Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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Numerous single nucleotide polymorphisms (SNPs) have been identified in the human genome, yet the functional significance of most is unknown. CYP3A4 is a key enzyme in the metabolism of numerous compounds. An A-->G substitution 290 bp upstream of the CYP3A4 transcription start site (CYP3A4*1B) has been associated with cancer phenotypes, but its phenotypic effects are unclear. To investigate the functional significance of CYP3A4*1B, we generated two luciferase reporter constructs: 1-kb (denoted L, long) and 0.5-kb (denoted S, short) promoter fragments containing either the variant (V(L),V(S)) or the wild-type (W(L), W(S)) sequences. We evaluated the effect of the variant sequence in the HepG2 and MCF-7 cell lines, and in primary human hepatocytes from three donors. Reporter constructs with the variant sequence had 1.2- to 1.9-fold higher luciferase activity than constructs with wild-type sequence in the cell lines (P < 0.0001) and hepatocytes (P = 0.021, P = 0.027, P = 0.061). The ratio of transcriptional activity for V(S):W(S) was similar to the V(L):W(L) ratio in HepG2 cells, but the V(S):W(S) ratio was consistently less than the V(L):W(L) ratio in MCF-7 cells. This suggests that CYP3A4 expression is higher from the variant promoter and that a repressor sequence may exist in the longer constructs. Electrophoretic mobility shift assays demonstrated specific binding of a component of HepG2 nuclear extract to both wild-type and variant promoters with consistently higher binding affinities to the wild-type sequence. This suggests the existence of a transcriptional repressor responsible for the lower CYP3A4*1A activity. Therefore, the phenotypic effects of the variant CYP3A4*1B may be associated with enhanced CYP3A4 expression due to reduced binding of a transcriptional repressor.
Environ Mol Mutagen 2003
PMID:Increased transcriptional activity of the CYP3A4*1B promoter variant. 1467 75

Several known anti-cancer agents have been shown to lead to increased expression of phase 2 metabolic enzymes without affecting phase 1 enzymes. Phase 1 cytochrome P450 (CYP) enzymes are relevant in cancer studies in that they are involved in oxidative metabolism, biotransformation and detoxification, whereas phase 2 enzyme catalysis leads to clearance. In this study, we obtained semi-quantitative measurements of cytochrome P450 (phase 1) and phase 2 microsomal epoxide hydrolase (mEH) gene expression levels in response to treatment of MCF-7 breast cancer cells with water-soluble Vernonia amygdalina (V.A.) extract. V.A., a vegetable grown in Nigeria, has potential as an anti-cancer agent. The results of Western blot and RT-PCR analyses show that V.A. extract acts as a monofunctional inducer within exposure times ranging from 2-16 hr and dose ranges from 3-100 microg/ml of V.A. Exposure of cells to low doses of V.A. did not affect expression levels of CYP1A1/1A2 mRNA, but lead to induction of mEH, thus supporting the chemotherapeutic potential of VA. However, in parallel studies, CYP3A4 gene expression was also induced, suggesting potential intermediates which influence drug-drug interactions. These data are useful toward further validating V.A. extract as a potential clinically useful natural anti-cancer agent and provide some support for the concept that modulation in CYP3A4 expression in response to treatment is relevant to prognosis.
Cell Mol Biol (Noisy-le-grand) 2003 Nov
PMID:Time and dose-dependent modulation of phase 1 and phase 2 gene expression in response to treatment of MCF-7 cells with a natural anti-cancer agent. 1468 87

CYP3A4, the most abundant form of cytochrome P450 in the human adult liver, shows wide interindividual variation in its activity. This variability is thought to be caused largely by transcriptional and genetic factors, yet the underlying mechanisms are poorly understood. The purpose of this study was to clarify the mechanisms controlling the CYP3A4 gene transcription and to search for genetic polymorphisms in the 5'-flanking region of the CYP3A4 gene. Transient transfection of human hepatoma HepG2 cells and of normal human hepatocytes with a series of CYP3A4 promoter-luciferase reporter plasmids revealed that a region from -11.4 to -10.5 kilobases, designated the constitutive liver enhancer module of CYP3A4 (CLEM4), was important for the constitutive activation of the CYP3A4 gene. Gel shift assay using nuclear extracts prepared from HepG2 cells showed that HNF-1alpha, HNF-4alpha, USF1, and AP-1 interacted with CLEM4. Furthermore, the introduction of mutations into their binding sites demonstrated that essentially all sites were required for the maximal enhancer activity. Screening for genetic polymorphisms within CLEM4 in genomic DNA from French persons, we identified the novel variant, TGT insertion between -11,129 and -11,128 (-11,129_-11,128insTGT), whose allele frequency was 3.1%. The -11,129_-11,128insTGT resulted in the disruption of USF1 binding and a 36% reduction of the enhancer activity. These results suggest that CLEM4 is a constitutive enhancer of the CYP3A4 gene in the liver and that -11,129_-11,128insTGT may at least partly contribute to the interindividual variability of CYP3A4 expression.
Mol Pharmacol 2004 Feb
PMID:Identification of a novel polymorphic enhancer of the human CYP3A4 gene. 1474 74

Human constitutive androstane (or active) receptor (hCAR), a member of the nuclear receptor superfamily NR1I3, regulates the expression of several genes that are mainly involved in the metabolism of endogenous and xenobiotic compounds (e.g., CYP2B6, CYP3A4, and UGT1A1). We found four novel splice variants in the ligand-binding domain (LBD) of hCAR (NCBI reference sequence, NM_005122; designated SV0 herein). The variants designated SV1 and SV2 contained in-frame 12- and 15-base pair (bp) insertions, respectively. SV3 carried both of the insertions, and SV4 contained an in-frame 117-bp deletion. The insertion site of SV1 is located in the alpha6 helix of hCAR LBD, which makes up the ligand-binding cavity, and that of SV2 is located in the highly conserved loop between helices alpha8 and alpha9. SYBR Green real-time reverse transcription-polymerase chain reaction analysis of each splice variant revealed that the hepatic expression of SV2 was almost comparable with that of SV0 (approximately 40%), whereas other variants accounted for 6 to 10% of the total hCAR transcripts. In the reporter gene assays employing the phenobarbital-responsible enhancer module (PBREM) from CYP2B6 and UGT1A1 genes, the splice variants, except for SV1, were inactive, whereas SV1 transactivated the CYP2B6 PBREM but not the UGT1A1 PBREM reporter. A nuclear translocation assay in rat hepatocytes revealed that all the splice variants lack the responsiveness toward phenobarbital and 6-(4-chloropheny-l)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime (CITCO) in terms of the ligand-dependent nuclear translocation. Further characterization, such as the identification of specific ligands, will help elucidate physiological implication of these hCAR splice variants.
Mol Pharmacol 2004 Mar
PMID:Identification of novel alternative splice variants of human constitutive androstane receptor and characterization of their expression in the liver. 1497 27

Many cytochrome P450 isoforms are known to be drug-inducible. The anticonvulsant phenytoin has been reported to be an inducer of human CYP2B6, CYP3A4, and murine CYP2C29. However, the molecular mechanism mediating phenytoin induction remains unclear. Herein, we used in vivo and in vitro gene reporter assays of the Cyp2c29 promoter to delineate the phenytoin-response activity to a phenytoin-responsive module located at -1371 kb upstream of the Cyp2c29 translation start site. The phenytoin-responsive module, consisting of two motifs of two imperfect direct repeat hexamers spaced by four nucleotides and a putative CCAAT/enhancer-binding protein-binding site, mediated luciferase reporter induction by phenytoin in mouse livers in vivo and was activated by CAR in HepG2 cells. Hepatic CYP2C29 mRNA was induced by phenytoin in wild-type but not in CAR-null mice, indicating that constitutive active or androstane receptor (CAR) regulates phenytoin-induced transcription of the Cyp2c29 gene. Furthermore, the constitutive levels of CYP2C29 mRNA were reduced approximately 77-fold in CAR-null mice compared with those in the wild-type mice, suggesting that CAR may also regulate the constitutive expression of the Cyp2c29 gene either directly or indirectly.
Mol Pharmacol 2004 Jun
PMID:The constitutive active/androstane receptor regulates phenytoin induction of Cyp2c29. 1515 33

An analysis of the cytochrome P450 3A subfamily (CYP3A) was undertaken in order to define relationships across species among subfamily members. Some members were excluded due to incomplete sequences, while others were held in abeyance because of their almost complete homology. This is the first publication of five chimpanzee CYP3A genes-CYP3A4, CYP3A5, CYP3A7, CYP3A43, and CYP3A67. This project utilized two approaches for characterizing possible relationships-phylogenetic analysis and genomic structure. For the phylogenetic analysis, both nucleotide and amino acid sequences were aligned in silico using the CLUSTAL algorithm, and then visually inspected for accuracy. Three different computer software packages were utilized: MEGA 2.1, TREECON 1.3b, and PHYLIP 3.5. Multiple methods were used: neighbor-joining (NJ), minimum evolution (ME), maximum parsimony (MP), and maximum likelihood (ML). The resulting topologies were compared against each other to define the consensus topology. In addition, the chimpanzee, human, mouse, and rat genome databases were searched for intron/exon information pertaining to the included genes. Both methods suggest the same conclusion, defining orthologs is plausible between similar species (i.e., mouse and rat), but is less useful between species of different orders (i.e., primate and rodent) or classes (i.e., mammal and avian).
Mol Phylogenet Evol 2004 Nov
PMID:Defining relationships between the known members of the cytochrome P450 3A subfamily, including five putative chimpanzee members. 1533 65

A number of computational approaches are being proposed for an early optimization of ADME (absorption, distribution, metabolism and excretion) properties to increase the success rate in drug discovery. The present study describes the development of an in silico model able to estimate, from the three-dimensional structure of a molecule, the stability of a compound with respect to the human cytochrome P450 (CYP) 3A4 enzyme activity. Stability data were obtained by measuring the amount of unchanged compound remaining after a standardized incubation with human cDNA-expressed CYP3A4. The computational method transforms the three-dimensional molecular interaction fields (MIFs) generated from the molecular structure into descriptors (VolSurf and Almond procedures). The descriptors were correlated to the experimental metabolic stability classes by a partial least squares discriminant procedure. The model was trained using a set of 1800 compounds from the Pharmacia collection and was validated using two test sets: the first one including 825 compounds from the Pharmacia collection and the second one consisting of 20 known drugs. This model correctly predicted 75% of the first and 85% of the second test set and showed a precision above 86% to correctly select metabolically stable compounds. The model appears a valuable tool in the design of virtual libraries to bias the selection toward more stable compounds.
J Comput Aided Mol Des 2004 Mar
PMID:Model based on GRID-derived descriptors for estimating CYP3A4 enzyme stability of potential drug candidates. 1536 16

Cytochrome P450 (CYP) 3A is responsible for about 50% of drug metabolizing activity in the liver. The present study was undertaken to establish a CYP3A4-active model for in vitro analysis of human drug metabolism. The cells used were immortalized normal human fetal hepatocytes (OUMS-29) and its HNF4alpha-introduced subline (OUMS-29/H-11). The cells were cultivated under high-density three-dimensional conditions in a radial-flow bioreactor (RFB). The number of OUMS-29 cells increased 15-fold over 49 days and their apical surfaces were covered with abundant microvilli, a characteristic of hepatocytes in vivo. The amount of albumin secreted by OUMS-29 cells in the three-dimensional RFB culture was 6-fold higher than those in a monolayer culture. CYP3A4 protein and an intermediate metabolite of testosterone by CYP3A4 were detected in OUMS-29/H11 cells cultivated in RFB >29 days. These results indicate that the RFB culture of OUMS-29/H-11 cells is useful for screening and developing new drugs.
Int J Mol Med 2004 Oct
PMID:Expression of CYP3A4 by an immortalized human hepatocyte line in a three-dimensional culture using a radial-flow bioreactor. 1537 99

Methoxymorpholinyl doxorubicin (MMDX) is a novel liver cytochrome P450 (P450)-activated anticancer prodrug whose toxicity toward cultured tumor cells can be potentiated up to 100-fold by incubation with liver microsomes and NADPH. In the present study, a panel of human liver microsomes activated MMDX with potentiation ratios directly correlated to the CYP3A-dependent testosterone 6beta-hydroxylase activity of each liver sample. Microsome-activated MMDX exhibited nanomolar IC(50) values in growth-inhibition assays of human tumor cell lines representing multiple tissues of origin: lung (A549 cells), brain (U251 cells), colon (LS180 cells), and breast (MCF-7 cells). Analysis of individual cDNA-expressed CYP3A enzymes revealed that rat CYP3A1 and human CYP3A4 activated MMDX more efficiently than rat CYP3A2 and that human P450s 3A5 and 3A7 displayed little or no activity. MMDX cytotoxicity was substantially increased in Chinese hamster ovary cells after stable expression of CYP3A4 in combination with P450 reductase. CYP3A activation of MMDX abolished the parent drug's residual cross-resistance in a doxorubicin-resistant MCF-7 cell line that overexpresses P-glycoprotein. CYP3A-activated MMDX displayed a comparatively high intrinsic stability, with a t(1/2) of approximately 5.5 h at 37 degrees C. MMDX was rapidly activated by CYP3A at low ( approximately 1-5 nM) prodrug concentrations, with 100% tumor cell kill obtained after as short as a 2-h exposure to the activated metabolite. These findings demonstrate that MMDX can be activated by CYP3A metabolism to a potent, long-lived, and cell-permeable cytotoxic metabolite and suggest that this anthracycline prodrug may be used in combination with CYP3A4 in a P450 prodrug activation-based gene therapy for cancer treatment.
Mol Pharmacol 2005 Jan
PMID:Antitumor activity of methoxymorpholinyl doxorubicin: potentiation by cytochrome P450 3A metabolism. 1546 24

Coimmunoprecipitation was used to investigate protein-protein interactions between several UDP-glucuronosyltransferase (UGT) isoforms and cytochrome P450 3A4. Solubilized human liver microsomes were incubated with specific antibodies to UGT2B7, UGT1A6, UGT1A1, and CYP3A4, and the immunoprecipitates were run on SDS-polyacrylamide gel electrophoresis. Western blots showed that UGT2B7, UGT1A6, UGT1A1, and CYP3A4 were successfully immunoprecipitated with the specific antibodies for each enzyme. Upon immunoprecipitating UGT2B7, the corresponding immunoblot showed that UGT1A6, UGT1A1, and CYP3A4 were immunoprecipitated. Similar studies found that different UGT isoforms or CYP3A4 immunoprecipitated along with the original immunoprecipitating enzyme. These data suggest that UGT isoforms may form complexes (dimers, tetramers, etc.) with each other in the endoplasmic reticulum and nuclear envelope. In addition, the UGT isoforms tested here may have interacted with CYP3A4 in the endoplasmic reticulum, suggesting that these enzymes may cooperate in the excretion of compounds in a multistep metabolic process.
Mol Pharmacol 2005 Jan
PMID:Coimmunoprecipitation of UDP-glucuronosyltransferase isoforms and cytochrome P450 3A4. 1548 48


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