse hepatoma line, and two mutant Hepa 1c1c7 lines, Class I and Class II. Suspension of wild-type Hepa 1c1c7 cells for 4 h led to an induction of steady state levels of CYP1A1 mRNA, similar to that obtained following treatment of adherent cells with 10(-9) M 2,3,7,8-tetrachlorodibenzo-p-dioxin. Mutants of the Hepa 1c1c7 cells defective in different aspects of the Ah receptor signal transduction pathway exhibited negligible (Class I) or no (Class II) suspension-mediated induction of CYP1A1 mRNA. Gel mobility shift analysis of nuclear extracts from suspended or 2,3,7,8-tetrachlorodibenzo-p-dioxin-treated wild-type cells showed that both treatments produced identical shifts in the mobility of an XRE-containing probe. Antibody supershift experiments confirmed that the Ah receptor was a component of the DNA-protein complex from suspended wild-type Hepa 1c1c7 cells. These data directly demonstrate that suspension of wild-type Hepa 1c1c7 cells leads to nuclear localization and activation of the Ah receptor to a DNA-binding form.
我们最近证实，正常人类上皮细胞从细胞-基质和/或细胞-细胞黏附中脱离会产生细胞信号，在没有外源性多环芳烃的情况下诱导CYP1A1的表达（萨德克，C.M.，和艾伦-霍夫曼，B.L.（1994年）《生物化学杂志》169，16067 - 16074）。为了直接检测上皮细胞悬浮后Ah受体信号转导途径在CYP1A1诱导中的作用，我们分析了野生型Hepa 1c1c7细胞（Hepa - 1c1小鼠肝癌细胞系的一个亚克隆）以及两个突变的Hepa 1c1c7细胞系，I类和II类。野生型Hepa 1c1c7细胞悬浮4小时导致CYP1A1 mRNA稳态水平的诱导，类似于用10⁻⁹ M 2,3,7,8 - 四氯二苯并 - p - 二噁英处理贴壁细胞后获得的水平。在Ah受体信号转导途径不同方面存在缺陷的Hepa 1c1c7细胞突变体，表现出可忽略不计（I类）或没有（II类）悬浮介导的CYP1A1 mRNA诱导。对悬浮或经2,3,7,8 - 四氯二苯并 - p - 二噁英处理的野生型细胞的核提取物进行凝胶迁移率变动分析表明，两种处理在含XRE探针的迁移率上产生相同的变动。抗体超迁移实验证实，Ah受体是悬浮的野生型Hepa 1c1c7细胞核蛋白复合物的一个组成部分。这些数据直接表明，野生型Hepa 1c1c7细胞的悬浮导致Ah受体核定位并激活为DNA结合形式。
