Ciaccio P J, Jaiswal A K, Tew K D
Department of Pharmacology, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111.
J Biol Chem. 1994 Jun 3;269(22):15558-62.
A human oxidoreductase (H-37) that is overexpressed in ethacrynic acid-resistant HT29 colon cells (Ciaccio, P. J., Stuart, J.E., and Tew, K.D. (1993) Mol. Pharmacol. 43, 845-853) has been identified as a dihydrodiol dehydrogenase. Translated protein from a dihydrodiol dehydrogenase cDNA isolated from a library prepared from ethacrynic acid-resistant HT29 cell poly(A+) RNA was recognized by anti-H-37 IgG and was identical in molecular weight with H-37. The isolated cDNA was identical in both nucleotide and amino acid sequences with the recently cloned liver dihydrodiol dehydrogenase (Stolz, A., Hammond, L., Lou, H., Takikawa, H., Ronk, M., and Shively, J.E. (1993) J. Biol. Chem. 268, 10448-10457). Using this cDNA as probe, we have examined its induction by Michael acceptors. The steady state dihydrodiol dehydrogenase mRNA level in the ethacrynic acid-resistant line was increased 30-fold relative to that of wild-type cells. Twenty-four hour treatment of wild-type cells with ethacrynic acid or dimethyl maleate increased mRNA 10-fold and 5-fold, respectively. These changes are accompanied by both increased protein expression and increased NADP-dependent 1-acenaphthenol oxidative activity in cell cytosol. In gel shift assays, compared to wild type controls, increased binding of NAD(P)H quinone oxidoreductase human antioxidant response element (hARE) DNA to redox labile protein complexes present in treated and resistant cell nuclear extract was observed. Ethacrynic acid induced CAT activity 2-fold in Hepa1 cells stably transfected with NAD(P)H quinone oxidoreductase hARE-tk-CAT chimeric gene construct. Thus, dihydrodiol dehydrogenase protein is inducible by de novo synthesis from mRNA by structurally related monofunctional inducer Michael acceptors. Altered in vitro binding of nuclear protein to the hARE is indirect evidence for the involvement of an element similar to hARE in the regulation of dihydrodiol dehydrogenase by these agents.
一种在对依他尼酸耐药的HT29结肠癌细胞中过表达的人氧化还原酶（H-37）（恰乔，P.J.，斯图尔特，J.E.，以及图，K.D.（1993年）《分子药理学》43卷，第845 - 853页）已被鉴定为二氢二醇脱氢酶。从由对依他尼酸耐药的HT29细胞多聚腺苷酸加尾RNA制备的文库中分离出的二氢二醇脱氢酶cDNA翻译的蛋白质能被抗H-37 IgG识别，并且分子量与H-37相同。分离出的cDNA在核苷酸和氨基酸序列上与最近克隆的肝脏二氢二醇脱氢酶（斯托尔兹，A.，哈蒙德，L.，娄，H.，泷川，H.，罗恩克，M.，以及什利夫利，J.E.（1993年）《生物化学杂志》268卷，第10448 - 10457页）相同。以该cDNA为探针，我们研究了其受迈克尔受体诱导的情况。相对于野生型细胞，对依他尼酸耐药细胞系中二氢二醇脱氢酶mRNA的稳态水平增加了30倍。用依他尼酸或马来酸二甲酯对野生型细胞处理24小时，mRNA分别增加了10倍和5倍。这些变化伴随着细胞胞质溶胶中蛋白质表达增加以及NADP依赖的1 - 苊醇氧化活性增强。在凝胶迁移实验中，与野生型对照相比，观察到在处理过的和耐药细胞核提取物中，NAD(P)H醌氧化还原酶人抗氧化反应元件（hARE）DNA与氧化还原不稳定蛋白复合物的结合增加。依他尼酸在稳定转染了NAD(P)H醌氧化还原酶hARE - tk - CAT嵌合基因构建体的Hepa1细胞中诱导CAT活性增加了2倍。因此，二氢二醇脱氢酶蛋白可通过与结构相关的单功能诱导剂迈克尔受体从头合成mRNA来诱导。核蛋白与hARE的体外结合改变是类似hARE的元件参与这些药物对二氢二醇脱氢酶调控的间接证据。
