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C R Wolf

Publications and source records attributed to C R Wolf.

At least 73 records · Page 4Linked to original sources
PubMed1998

Determinants of the substrate specificity of human cytochrome P-450 CYP2D6: design and construction of a mutant with testosterone hydroxylase activity.

Cytochrome P-450 CYP2D6, human debrisoquine hydroxylase, metabolizes more than 30 prescribed drugs, the vast majority of which are small molecules containing a basic nitrogen atom. In contrast, the similar mouse protein Cyp2d-9 was first characterized as a testosterone 16alpha-hydroxylase. No common substrates have been reported for the two enzymes. Here we investigate the structural basis of this difference in substrate specificity. We have earlier used a combination of NMR data and homology modelling to generate a three-dimensional model of CYP2D6 [Modi, Paine, Sutcliffe, Lian, Primrose, Wolf, C.R. and Roberts (1996) Biochemistry 35, 4541-4550]. We have now generated a homology model of Cyp2d-9 and compared the two models to identify specific amino acid residues that we believe form the substrate-binding site in each protein and therefore influence catalytic selectivity. Although there are many similarities in active site structure, the most notable difference is a phenylalanine residue (Phe-483) in CYP2D6, which in the model is located such that the bulky phenyl ring is positioned across the channel mouth, thus limiting the size of substrate that can access the active site. In Cyp2d-9, the corresponding position is occupied by an isoleucine residue, which imposes fewer steric restraints on the size of substrate that can access the active site. To investigate whether the amino acid residue at this position does indeed influence the catalytic selectivity of these enzymes, site-directed mutagenesis was used to change Phe-483 in CYP2D6 to isoleucine and also to tryptophan. CYP2D6, Cyp2d-9 and both mutant CYP2D6 proteins were co-expressed with NADPH cytochrome P-450 reductase as a functional mono-oxygenase system in Escherichia coli and their relative catalytic activities towards bufuralol and testosterone were determined. All four proteins exhibited catalytic activity towards bufuralol but only Cyp2d-9 catalysed the formation of 16alpha-hydroxytesterone. Uniquely, the CYP2D6F483I mutant acquired the ability to metabolize testosterone to a novel product, which was identified by MS and proton NMR spectroscopy as 15alpha-hydroxytestosterone. NMR spin relaxation experiments were used to measure distances between the haem iron and protons of testosterone bound to the CYP2D6F483I mutant. These experiments demonstrate that very minor modifications to the active site structure of CYP2D6 can have a profound influence on the substrate specificity of the enzyme.

Amino Acid Sequence↗
PubMed1998

Increased skin tumorigenesis in mice lacking pi class glutathione S-transferases.

The activity of chemical carcinogens is a complex balance between metabolic activation by cytochrome P450 monooxygenases and detoxification by enzymes such as glutathione S-transferase (GST). Regulation of these proteins may have profound effects on carcinogenic activity, although it has proved impossible to ascribe the observed effects to the activity of a single protein. GstP appears to play a very important role in carcinogenesis, although the precise nature of its involvement is unclear. We have deleted the murine GstP gene cluster and established the effects on skin tumorigenesis induced by the polycyclic aromatic hydrocarbon 7, 12-dimethylbenz anthracene and the tumor promoting agent 12-O-tetradecanoylphorbol-13-acetate. After 20 weeks, a highly significant increase in the number of papillomas was found in the GstP1/P2 null mice [GstP1/P2(-/-) mice, 179 papillomas, mean 9.94 per animal vs. GstP1/P2(+/+) mice, 55 papillomas, mean 2.89 per animal, (P < 0.001)]. This difference in tumor incidence provides direct evidence that a single gene involved in drug metabolism can have a profound effect on tumorigenicity, and demonstrates that GstP may be an important determinant in cancer susceptibility, particularly in diseases where exposure to polycyclic aromatic hydrocarbons is involved, for instance in cigarette smoke-induced lung cancer.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1998

Pi-class glutathione S-transferase: regulation and function.

Our laboratory has been involved in the study of Glutathione S-transferase pi (GST pi) for many years, both in terms of regulation of gene expression and in trying to understand the endogenous function(s) of this enzyme and also what role it may play in the carcinogenic process [1]. Over-expression of GST pi has been associated with carcinogenesis and the development of many different human tumours, for example testis [2], ovarian [3] and colorectal [4] and is often inversely correlated with prognosis or patient survival [5,6]. In addition, GST Pi has been implicated in the acquisition of antineoplastic drug resistance [7-9]. In order to study the transcriptional regulation of this gene, we have utilised a multi-drug resistant derivative (VCREMS) of the human mammary carcinoma cell line, MCF7, in which GST P1 mRNA and protein are significantly elevated in the absence of gene amplification [10-13]. Interestingly, we have recently reported the discovery of polymorphisms at the GSTP1 locus, resulting in two alleles GSTP1a and GSTP1b. In the study, the GSTP1b allele was found with increased frequency in bladder and testicular cancer, while the GSTP1a allele was significantly decreased in cases of prostate cancer [14]. In an attempt to elucidate the endogenous role(s) of GST pi, we have used homologous recombination in embryonic stem (ES) cells to inactivate both murine GST Pi genes and create a mouse strain completely deficient in the expression of this enzyme. This provides us with a unique animal model with which to study the effects of the absence of GST pi expression on the metabolism and pharmacokinetics of xenobiotics.

Alleles↗
PubMed1998

Induction of CYP102 (cytochrome P450BM-3) in Bacillus megaterium by 17 beta-estradiol and 4-sec-butylphenol.

CYP102 (Cytochrome P450BM-3) is induced in Bacillus megaterium by barbiturates, peroxisome proliferators, and nonsteroidal anti-inflammatory drugs. We now describe the induction of CYP102 in B. megaterium by 17 beta-estradiol and by 4-sec-butylphenol. These estrogens interact with the repressor protein Bm3R1, causing it to dissociate with the operator of the CYP102 gene and allowing transcription to occur. We have developed a stable transfection of a construct into B. megaterium of a truncated CYP102 gene coupled with the luciferase gene in a promoterless plasmid and have used this construct to test the induction of CYP102 by these estrogens. Estradiol demonstrated a dose-dependent induction of CYP102 which saturated at a 2-fold increase at 150 microM 4 hr post-addition. 4-sec-Butylphenol produced a dose-dependent and time-dependent induction up to 300 microM and 6 hr post-induction.

Bacillus megaterium↗
PubMed1998

The glutathione S-transferase GSTP1 polymorphism: effects on susceptibility to oral/pharyngeal and laryngeal carcinomas.

We have examined the hypothesis that the polymorphic, glutathione S-transferase GSTP1 gene is a susceptibility candidate for squamous cell cancer of the oral/pharynx and larynx. We describe GSTP1 genotype frequencies in 380 cases and 180 controls. We found a lower frequency of GSTP1 AA in the oral/pharyngeal cases compared with controls (p = 0.003, odds ratio = 0.47) after correction for age and gender. We used an immunohistochemical approach to show widespread expression of the GSTP1 subunit throughout the pharynx and larynx. In uninfiltrated tissue, strong positivity was found throughout the squamous cell epithelium with the exception of the basal cell layer. The cilia of the respiratory epithelium of the larynx also showed positivity for GSTP1. In tumour tissue, expression of GSTP1 was similar in pharyngeal and laryngeal samples. These data are the first to show that polymorphism at GSTP1 mediates susceptibility to squamous cell cancer of the upper aerodigestive tract. No significant interactions were identified between GSTP1 and GSTM1, GSTM3, GSTT1 and the cytochrome P450 CYP1A1, CYP2D6 and CYP1A1 genotypes.

Alcohol Drinking↗
PubMed1998

Functional co-expression of CYP2D6 and human NADPH-cytochrome P450 reductase in Escherichia coli.

The polymorphic human CYP2D6 has been co-expressed with human NADPH-cytochrome P450 oxidoreductase in Escherichia coli in order to generate a functional recombinant monooxygenase system for the study of xenobiotic metabolism. The two cDNAs were co-expressed from separate, compatible plasmids with different antibiotic selection markers. The CYP2D6 could be detected in bacterial cells at levels up to 700 nmol I-1 culture by Fe(2+)-CO versus Fe2+ difference spectroscopy, exhibiting the characteristic absorbance peak at 450 nm. Immunoblotting demonstrated the presence of both proteins in bacterial membranes, where they were expressed at levels significantly higher than those found in human liver microsomes. Membrane content was 150-200 pmol CYP2D6 (determined spectrally) and 100-230 pmol CYP-reductase (determined enzymatically) per mg protein. Critically, the two co-expressed proteins were able to couple to form a NADPH-dependent monooxygenase which metabolized the CYP2D6 substrate bufuralol (Vmax 3.30 nmol min-1 mg-1 protein; K(m) 11.1 microM) in isolated membrane fractions. This K(m) value was similar to the K(m) determined in human liver microsomes. Activity could be inhibited by the specific inhibitor quinidine. Of greater significance however, was the finding that intact E. coli cells, even in the absence of exogenous NADPH, were able to metabolize bufuralol at rates almost as high as those measured in membranes (4.6 +/- 0.4 min-1 versus 5.7 +/- 0.2 min-1 at 50 microM substrate). Such recombinant strains will greatly facilitate the molecular characterization of allelic variants of cytochrome P450 isoenzymes.

Adrenergic beta-Antagonists↗
PubMed1998

Susceptibility and outcome in oral cancer: preliminary data showing an association with polymorphism in cytochrome P450 CYP2D6.

Members of the cytochrome P450 and glutathione S-transferase supergene families are candidates for susceptibility and outcome in oral squamous cell cancer. We determined GSTM1, GSTM3, GSTT1, CYP1A1 and CYP2D6 genotypes in 100 Caucasian cases and 467 control individuals. The frequency of homozygosity for mutant CYP2D6 alleles was higher in the cases (P = 0.001, OR = 3.2, 95% CI = 1.6-6.5) than control individuals. In the cases, the frequency of homozygosity for mutant alleles was greater and that of homozygosity for wild-type CYP2D6 alleles was lower in those diagnosed at > or = 65 years (P = 0.009) than in those diagnosed at < or = 64 years. The older cases included relatively more women and patients who did not consume tobacco or alcohol. The association of CYP2D6 with outcome was assessed using the Cox's proportional hazards model. The time to first cervical node metastasis was shorter in heterozygotes and homozygotes for mutant CYP2D6 alleles compared with homozygotes for wild-type alleles after correction for age at diagnosis, gender, alcohol and tobacco consumption and tumour differentiation (P = 0.04, hazard ratio 3.6, 95% CI 1.1-12.5). The mechanism for the association of CYP2D6 alleles with susceptibility and outcome is unclear though the data are compatible with the view that homozygosity for mutant alleles confers impaired detoxication of an unknown carcinogen. No associations between GSTM1, GSTM3, GSTT1 or CYP1A1 genotypes and susceptibility or, time to node metastases were identified. We previously showed that CYP2D6 genotypes were not associated with susceptibility to squamous cell cancer in the pharynx or larynx. Therefore, the data presented suggest that susceptibility to squamous cell cancer in the various parts of the upper aerodigestive tract is associated with different genes and allelic variants.

Adult↗
PubMed1998

Use of transgenic animals in understanding molecular mechanisms of toxicity.

Understanding molecular mechanisms of chemical toxicity and the potential risks of drugs to man is a pivotal part of the drug development process. With the dramatic increase in the number of new chemical entities arising from high throughput screening, there is an urgent need to develop systems for the rapid evaluation of potential drugs so that those agents which are most likely to be free of adverse effects can be identified at the earliest possible stage in drug development. The complex mechanisms of action of chemical toxins has made it extremely difficult to evaluate the precise toxic mechanism and also the relative role of specific genes in either potentiating or ameliorating the toxic effect. This problem can be addressed by the application of genetic strategies. Such strategies can exploit strain differences in susceptibility to specific toxic agents or, with the rapidly developing technologies, can exploit the use of transgenic animals where specific genes can be manipulated and subsequent effects on chemical toxicity evaluated. Transgenic animals can be exploited in a variety of ways to understand mechanisms of chemical toxicity. For example, a human gene encoding a drug metabolizing enzyme can be directly introduced and the effects on toxic response evaluated. Alternatively, specific genes can be deleted from the mouse genome and the consequences on toxicological response determined. Many toxic chemical agents modulate patterns of gene expression within target cells. This can be used to screen for responses to different types of toxic insult. In such experiments the promotor of a stress-regulated gene can be ligated to a suitable reporter gene, such as lacZ, or green fluorescent protein, and inserted into the genome of an appropriate test species. On administration of a chemical agent, cells which are sensitive to the toxic effects of that chemical will express the reporter, which can then be identified using an appropriate assay system. This latter strategy provides the potential for screening a large number of compounds rapidly for their potential toxic effects and also provides information on tissue and cellular specificity. Experiments using transgenic animals can be complex, and care must be taken to ensure that the results are not affected by background activities within the species being used. For example, the introduction of a specific human cytochrome P450 gene may have no effect on the metabolic disposition of a drug or toxin because of the background activity within the mouse. As the toxicity of a chemical agent is determined by a wide range of different factors including drug uptake, metabolism, detoxification and repair, differences between man and the species being used could potentially generate a toxic response in the animal model whereas no toxicity may be observed in man. In spite of these confounding factors, the application of transgenic animals to toxicological issues has enormous potential for speeding up the drug discovery process and will undoubtedly become part of this process in the future.

Animals↗
PubMed1997

High levels of recombinant CYP3A4 expression in Chinese hamster ovary cells are modulated by coexpressed human P450 reductase and hemin supplementation.

Expression of recombinant cytochrome P450s (P450s) in mammalian cells has been used as a powerful tool to study these enzymes. However, the activity of CYP3A4 expressed in several stable mammalian cell lines was much lower than native enzyme in human liver. The low level of recombinant CYP3A4 may have been due to the low copy number of the cDNA. In addition, the low activity is caused by the low level of P450 reductase in these cells. To achieve high levels of CYP3A4 expression, we employed gene amplification of the CYP3A4 cDNA in Chinese hamster ovary (CHO) cells followed by transfection of the P450 reductase cDNA. Using this strategy, we have obtained a cell line, designated D3A4, with high levels of recombinant CYP3A4. The content of spectrally active P450 was 14 pmol/mg total cellular protein. Hemin treatment increased the P450 content 2-fold. Upon coexpression of P450 reductase in DHR/3A4 cells, enzyme activity of CYP3A4 was stimulated 15-fold, despite a 40% decrease in spectrally active P450. Interestingly, the latter effect was not due to a decrease in CYP3A4 mRNA. Treatment of these cells with hemin, however, counteracted the P450 reductase-mediated decrease of spectrally active P450. These data demonstrate that P450 reductase has a strong influence on the levels of recombinant P450 holoenzyme, possibly by modulating the level of heme in CHO cells. Concomitantly our results show that the gene amplification strategy provides a powerful approach to obtain a high level of functional recombinant P450.

Animals↗
PubMed1997

Fatty acid signals in Bacillus megaterium are attenuated by cytochrome P-450-mediated hydroxylation.

In previous publications [English, Hughes and Wolf (1994) J. Biol. Chem. 269, 26836-26841; English, Hughes and Wolf (1996) Biochem. J. 316, 279-283], we have demonstrated that peroxisome proliferators and non-steroidal anti-inflammatory drugs are inducers of the cytochrome P-450BM-3 gene in Bacillus megaterium ATCC14581. Their mechanism of action involves binding to and subsequent displacement of the transcriptional repressor, Bm3R1, from its operator site, which results in the activation of cytochrome P-450BM-3 gene transcription. We now present evidence that the branched-chain fatty acid, phytanic acid, is a potent inducer of cytochrome P-450BM-3. We have also observed that phytanic acid and peroxisome proliferators are inducers of Bm3R1 protein accumulation and associated DNA-binding activity. In contrast, several barbiturates, although capable of inducing cytochrome P-450BM-3 and Bm3R1 gene transcription, were unable to induce the Bm3R1 protein. We also demonstrate that cytochrome P-450BM-3 readily oxidizes phytanic acid, and provide evidence that, although the omega-1 hydroxy acid derivatives of phytanic acid can associate with Bm3R1, they do so with an affinity two orders of magnitude lower than the unmodified fatty acid. As a consequence, the ability of the hydroxylated product to induce cytochrome P-450BM-3 gene expression in vivo is markedly reduced. These data collectively suggest that metabolism of fatty acids by cytochrome P-450BM-3 leads to an attenuation of their ability to activate the transcription of the BM-3 operon. This work places the action of bacterial fatty acid hydroxylases in an autoregulatory loop where they may be responsible for the inactivation or clearance of the inducing fatty acid signal.

Bacillus megaterium↗
PubMed1997

A general strategy for the expression of recombinant human cytochrome P450s in Escherichia coli using bacterial signal peptides: expression of CYP3A4, CYP2A6, and CYP2E1.

Heterologous expression of unmodified recombinant human cytochrome P450 enzymes (P450s) in Escherichia coli has proved to be extremely difficult. To date, high-level expression has only been achieved after altering the 5'-end of the native cDNA, resulting in amino acid changes within the P450 protein chain. We have devised a strategy whereby unmodified P450s can be expressed to high levels in E. coli, by making NH2-terminal translational fusions to bacterial leader sequences. Using this approach, we initially tested two leader sequences, pelB and ompA, fused to CYP3A4. These were compared with an expression construct producing a conventional NH2-terminally modified CYP3A4 (17alpha-3A4). Both leader constructs produced spectrally active, functional protein. Furthermore, the ompA-3A4 fusion gave higher levels of expression, and a marked improvement in the recovery of active P450 in bacterial membrane fractions, when compared with 17alpha-3A4. We then tested the ompA leader with CYP2A6 and CYP2E1, again comparing with the conventional (17alpha-) approach. As before, the leader construct produced active enzyme, and, for CYP2E1 at least, gave a higher level of expression than the 17alpha-construct. The ompA fusion strategy thus appears to represent a significant advance for the expression of P450s in E. coli, circumventing the previous need for individual optimization of P450 sequences for expression.

Aminolevulinic Acid↗
PubMed1997

Transcriptional and post-transcriptional mechanisms can regulate cell-specific expression of the human Pi-class glutathione S-transferase gene.

Previous studies from this laboratory have identified transcriptional mechanisms that are utilized to increase expression of the human glutathione S-transferase gene GSTP1 in a multidrug-resistant derivative (VCREMS) of the human mammary carcinoma cell line MCF7 [Moffat, McLaren and Wolf (1994) J. Biol. Chem. 269, 16397-16402]. The data presented here provide strong evidence that post-transcriptional mechanisms can also play an important role in determining cell-specific expression of the GSTP1 gene. GSTP1 mRNA levels were shown to be elevated 3.1-fold in the human bladder carcinoma cell line EJ compared with VCREMS cells. Despite this observation, transient transfection assays revealed a decreased rate of GSTP1 promoter activity in EJ cells. Indeed, GSTP1 transcriptional repressor activity, mediated by a region located between nucleotides -105 and -86 (as we have previously described in MCF7 cells), was observed in EJ cells. However, in contrast with our results in MCF7 cells, the EJ repressor activity did not displace the essential nuclear complex bound to the C1 promoter element (-73 to -54) in vitro. In addition, competition experiments indicated that an AP-1-like protein is an integral component of the C1-bound complex in EJ cells. Interestingly, experiments utilizing actinomycin D to inhibit transcription demonstrated significantly greater stability of GSTP1 mRNA in EJ cells than in VCREMS cells. These findings suggest that cell-specific differences in the rates of GSTP1 mRNA decay provide the predominant mechanism responsible for elevated expression of the GSTP1 gene in EJ cells.

Base Sequence↗
PubMed1997

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine as a substrate of cytochrome P450 2D6: allosteric effects of NADPH-cytochrome P450 reductase.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a neurotoxin that produces Parkinsonism symptoms in man, has been examined as a substrate of recombinant human cytochrome P450 2D6. When cumene hydroperoxide is used as an oxygen and electron donor, a single product is formed, identified as 4-phenyl-1,2,3,6-tetrahydropyridine. The K(m) for formation of this product (130 microM) is in agreement with the dissociation constants for MPTP binding to the enzyme determined by optical and nuclear magnetic resonance (NMR) spectroscopy. When the reaction is carried out with nicotinamide adenine dinucleotide phosphate (reduced) (NADPH) and recombinant human NADPH-cytochrome P450 reductase, a second product, identified as 1-methyl-4-(4'-hydroxyphenyl)-1,2,3,6-tetrahydropyridine, is formed in addition to 4-phenyl-1,2,3,6-tetrahydropyridine. The K(m) values for formation of these two products are 19 microM and 120 microM, respectively. Paramagnetic relaxation experiments have been used to measure distances between the protons of bound MPTP and the heme iron, and these have been used to construct models for the position and orientation of MPTP in the active site. For the cytochrome alone, a single mode of binding was observed, with the N-methyl close to the heme iron in a position appropriate for the observed N-demethylation reaction. In the presence of the reductase, the data were not consistent with a single mode of binding but could be explained by the existence of two alternative orientations of MPTP in the active site. One of these, characterized by a dissociation constant of 150 microM, is essentially identical to that observed in the absence of the reductase. In the second, which has a K(d) of 25 microM, the MPTP is oriented so that the aromatic ring is close to the heme iron, in a position appropriate for p-hydroxylation leading to the formation of the product seen only in the presence of the reductase. In the case of codeine, another substrate for cytochrome P450 2D6, the addition of reductase had no effect on the nature of the product formed, the dissociation constant, or the orientation in the binding site. These observations show that NADPH-cytochrome P450 reductase has an allosteric effect on the active site of cytochrome P450 2D6 that affects the binding of some substrates but not others.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
PubMed1997

1H, 15N and 13C NMR resonance assignment, secondary structure and global fold of the FMN-binding domain of human cytochrome P450 reductase.

The FMN-binding domain of human NADPH-cytochrome P450 reductase, corresponding to exons 3-7, has been expressed at high level in an active form and labelled with 13C and 15N. Most of the backbone and aliphatic side-chain 1H, 15N and 13C resonances have been assigned using heteronuclear double- and triple-resonance methods, together with a semiautomatic assignment strategy. The secondary structure as estimated from the chemical shift index and NOE connectivities consists of six alpha-helices and five beta-strands. The global fold was deduced from the long-range NOEs unambiguously assigned in a 4D 13C-resolved HMQC-NOESY-HMQC spectrum. The fold is of the alternating alpha/beta type, with the five beta-strands arranged into a parallel beta-sheet. The secondary structure and global fold are very similar to those of the bacterial flavodoxins, but the FMN-binding domain has an extra short helix in place of a loop, and an extra helix at the N-terminus (leading to the membrane anchor domain in the intact P450 reductase). The experimental constraints were combined with homology modelling to obtain a structure of the FMN-binding domain satisfying the observed NOE constraints. Chemical shift comparisons showed that the effects of FMN binding and of FMN reduction are largely localised at the binding site.

Amino Acid Sequence↗
PubMed1997

Menadione-resistant Chinese hamster ovary cells have an increased capacity for glutathione synthesis.

A cell line (MRc40) resistant to the model quinone compound, menadione, has been isolated from a parental Chinese hamster ovary cell line (CHO-K1). The known relationship between menadione toxicity and glutathione (GSH) depletion led us to investigate whether the mechanism of resistance of MRc40 was related to alteration in GSH homeostasis. Intracellular concentrations of GSH and cysteine (CySH) were twofold and 3.2-fold greater in MRc40 than in CHO-K1. Following exposure to menadione, GSH and CySH were depleted, but subsequent recovery of thiols was more rapid and of greater magnitude in MRc40 than in CHO-K1. Twelve hours after exposure to menadione, the concentrations of GSH and CySH were 9.7- and 4.2-fold greater in MRc40 than in CHO-K1. Using nuclear magnetic resonance (NMR) spectroscopy, we observed the in situ removal of menadione from cell suspensions of CHO-K1 and MRc40. However, only in CHO-K1 did we observe concomitant depletion of NMR-visible GSH. We conclude that the perturbation of GSH metabolism contributes to the resistant phenotype and is an important characteristic of menadione-resistant CHO cells.

Animals↗