Puga A, Raychaudhuri B, Salata K, Zhang Y H, Nebert D W
Laboratory of Developmental Pharmacology, National Institute of Child Health and Human Development, Bethesda, MD 20892.
DNA Cell Biol. 1990 Jul-Aug;9(6):425-36. doi: 10.1089/dna.1990.9.425.
Using the mouse hepatoma Hepa-1c1c7 c37 mutant cell line that exhibits negligible benzo[a]pyrene hydroxylase (Cyp1a1) and acetanilide 4-hydroxylase (Cyp1a2) enzyme activities, we developed stable transfectants of plasmids containing the murine Cyp1a1 (cytochrome P(1)450) and the human CYP1A2 (P(3)450) cDNAs. We show that the assay measuring metabolism of ethoxyfluorescein ethyl ester (EFEE) was invaluable in screening large numbers of individual cell lines for high Cyp1a1 enzyme activity. Nine different plasmid constructs containing various combinations of promoter and enhancer sequences were compared, including: the Drosophila heat shock promoter, the mouse mammary tumor virus long terminal repeat (MMTV LTR) carrying the glucocorticoid-responsive element (GRE), enhancer sequences from simian virus 40 (SV40) and herpes simplex virus type 1 (HSV-1), and the aromatic hydrocarbon-responsive domain (AhRD) of the murine Cyp1a1 gene. Interestingly, only those constructs containing the AhRD produced high levels of Cyp1a1 enzyme activity. In contrast, high levels of CYP1A2 activity were obtained with plasmids carrying the HSV-1 enhancer, as well as the AhRD. These studies suggest that the AhRD, which responds to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), provides a post-transcriptional signal necessary for the induction of functional Cyp1a1 enzyme activity. Although untransfected c37 cells exhibit markedly elevated levels of endogenous Cyp1a1 mRNA, the expression of exogenous Cyp1a1 or CYP1A2 enzyme activity in these cells decreases the concentration of this endogenous Cyp1a1 mRNA to negligible levels and restores Cyp1a1 mRNA inducibility by TCDD; these data indicate that the functional product of either the Cyp1a1 gene or the CYP1A2 gene might have a role in an autoregulatory loop controlling the constitutive expression of the Cyp1a1 gene. The cell lines described herein should be valuable in assessing the contribution of these two P450 enzymes to the processes of cytotoxicity, mutagenesis, and carcinogenesis.
我们使用了苯并[a]芘羟化酶（Cyp1a1）和乙酰苯胺4-羟化酶（Cyp1a2）酶活性可忽略不计的小鼠肝癌Hepa-1c1c7 c37突变细胞系，构建了含有小鼠Cyp1a1（细胞色素P(1)450）和人CYP1A2（P(3)450）cDNA的质粒稳定转染细胞系。我们发现，用于检测乙氧基荧光素乙酯（EFEE）代谢的试验对于筛选大量具有高Cyp1a1酶活性的单个细胞系非常有价值。我们比较了九种不同的含有启动子和增强子序列不同组合的质粒构建体，包括：果蝇热休克启动子、携带糖皮质激素反应元件（GRE）的小鼠乳腺肿瘤病毒长末端重复序列（MMTV LTR）、猿猴病毒40（SV40）和单纯疱疹病毒1型（HSV-1）的增强子序列，以及小鼠Cyp1a1基因的芳烃反应域（AhRD）。有趣的是，只有那些含有AhRD的构建体产生了高水平的Cyp1a1酶活性。相反，携带HSV-1增强子以及AhRD的质粒获得了高水平的CYP1A2活性。这些研究表明，对2,3,7,8-四氯二苯并对二恶英（TCDD）有反应的AhRD提供了诱导功能性Cyp1a1酶活性所需的转录后信号。尽管未转染的c37细胞内源性Cyp1a1 mRNA水平显著升高，但这些细胞中外源Cyp1a1或CYP1A2酶活性的表达将这种内源性Cyp1a1 mRNA的浓度降低到可忽略不计的水平，并恢复了TCDD对Cyp1a1 mRNA的诱导能力；这些数据表明，Cyp1a1基因或CYP1A2基因的功能性产物可能在控制Cyp1a1基因组成型表达的自动调节回路中起作用。本文所述的细胞系对于评估这两种P450酶在细胞毒性、诱变和致癌过程中的作用应该是有价值的。
