Abstract
Cytochrome P-450 (CYP) 1B1 expression in mouse hepatoma (Hepa-1) wild-type (WT) cells was compared with responses in Hepa-1 variants LA1 and LA2, which, respectively, exhibit low aryl hydrocarbon receptor (AhR) level and defective AhR nuclear translocator (ARNT) protein. 10T1/2 mouse embryo fibroblasts express predominantly CYP1B1 and at a 100 times higher level than in Hepa-1 cells, whereas they express about 300-fold lower CYP1A1 than Hepa-1 cells. The expression of CYP1B1 in WT and LA1 variant, although at a much lower level, follows that of CYP1A1, reflecting their common regulation through the AhR. The LA2 (ARNT-defective) cells showed a major difference between CYP1B1 and CYP1A1 expression. Although CYP1A1 mRNA levels in LA2 were extremely low and unresponsive to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), basal CYP1B1 mRNA and protein were expressed at levels similar to those seen in TCDD-induced WT. The elevated basal CYP1B1 mRNA in LA2 cells decreased by 50% after transient transfection of ARNT cDNA, in parallel with substantial restoration of CYP1A1 induction. This implicates ARNT as a suppressor of CYP1B1 basal expression in Hepa cells. In transient CYP1B1-luciferase constructs in LA2 cells, ARNT shows stimulatory effects in the enhancer region but an inhibitory effect on the proximal promoter. Two CYP1B1 enhancer elements [xenobiotic-responsive element (XRE) 1/2 and XRE4] formed TCDD-unresponsive complexes of similar mobility to TCDD-stimulated AhR-ARNT complex with XRE5. However, because these two complexes were formed to the same extent in LA2 as in WT cells, they cannot be due to ARNT or contribute to ARNT-regulated suppression.
Footnotes
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Send reprint requests to: Dr. Sakina E. Eltom, Department of Pharmacology, University of Wisconsin Medical School, 1300 University Ave., Madison, WI 53706. E-mail:seeltom{at}facstaff.wisc.edu
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This work was supported by National Institutes of Health Grant CA16265. S.E.E. is supported by Grant T32 ES07015 from the National Institute of Environmental Health Sciences, National Institutes of Health and Contribution #323; Environmental Toxicology Center, University of Wisconsin, Madison, WI 53706. Part of this work was presented at the XI International Symposium on Microsomes and Drug Oxidations, Los Angeles, CA, July 21–24, 1996, and part of it was presented at the 37th Society of Toxicology Annual Meeting, Seattle, WA, March 1–5, 1998.
- Abbreviations:
- PAH
- polycyclic aromatic hydrocarbon
- AhR
- aryl hydrocarbon receptor
- ARNT
- AhR nuclear-translocating protein
- Hepa-1
- mouse hepatoma cell line
- WT
- wild type
- LA1
- low aryl hydrocarbon hydroxylase activity, class I variant
- LA2
- low aryl hydrocarbon hydroxylase activity, class II variant
- CYP
- cytochrome P-450
- TCDD
- 2,3,7,8-tetrachlorodibenzo-p-dioxin
- BA
- benz[a]anthracene
- DMBA
- 7,12-dimethylbenz[a]anthracene
- RT-PCR
- reverse transcription-polymerase chain reaction
- GAPDH
- glyceraldehyde-3-phosphate dehydrogenase
- ECL
- enhanced chemiluminescence
- XRE
- xenobiotic-responsive element
- EMSA
- electrophoretic mobility-shift assay
- bHLH
- basic helix-loop-helix
- DTT
- dithiothreitol
- Received April 27, 1998.
- Accepted November 19, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
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