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PubMed2002

Core-binding factor beta interacts with Runx2 and is required for skeletal development.

Core-binding factor beta (CBFbeta, also called polyomavirus enhancer binding protein 2beta (PEBP2B)) is associated with an inversion of chromosome 16 and is associated with acute myeloid leukemia in humans. CBFbeta forms a heterodimer with RUNX1 (runt-related transcription factor 1), which has a DNA binding domain homologous to the pair-rule protein runt in Drosophila melanogaster. Both RUNX1 and CBFbeta are essential for hematopoiesis. Haploinsufficiency of another runt-related protein, RUNX2 (also called CBFA1), causes cleidocranial dysplasia in humans and is essential in skeletal development by regulating osteoblast differentiation and chondrocyte maturation. Mice deficient in Cbfb (Cbfb(-/-)) die at midgestation, so the function of Cbfbeta in skeletal development has yet to be ascertained. To investigate this issue, we rescued hematopoiesis of Cbfb(-/-) mice by introducing Cbfb using the Gata1 promoter. The rescued Cbfb(-/-) mice recapitulated fetal liver hematopoiesis in erythroid and megakaryocytic lineages and survived until birth, but showed severely delayed bone formation. Although mesenchymal cells differentiated into immature osteoblasts, intramembranous bones were poorly formed. The maturation of chondrocytes into hypertrophic cells was markedly delayed, and no endochondral bones were formed. Electrophoretic mobility shift assays and reporter assays showed that Cbfbeta was necessary for the efficient DNA binding of Runx2 and for Runx2-dependent transcriptional activation. These findings indicate that Cbfbeta is required for the function of Runx2 in skeletal development.

Animals↗
PubMed2000

An allelic variation in the human prodynorphin gene promoter alters stimulus-induced expression.

Prodynorphin, the precursor of the dynorphin opioid peptides, has been shown to play an important role in several aspects of human diseases and complex traits, e.g., drug abuse, epilepsy, and mood disorders. The objective of this study was to identify polymorphisms in the 5' control region of the human prodynorphin gene and to relate these polymorphisms to prodynorphin gene expression. Within the core promoter region, a 68-bp sequence was found to occur as a polymorphic element, either singular or as tandemly repeated element two, three, or four times. This 68-bp repeat element contains an AP-1 transcription factor binding site as demonstrated by electrophoretic mobility shift assay. Reporter gene assays were performed and provided evidence for allele dependent different promoter activity. Dynorphin was found to be involved in many pathophysiological processes so that the described prodynorphin alleles may correlate with the occurrence of several diseases, for example, drug addiction. However, prodynorphin allelic distributions were not significantly different in heroin addicts and control subjects.

Alleles↗
PubMed2004

DNA from probiotic bacteria modulates murine and human epithelial and immune function.

BACKGROUND & AIMS: The intestinal epithelium must discriminate between pathogenic and nonpathogenic bacteria and respond accordingly. The aim of this study was to examine whether bacterial DNA can serve as the molecular basis for bacterial recognition. METHODS: HT-29 monolayers were treated with various bacterial DNA and interleukin (IL)-8 secretion measured by enzyme-linked immunosorbent assay, nuclear factor kappaB activation by electrophoretic mobility shift assay and reporter assays, and IkappaB levels by Western blotting. Cytokine secretion in response to bacterial DNA was measured in murine colonic segments and splenocytes. IL-10-deficient mice were fed DNA from VSL probiotic compound daily for 2 weeks. Colons were removed and analyzed for cytokine production and inflammation. RESULTS: HT-29 cells responded with IL-8 secretion to bacterial DNA in a differential manner. In the presence of proinflammatory stimuli, VSL3 DNA inhibited IL-8 secretion, reduced p38 mitogen-activated protein kinase activation, delayed nuclear factor kappaB activation, stabilized levels of IkappaB, and inhibited proteasome function. VSL3 DNA inhibited colonic interferon (IFN)-gamma secretion in mouse colons and also attenuated a Bacteroides vulgatus-induced IFN-gamma release from murine splenocytes. In mice, VSL3 DNA attenuated a systemic release of tumor necrosis factor alpha in response to Escherichia coli DNA injection. Treatment of IL-10-deficient mice with oral VSL3 DNA resulted in a reduction in mucosal secretion of tumor necrosis factor alpha and IFN-gamma and an improvement in histologic disease. CONCLUSIONS: DNA from probiotic bacteria can limit epithelial proinflammatory responses in vivo and in vitro. Systemic and oral administration of VSL3 DNA ameliorates inflammatory responses.

Animals↗
PubMed2004

KLF11-mediated repression antagonizes Sp1/sterol-responsive element-binding protein-induced transcriptional activation of caveolin-1 in response to cholesterol signaling.

Cholesterol is a potent regulator of gene expression via a canonical pathway co-regulated by SREBP and Sp1. Here we establish the caveolin-1 gene promoter as a cell type-specific model for SREBP/Sp1 regulation whereby lipoprotein cholesterol depletion activates caveolin-1 transcription in endothelial type cells, but not in fibroblasts, both in vitro and in vivo. By extending this model, we describe a novel pathway distinct from the prototypical SREBP/Sp1 regulatory loop involving the Sp1-like protein, KLF11. Through a combination of RNA interference, chromatin immunoprecipitation assays, electrophoretic mobility shift assays, and reporter assays, we demonstrate that in the presence of cholesterol, KLF11 acts as a dominant repressor of the caveolin-1 gene. Mechanistically, cholesterol depletion results in displacement of KLF11 from an Sp1 site flanking an SRE, indicating that activation by SREBP/Sp1 requires antagonism of KLF11 repression. The displacement of KLF11 results from both a down-regulation of its expression and competition by Sp1 for DNA binding. Therefore, these studies identify a novel pathway whereby KLF11 repression is coordinated with Sp1/SREBP activation of cholesterol-dependent gene expression in a cell type-specific manner and outline the mechanisms by which these functions are achieved.

Animals↗
PubMed2006

Distinct structural elements in the first membrane-spanning segment of the epithelial sodium channel.

Epithelial Na+ channels (ENaCs) comprise three subunits that have been proposed to be arranged in either an alpha2betagamma or a higher ordered configuration. Each subunit has two putative membrane-spanning segments (M1 and M2), intracellular amino and carboxyl termini, and a large extracellular loop. We have used the TOXCAT assay (a reporter assay for transmembrane segment homodimerization) to identify residues within the transmembrane segments of ENaC that may participate in important structural interactions within ENaC, with which we identified a candidate site within alphaM1. We performed site-directed mutagenesis at this site and found that, although the mutants reduced channel activity, ENaC protein expression at the plasma membrane was unaffected. To deduce the role of alphaM1 in the pore structure of ENaC, we performed tryptophan-scanning mutagenesis throughout alphaM1 (residues 110-130). We found that mutations within the amino-terminal part of alphaM1 had effects on activity and selectivity with a periodicity consistent with a helical structure but no effect on channel surface expression. We also observed that mutations within the carboxyl-terminal part of alphaM1 had effects on activity and selectivity but with no apparent periodicity. Additionally, these mutants reduced channel surface expression. Our data support a model in which the amino-terminal half of alphaM1 is alpha-helical and packs against structural element(s) that contribute to the ENaC pore. Furthermore, these data suggest that the carboxyl-terminal half of alphaM1 may be helical or assume a different conformation and may be involved in tertiary interactions essential to proper channel folding or assembly. Together, our data suggest that alphaM1 is divided into two distinct regions.

Amino Acid Sequence↗
PubMed2002

Deletion of dinucleotide repeat (Delta 14 allele) in the methylthioadenosine phosphorylase (MTAP) promoter and the allelotype of MTAP promoter in the Japanese population.

5'-deoxy-5'-methylthioadenosine phosphorylase (MTAP) is an enzyme involved in purine and polyamine metabolism and is ubiquitously expressed in normal human tissues and cells. However, this enzyme has been found to be deficient in a variety of human cancers. Although the enzyme deficiency is known to be caused by MTAP gene deletion, human diffuse histiocytic lymphoma cell line DHL-9 without any detectable MTAP activity has been found to possess the intact MTAP gene. These lines of evidence suggested that promoter abnormality might cause the MTAP deficiency in DHL-9. Therefore, we analyzed the MTAP promoter region of DHL-9 and found the deletion of 14 bases in its sequence. We designated the allele lacking (GT)(6)GC as dinucleotide repeat deletion (Delta 14 allele) and determined the effect of the Delta 14 allele on the MTAP promoter activity by a luciferase reporter assay. We have also analyzed the distribution of the Delta 14 allele and wild-type (WT) allele in the Japanese population by PCR assay. A reporter plasmid harboring the Delta 14 allele exhibited luciferase activity comparable to that of a plasmid containing the WT allele. Forty-six (22%) out of 210 people were homozygous for WT allele in the MTAP promoter, whereas 43 (20.5%) were homozygous for Delta 14 allele. The remaining 121 people (57.5%) possessed Delta 14/WT alleles in the MTAP promoter region. These results indicated that the Delta 14 allele has nothing to do with MTAP deficiency in DHL-9. The Delta 14 allele is distributed among the general population irrespective of gender.

Alleles↗
PubMed2005

Regulation of expression of the Epstein-Barr virus BamHI-A rightward transcripts.

The Epstein-Barr virus (EBV) BamHI-A rightward transcripts, or BARTs, are a family of mRNAs expressed in all EBV latency programs, including EBV-infected B cells in healthy carriers. Despite their ubiquitous expression, the regulation and biological function of BARTs are still unclear. In this study, the BART 5' termini were characterized by using a procedure that selects capped, full-length mRNAs. Two TATA-less promoter regions, designated P1 and P2, were mapped. P1 had relatively high basal activity in both epithelial and B cells, whereas P2 exhibited higher activity in epithelial cells. Upon EBV infection of B cells, transcription from P1 was detected soon after infection, while expression from P2 was delayed. Promoter-reporter assays in transiently transfected cells revealed that P1 and P2 were differentially regulated. Interferon regulatory factor 7 (IRF7) and IRF5 negatively regulated P1 activity. c-Myc and C/EBP family members positively regulated P2. Regulation of P2 by C/EBPs was characterized by electrophoretic mobility shift assay, chromatin immunoprecipitation, and reporter assays. More-abundant BART expression in epithelial cells correlated with the relative expression of positive and negative regulators in these cells.

B-Lymphocytes↗
PubMed2004

Emodin enhances arsenic trioxide-induced apoptosis via generation of reactive oxygen species and inhibition of survival signaling.

Although arsenic trioxide (As(2)O(3)) induces apoptosis in a relatively wide spectrum of tumors, the sensitivity of different cell types to this treatment varies to a great extent. Because reactive oxygen species (ROS) are critically involved in As(2)O(3)-induced apoptosis, we attempted to explore the possibility that elevating the cellular ROS level might be an approach to facilitate As(2)O(3)-induced apoptosis. Emodin, a natural anthraquinone derivative, was selected because its semiquinone structure is likely to increase the generation of intracellular ROS. Its independent and synergistic effects with As(2)O(3) in cytotoxicity were studied, and the plausible signaling mechanism was investigated in HeLa cells. Cell Proliferation Assay and flow cytometry were used to assess cell viability and apoptosis. Electrophoretic mobility shift assay, luciferase reporter assay, and Western blotting were performed to analyze signaling alteration. The results demonstrated that coadministration of emodin, at low doses of 0.5-10 micro M, with As(2)O(3) enhanced As(2)O(3)-rendered cytotoxicity on tumor cells, whereas these treatments caused no detectable proproliferative or proapoptotic effects on nontumor cells. ROS generation was increased, and activation of nuclear factor kappaB and activator protein 1 was suppressed by coadministration. All enhancements by emodin could be abolished by the antioxidant N-acetyl-L-cysteine. Therefore, we concluded that emodin sensitized HeLa cells to As(2)O(3) via generation of ROS and ROS-mediated inhibition on two major prosurvival transcription factors, nuclear factor kappaB and activator protein 1. This result allows us to propose a novel strategy in chemotherapy that uses mild ROS generators to facilitate apoptosis-inducing drugs whose efficacy depends on ROS.

Apoptosis↗
PubMed1996

Platelet-activating factor receptor. Gene structure and tissue-specific regulation.

The human platelet-activating factor receptor gene exists as a single copy on chromosome 1. Two 5'-noncoding exons (Exon 1 and 2) has distinct transcription initiation sites and promoters. These exons are alternatively spliced to a common splice acceptor site on exon 3 that contains a total coding regions. The transcript 1 is expressed ubiquitously with an emphasis of differentiated eosinophilic cell line (Eol-1), and leukocytes. On the other hand, the transcript 2 is expressed tissue-specifically. The latter is not expressed in leukocytes or brain. The transcript 1 has three tandem repeats of NF-kappa B, and SP-1 site, and responded to various inflammatory reagents including PAF itself, lipopolysaccharide, or phorbol ester. By northern blotting of tissue or cells with various nutritional or hormonal treatments, the PAF receptor messages are up-regulated. Estrogen increased the expression of the PAF receptor in human endometrial glandular cells, and vitamin A (retinoic acid) or thyroid hormone treatment up-regulates the PAF receptor expression only tissues with transcript 2 By various in vivo and in vitro transcriptional assays (CAT reporter assay, gel mobility shift assay), we identified estrogen responsible element, and hormone responsive element. The PAF receptor hormone responsive element is composed of three direct repeated TGACCT-like hexamer motifs with 2 and 4 bp spaces, and the two upstream and two downstream motifs were identified as response elements for RA and T.

Animals↗
PubMed2005

Androgen receptor activities of p,p'-DDE, fenvalerate and phoxim detected by androgen receptor reporter gene assay.

In this study, we have developed a transient human androgen receptor (hAR) reporter gene assay using African monkey kidney cell line CV-1. The assay displayed appropriate response to the known androgen receptor (AR) agonist 5alpha-dihydrotestosterone (DHT) and AR antagonist nilutamide. DHT induced AR-mediated transcriptional activity in a concentration-dependent manner with median effective concentration (EC(50)) value of 3.90 x 10(-10) M. Nilutamide exhibited potent antiandrogenic activity with median inhibitory concentration (IC(50)) value of 8.90 x 10(-7) M. The transcription could not be activated by glucocorticoid receptor (GR) agonist dexamethasone, which suggested that the assay system be highly specific to androgenic compounds without cross-talk to GR agonist. The assay shows acceptable repeatability to DHT with intra coefficient of variability (CV) of 9.3% and inter CV of 10.9%. We applied this assay to evaluate the androgenic and antiandrogenic activities of some pesticides including organochlorine p,p'-dichlorodiphenyldichloroethylene (DDE), pyrethroid fenvalerate and organophosphorus phoxim. The results showed that p,p'-DDE exhibited potent antiandrogenic activity. Fenvalerate acted as weak AR antagonist and phoxim did not show AR antagonistic activity. We failed to find AR agonistic effects of the three pesticides. The present study provided a promising tool to screen for AR agonists and antagonists.

Androgen Antagonists↗
PubMed2001

An in vitro reporter gene assay method incorporating metabolic activation with human and rat S9 or liver microsomes.

A metabolic activation system with an S9 fraction or liver microsomes was applied to a reporter gene assay in vitro for the screening of estrogenicity of chemicals. The endpoint (luciferase) was luciferase induction in cells transfected with a reporter plasmid containing an estrogen-responsive element linked to the luciferase gene. Compounds were applied to the reporter gene assay system after pretreatment or simultaneous treatment with an S9 fraction or liver microsomes. Both trans-stilbene and methoxychlor themselves showed no or little estrogenicity, but when they were treated with an S9 fraction or liver microsomes, they demonstrated strong effects, indicating their metabolites to be estrogenic. When four pyrethroid insecticides were subjected to this assay system, however, they showed no estrogenicity even with liver microsome or S9 mix treatment.

Animals↗
PubMed2006

Evaluation of human liver slices and reporter gene assays as systems for predicting the cytochrome p450 induction potential of drugs in vivo in humans.

PURPOSE: The aim of the study was to investigate the feasibility of predicting human in vivo cytochrome P450 (CYP) induction properties of drugs using in vitro methods. METHODS: The CYP induction potential of compounds was tested in human liver slices and in reporter gene assays for the aryl hydrocarbon receptor (AhR) and the pregnane X receptor (PXR). RESULTS: In human liver slices, CYP activities decreased dramatically over the experimental period, whereas mRNA levels could reliably be used to investigate CYP1A, 2C9, and 3A4 induction. However, the interindividual variations and demanding experimentation limit the use of liver slices in screening programs. Reporter gene assays are robust and reliable assays, amenable to high throughput screening. Several compounds activated AhR. The relevance of this activation, however, needs to be further investigated since there are no clear reports on drugs inducing CYP1A in vivo. The results from the PXR assay could be used to correctly classify compounds with known CYP3A induction properties when relating in vivo AUCtot to PXR EC50 values. CONCLUSIONS: Liver slices are a valuable model to study the regulation of a larger number of enzymes by single compounds. The PXR reporter gene assay could be used as a reliable screening method to predict CYP3A induction in vivo.

Aged↗
PubMed2004

Quantification of Atlantic salmon type-I interferon using an Mx1 promoter reporter gene assay.

We here describe an assay for the detection of interferon-like activity in Atlantic salmon based on the transient transfection of chinook salmon embryo cells (CHSE-214 cells) with a rainbow trout Mx1 promoter linked to a luciferase reporter. A beta-galactosidase gene under the control of a constitutively expressed beta-actin promoter was used as a transfection standard, and luciferase and beta gal expression were measured by a commercially available kit. Interferon containing supernatants from poly I:C- or CpG-stimulated leucocytes added to transfected CHSE-cells induced high luciferase expression (>60-fold induction compared to supernatants from non-stimulated cells). There was no response to supernatants from LPS- and ConA/PMA-stimulated leucocytes, demonstrating the specificity for type I interferon-like activity. Duplicate samples analysed using a cell protection assay for detection of antiviral activity correlated well with levels obtained by the Mx1 promoter reporter gene assay (R2=0.97), confirming the reporter assay as a reliable substitute for the standard antiviral assay. The Mx reporter gene assay also has advantages in terms of sensitivity, high dynamic range and reliability over the conventional cell protection assay.

Animals↗
PubMed1999

Rapid fluorescence-based reporter-gene assays to evaluate the cytotoxicity and antitumor drug potential of platinum complexes.

BACKGROUND: The need for new platinum antitumor drugs is underscored by the usefulness of cisplatin and carboplatin in chemotherapy and the resistance of many tumors to these compounds. Combinatorial chemistry could aid in the search for cisplatin analogs if fast, high-throughput assays were available. Our goal was to develop rapid cell-based assays suitable for high-throughput screening that accurately predict the cytotoxicity of platinum complexes. We examined the effects of platinum complexes and other agents on reporter-gene expression in cancer cells. RESULTS: HeLa Tet-On cells with inducible enhanced green fluorescent protein (EGFP) were prepared. Cisplatin and other cis-disubstituted platinum complexes inhibited EGFP expression, with a strong positive correlation between EGFP inhibition and cytotoxicity. By contrast, trans-[Pt(NH(3))(2)Cl(2)], other trans-platinum complexes, methyl methanesulfonate or heat shock stimulated EGFP expression. Northern and nuclear run-on analyses revealed that the changes in EGFP expression were at the level of transcription. In another reporter-gene assay in Jurkat cells, cisplatin, but not trans-[Pt(NH(3))(2)Cl(2)] or K(2)[PtCl(4)], inhibited beta-lactamase expression, as measured by hydrolysis of the fluorescent substrate CCF2. CONCLUSIONS: The EGFP results indicate that cytotoxic stress enhances transcription from the inducible promoter, whereas compounds able to form the 1,2-intrastrand platinum-DNA cross-links repress transcription. Both fluorescence-based reporter-gene assays afford promising new approaches to platinum anticancer drug discovery.

Antineoplastic Agents↗
PubMed2001

RepGene: a spreadsheet template for the management of reporter gene assays.

One of the most powerful techniques in molecular biology is the controlled expression of specific proteins by transfection of eukaryotic cells. This method has become feasible and highly sensitive and, thus, suitable for high-throughput reporter gene assays in basic and applied research. Moreover, the limiting factors are neither the transfection efficiency nor the functional analysis, but rather the ability to manage complex experimental protocols when multiple genes are co-transfected and/or when the effects of several chemical compounds are investigated within the same experiment. Here, we describe an easy-to-use and highly flexible spreadsheet template intended to rationalize and expedite the organization and data management of multi-step reporter gene assays. The objectives of this spreadsheet template are the design of the transfection protocol, the coordination of the administration of test compounds, and the graphical presentation and statistical analysis of the results.

Data Display↗
PubMedDate not supplied

Hybridization among the ancient mariners: characterization of marine turtle hybrids with molecular genetic assays.

Reports of hybridization between marine turtle species (family Cheloniidae) have been difficult to authenticate based solely on morphological evidence. Here we employ molecular genetic assays to document the sporadic, natural occurrence of viable interspecific hybrids between species representing four of the five genera of cheloniid sea turtles. Using multiple DNA markers from single-copy nuclear loci, eight suspected hybrids (based on morphology) were confirmed to be the products of matings involving the loggerhead turtle (Caretta caretta) x Kemp's ridley (Lepidochelys kempii) (N = 1 specimen), loggerhead turtle x hawksbill (Eretmochelys imbricata) (N = 2), loggerhead turtle x green turtle (Chelonia mydas) (N = 4), and green turtle x hawksbill (N = 1). Molecular markers from mitochondrial DNA permitted identification of the maternal parental species in each cross. The species involved in these hybridization events represent evolutionary lineages thought to have separated 10-75 million years ago (mya) and thus may be among the oldest vertebrate lineages capable of producing viable hybrids in nature. In some cases, human intervention with the life cycles of marine turtles (e.g., through habitat alteration, captive rearing, or attempts to establish new breeding sites) may have increased the opportunities for interspecific hybridization.

Animals↗
PubMed1998

Assessment of polystyrene extract for estrogenic activity in the rat uterotrophic model and an in vitro recombinant receptor reporter gene assay.

The purpose of the study was to determine whether polystyrene used in food-contact applications would elicit an estrogenic response when extracts simulating exaggerated conditions of use were subjected to in vivo and in vitro tests. A sample of polystyrene was subjected to extraction conditions that simulate, or exaggerate, the actual food-contact uses of polystyrene to maximize the amount of low molecular weight polystyrene extractables. The food-simulating solvent and the time and temperature conditions recommended by the Food and Drug Administration (FDA) were selected to maximize the level of extractable components from polystyrene. The extract was examined for its estrogenic response in vivo using the immature rat uterotrophic assay and in vitro using an estrogen receptor (ER)-mediated recombinant receptor reporter gene assay. In vivo, the uterine weights of juvenile female Sprague Dawley rats (10 rats/group) were determined after oral gavage exposure to the extract (two dosage levels: one represents the maximum potential daily human exposure to polystyrene extractables and the other represents one-tenth of the maximum exposure level), vehicle control (sesame oil), or positive control [diethylstilbestrol (DES), at 200 micrograms/kg body weight]. In addition, five treatment groups were dosed by subcutaneous injection of either estradiol (1, 50, and 500 micrograms/kg body weight) or DES (2 and 200 micrograms/kg body weight). Dosing began on postnatal day (pnd) 21 and continued daily through pnd 23. Body weights were collected at study initiation (pnd 21) and at necropsy (pnd 24). Body weights were not different statistically between treatment groups at study initiation or at necropsy. Uterine wet weights and uterine weights relative to body weights were significantly increased (p < 0.05) for estradiol at 50 and 500 micrograms/kg, DES at 2 and 200 micrograms/kg, and DES at 200 micrograms/kg (oral) over vehicle control. The polystyrene extract had no effect on uterine wet weight or uterine weights relative to body weights at either level tested. An in vitro recombinant estrogen receptor/reporter gene assay that involved transiently transfecting MCF-7 human breast cancer cells with the chimeric human ER, Ga14-HEGO, consisting of the yeast Ga14 DNA binding domain linked to the ligand binding domain of the human ER and a Ga14 response element (17mer)-regulated reporter gene (17m5-G-Luc) was employed. Dose-dependent induction of the reporter gene, 17m5-G-Luc, was observed with the positive control, 17 beta-estradiol (E2). Induction of greater than 100-fold was obtained following incubation of transfected MCF-7 cells with 10 nM E2 for 24 hours. No induction of reporter gene activity was observed with the polystyrene extracts dissolved in dimethylsulfoxide (0.01, 0.1 or 0.01 mg/ml) using the same assay conditions. These results indicate that polystyrene extract does not elicit ER-mediated activity using the Ga14-HEGO/17m5-G-Luc recombinant receptor/reporter gene assay. In conclusion, extracts from polystyrene produced no estrogenic response in either the rat uterotrophic assay or the MCF-7 cell assay for estrogen receptor-mediated activity.

Animals↗
PubMed2004

A human and mouse pregnane X receptor reporter gene assay in combination with cytotoxicity measurements as a tool to evaluate species-specific CYP3A induction.

Recent studies have demonstrated that a member of the nuclear receptor family, pregnane X receptor (PXR) is a key regulator of the expression of cytochrome P450 3A (CYP3A) in humans and rodents. It is also known that species specificity in the induction of CYP3A by xenobiotics is likely a consequence of differences at the level of PXR activation. Because of the importance of CYP3A4 in drug metabolism, the development of rapid and accurate in vitro assays for predicting the effects of compounds on CYP3A4 expression or activity in humans has been a long-standing goal within pharmaceutical industries. PXR activation measurements using an in vitro reporter gene approach appears to provide a rapid and relatively inexpensive means for predicting whether compounds will induce CYP3A levels in vivo. In this study, using an HepG2 cell based human and mouse PXR reporter gene assay, 23 compounds were tested for their potential to activate hPXR or mPXR. Data demonstrated that potent activators of hPXR had virtually no activity on mPXR and efficient activators of mPXR had weak activity on hPXR. In addition, a third category of moderate/weak activators of both hPXR and mPXR was identified. Exemestane was a strong activator of mPXR ( approximately 22-fold activation) with only minor effect on hPXR ( approximately 5-fold activation). The importance of cell viability measurements as part of the PXR reporter gene assay was demonstrated as significant cytotoxicity or inhibition of cell proliferation might underestimate the potential for PXR activation.

Androstadienes↗