Abstract:
:The Ah receptor mediates the induction of cytochrome P450 1A1 (CYP1A1) and toxicities of 2,3,7,8tetrachlorodibanzo-p-dioxin (TCDD). It has been detected in tissues of many species and in murine and human hepatoma lines. We show that the human hepatoma line SK-Hep-1 has cytosolic Ah receptor detectable by specific binding of [3H]TCDD. Concentrations of Ah receptor were low (mean = 43 +/- 3 fmol/mg cytosol protein compared to 430 fmol/mg protein in Hepa-1); the estimated number of receptor sites per cell is approximately 9,000, compared to 35,000 in Hepa-1. Ah receptor in SK-Hep-1 cells was physicochemically similar to Ah receptor in C57BL/6 mouse liver and in other human hepatoma lines studied to date except that binding affinity for TCDD, the most avidly bound ligand, was lower (estimated Kd was 14 nM by Woolf plot analysis). Translocation of the Ah receptor-ligand complex to the nucleus was shown; binding of the activated Ah receptor-ligand complex to an XRE in the 5'-upstream region of the CYP1A1 gene was demonstrated by gel-shift analysis. However, after SK-Hep-1 cells were incubated with typical PAHs including 3-methylcholanthrene, benzanthracene, and dibenz(a,h)anthracene, each over a wide range of concentrations, no induction of aryl hydrocarbon hydroxylase activity was detectable. On Northern analysis, no message for human CYP1A1 was detected in mRNA prepared from noninduced SK-Hep-1 cells or from cells treated for 24 h with 13 microM dibenz(a,h)anthracene. Further analysis by RT-PCR did not detect the induction of CYP1A1, CYP1A2, or CYP1B1 message in response to 10(-7) M TCDD, 10(-5) M benzanthracene, or 10(-5) M 3-methylcholanthrene. Transient transfection of reporter constructs containing either a minimal promoter or the CYP1A1 promoter fused to a reporter gene (luciferase) did not show any expression in response to increasing concentrations of TCDD up to 10(-8) M. Estimation of the size of the transcripts for AhR and ARNT protein revealed normal sizes, 2.7 and 2.4 kb, respectively. Together, these data suggest that SK-Hep-1 cells express an Ah receptor defective at the level of trans-activation of gene expression. SK-Hep-1 is the first human hepatoma line described with a demonstrable defect in CYP1A1 or its regulation.
journal_name
Arch Biochem Biophysjournal_title
Archives of biochemistry and biophysicsauthors
Roberts EA,Harper PA,Wong JM,Wang Y,Yang Sdoi
10.1006/abbi.2000.2059subject
Has Abstractpub_date
2000-12-01 00:00:00pages
190-8issue
1eissn
0003-9861issn
1096-0384pii
S0003-9861(00)92059-9journal_volume
384pub_type
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