Cytochrome P450 3A4 Promoter Research Articles

No Influence of the MDR-1 C3435T Polymorphism or a CYP3A4 Promoter Polymorphism (CYP3A4-V Allele) on Dose-adjusted Cyclosporin A Trough Concentrations or Rejection Incidence in Stable Renal Transplant Recipients

A substantial proportion of the variability in the absorption and clearance of cyclosporin A (CsA) after oral administration has been attributed to variability in liver cytochrome P-450 3A4 (CYP3A4) activity and intestinal P-glycoprotein (P-gp) concentration. A polymorphism in the CYP3A4 promoter region, termed "variant" allele CYP3A4-V, was postulated to be associated with altered CYP3A4 enzyme activity. A polymorphism in exon 26 (C3435T) of the multidrug resistance-1 (MDR-1) gene was correlated with intestinal expression and in vivo activity of P-gp. We investigated the occurrence of both polymorphisms in 124 stable Caucasian renal transplant recipients (>6 months after transplantation) on CsA as the primary immunosuppressant. Real-time, rapid-cycle PCR methods were developed and used for genotyping. The estimated allele frequencies for the MDR-1 C3435T allele (54%) and the CYP3A4-V allele (4.8%) were similar to those reported for Caucasian populations. No significant differences were found for the CsA doses needed to maintain similar CsA trough concentrations in patients with and without the CYP3A4-V allele or in patients with different MDR-1 C3435T genotypes. Furthermore, neither of the polymorphisms investigated was associated with renal function as assessed by creatinine plasma concentration or, in a retrospective analysis, the incidence of acute rejection. These findings suggest that the MDR-1 C3435T mutation and the CYP3A4-V variant are not major determinants of CsA efficacy in renal transplant recipients.

Human arylhydrocarbon receptor repressor (AHRR) gene: genomic structure and analysis of polymorphism in endometriosis.

The diversity of biological effects resulting from exposure to dioxin may reflect the ability of this environmental pollutant to alter gene expression by binding to the arylhydrocarbon receptor (AHR) gene and related genes. AHR function may be regulated by structural variations in AHR itself, in the AHR repressor (AHRR), in the AHR nuclear translocator (ARNT), or in AHR target molecules such as cytochrome P-4501A1 (CYP1A1) and glutathione S-transferase. Analysis of the genomic organization of AHRR revealed an open reading frame consisting of a 2094-bp mRNA encoded by ten exons. We found one novel polymorphism, a substitution of Ala by Pro at codon 185 (GCC to CCC), in exon 5 of the AHRR gene; among 108 healthy unrelated Japanese women, genotypes Ala/Ala, Ala/Pro, and Pro/Pro were represented, respectively, by 20 (18.5%), 49 (45.4%), and 39 (36.1%) individuals. We did not detect previously published polymorphisms of ARNT (D511N) or the CYP1A1 promoter (G-469A and C-459T) in our subjects, suggesting that these polymorphisms are rare in the Japanese population. No association was found between uterine endometriosis and any polymorphisms in the AHRR, AHR, ARNT, or CYP1A1 genes analyzed in the present study.

Failure of Ah Receptor to Mediate Induction of Cytochromes P450 in the CYP1 Family in the Human Hepatoma Line SK-Hep-1

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 [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 ∼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 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 μM 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.

The flavonoid galangin is an inhibitor of CYP1A1 activity and an agonist/antagonist of the aryl hydrocarbon receptor.

The effect of the dietary flavonoid galangin on the metabolism of 7,12-dimethylbenz[a]anthracene (DMBA), the activity of cytochrome P 450 1A1 (CYP1A1), and the expression of CYP1A1 in MCF-7 human breast carcinoma cells was investigated. Galangin inhibited the catabolic breakdown of DMBA, as measured by thin-layer chromatography, in a dose-dependent manner. Galangin also inhibited the formation of DMBA-DNA adducts, and prevented DMBA-induced inhibition of cell growth. Galangin caused a potent, dose-dependent inhibition of CYP1A1 activity, as measured by ethoxyresorufin-O-deethylase activity, in intact cells and in microsomes isolated from DMBA-treated cells. Analysis of the inhibition kinetics by double-reciprocal plot demonstrated that galangin inhibited CYP1A1 activity in a non-competitive manner. Galangin caused an increase in the level of CYP1A1 mRNA, indicating that it may be an agonist of the aryl hydrocarbon receptor, but it inhibited the induction of CYP1A1 mRNA by DMBA or by 2,3,5,7-tetrachlorodibenzo-p-dioxin (TCDD). Galangin also inhibited the DMBA- or TCDD-induced transcription of a reporter vector containing the CYP1A1 promoter. Thus, galangin is a potent inhibitor of DMBA metabolism and an agonist/antagonist of the AhR, and may prove to be an effective chemopreventive agent. © 1999 Cancer Research Campaign

The gut-enriched Krüppel-like factor (GKLF) suppresses the promoter activity of the cytochrome P450 1A1 (CYP1A1) gene in a SP1-dependent fashion

Background: GKLF is a recently identified transcription factor that contains 3 C2H 2 zinc fingers with homology to the Drosophila Kriippel protein [Shields, Christy and Yang (1996) J. Biol. Chem. 271, 20009; Shields and Yang (1997) J. Biol. Chem. 277~2 18504]. Expression of GKLF is enriched in the gut epithelium and is associated with growth arrest. Recent studies have identified a consensus DNA sequence to which GKLF binds [Shields and Yang (1998) Nucl. Acids Res., in press]. This sequence is very similar to the basal transcription dement (BTE) this is essential for the promoter activity of many genes in the cytochrome P450 family, including CYP1A1. Aim: The aim of the present study is to investigate whether GKLF is involved in the regulation of the CYP1A1 promoter through the BTE. Methods: The binding of GKLF to BTE was analyzed by methylation interference and electrophoretic mobility shift assays (EMSA) using partially purified recombinant GKLF and a radioactively labeled double-stranded oligonucleotide containing the GKLF consensus binding sequence (named TDA) or BTE. The effect of GKLF on CYP1A1 promoter activity was examined by cotransfection experiments in Chinese hamster ovary (CHO) cells with an eukaryotic expression construct containing full-length or truncated GKLF and a chloramphenicol acetyl transferase (CAT) reporter gene driven by the CYP1A1 Y-flanking sequence including or not BTE. Results: EMSA of labeled TDA and BTE using recombinant GKLF demonstrated the formation of a single, identical DNA-protein complex. The minimal essential DNA sequences within TDA and BTE that bound GKLF were identical as defined by base-specific mutagenesis and methylation interference assays. In transiently transfected CHO cells, an expression construct containing full-length but not truncated GKLF was able to suppress, in a dose-dependent fashion, the activity of the CAT reporter driven by the wild-type CYP1A1 promoter but not by a mutated promoter in which the BTE was specifically deleted. As a deletion of the BTE from the CYP1A1 promoter also resulted in a significant decrease in the basal promoter activity, nuclear extracts from CHO cells were prepared and analyzed by EMSA. The results indicate that the primary protein in CHO cells binding to BTE was Spl as demonstrated by antibody super-shift experiments. Cotransfection of the CYP1A1 promoter-reporters with an expression construct containing Spl showed that Spl transactivated the CYP1A1 promoter in a BTE-dependent fashion. The further addition of GKLF to the above transfection condition resulted in a dose-dependent abrogation of the ability of Spl to activate CYP1A1 promoter. Conclusion: The results indicate that GKLF is an GASTROENTEROLOGY Vol. 114, No. 4

Regulation of Promoter-CAT Stress Genes in HepG2 Cells by Suspensions of Particles from Ambient Air

A panel of HepG2-derived cell lines (CAT-Tox [L] assay, Xenometrix), harboring stress genes consisting of a sequence for chloramphenicol acetyltransferase (CAT) under the transcriptional regulation from mammalian promoters and response elements, was exposed for 18-24 hr to aqueous suspensions of urban dusts (SRM-1648, SRM-1649, EHC-93) or PM2.5 particles (particulate matter < 2.5 micron). Expression of CAT protein was measured by enzyme-linked immunosorbent assay. Induction of the CAT genes was verified with benzo[a]pyrene (CYP1A1, cytochrome P450 1A1 promoter; GSTYa, glutathione transferase subunit Ya promoter; XRE, xenobiotic response element), cadmium sulfate, and copper sulfate (HMTIIa, metallothionein IIa promoter; HSP70, heat shock protein 70 promoter). The urban dust suspensions were active on CYP1A1, GSTYa, and XRE cell lines. SRM-1648 and SRM-1649 were twice as potent as EHC-93 per unit mass in inducing the xenobiotic-dependent responses, which correlated with contents in polycyclic aromatic hydrocarbons. These three reference particles, as well as six PM2.5 preparations collected on hi-vol filters in the Great Lakes basin, were also found to induce HMTIIa and HSP70, the magnitude of the responses correlating closely with the amount of soluble copper in the particulate preparations. The results indicate that bioavailable chemical species in the unfractionated particles can directly and quantitatively induce xenobiotic, metal, and stress-dependent responses in a target cell model, resulting in patterns of gene induction consistent with the chemical compositions of the environmental materials. We propose that cell culture models could be helpful for toxicodynamic inferences in adjunct to environmental monitoring and exposure assessments.

Cytochrome P450 1A1 promoter as a genetic switch for the regulatable and physiological expression of a plasma protein in transgenic mice.

Transgenic and gene knockout techniques allow for in vivo study of the consequences of adding or subtracting specific genes. However, in some instances, such as the study of lethal mutations or of the physiological consequences of changing gene expression, turning on and off an introduced gene at will would be advantageous. We have used cytochrome p450 1A1 promoter to drive expression of the human apolipoprotein E (apoE) gene in transgenic mice. In six independent lines, robust expression of the transgene depended upon injection of the inducer beta-naphthoflavone, whereas the seventh line had high basal expression that was augmented further by the inducer. The low level of basal expression in an inducer-dependent line was confirmed upon breeding the transgene onto the hypercholesterolemic apoE-deficient background. In the basal state transgene expression was physiologically insignificant, as these mice were as hypercholesterolemic as their nontransgenic apoE-deficient littermates. When injected with the inducer, plasma cholesterol levels of the transgenic mice decreased dramatically as apoE expression was induced to yield greater than physiological levels in plasma. The inducer could pass transplacentally from an injected mother to her fetuses with concomitant induction of fetal transgene mRNA. Inducer could also pass via breast milk from an injected mother to her suckling neonatal pups, giving rise to the induction of human apoE in neonate plasma. These finding suggest a strategy to temporarily ameliorate genetic deficiencies that would otherwise lead to fetal or neonatal lethality.