Failure of Ah receptor to mediate induction of cytochromes P450 in the CYP1 family in the human hepatoma line SK-Hep-1

摘要

The Ah receptor mediates the induction of cytochrome P450 1A1 (CYP1A1) and toxicities of 2,3,7,8-tetrachlorodibanzo-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 [H-3]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 similar to 9000, 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 K-d 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 SH-Hep-1 cells or from cells treated for 24 h with 13 muM 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. (C) 2000 Academic Press. [References: 44]