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Stable expression of mouse Cyp1a1 and human CYP1A2 cDNAs transfected into mouse hepatoma cells lacking detectable
A Puga1, B Raychaudhuri, K Salata
1Laboratory of Developmental Pharmacology, National Institute of Child Health and Human Development, Bethesda, MD 20892.
Abstract:
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.
Insights
Researchers developed cell lines to study cytochrome P450 enzymes (Cyp1a1 and CYP1A2). The aromatic hydrocarbon-responsive domain (AhRD) is crucial for functional Cyp1a1 enzyme activity and may regulate its own gene expression.
Area of Science:
- Molecular Biology
- Biochemistry
- Pharmacology
Background:
- The Hepa-1c1c7 c37 mutant cell line has negligible benzo[a]pyrene hydroxylase (Cyp1a1) and acetanilide 4-hydroxylase (Cyp1a2) activities.
- Understanding the regulation and function of cytochrome P450 enzymes is critical for assessing cytotoxicity, mutagenesis, and carcinogenesis.
Purpose of the Study:
- To develop stable transfectants expressing murine Cyp1a1 and human CYP1A2 cDNAs in the c37 mutant cell line.
- To compare the efficacy of various promoter and enhancer sequences in driving the expression of these P450 enzymes.
- To investigate the role of the aromatic hydrocarbon-responsive domain (AhRD) in Cyp1a1 and CYP1A2 activity and regulation.
Main Methods:
- Stable transfection of Hepa-1c1c7 c37 cells with plasmids encoding Cyp1a1 and CYP1A2.
- Utilized ethoxyfluorescein ethyl ester (EFEE) metabolism assay for screening high Cyp1a1 activity.
- Compared nine plasmid constructs with different promoter/enhancer elements, including AhRD, MMTV LTR, SV40, and HSV-1.
Main Results:
- Only constructs containing the AhRD yielded high Cyp1a1 enzyme activity.
- High CYP1A2 activity was achieved with plasmids containing the HSV-1 enhancer and AhRD.
- Expression of exogenous Cyp1a1 or CYP1A2 reduced endogenous Cyp1a1 mRNA levels and restored inducibility by TCDD, suggesting an autoregulatory loop.
Conclusions:
- The AhRD is essential for inducing functional Cyp1a1 enzyme activity, potentially via a post-transcriptional mechanism.
- The functional products of Cyp1a1 or CYP1A2 may participate in an autoregulatory feedback loop controlling constitutive Cyp1a1 gene expression.
- The developed cell lines are valuable tools for studying the roles of Cyp1a1 and CYP1A2 in cytotoxicity, mutagenesis, and carcinogenesis.