Human cytochromes P450 expressed in Escherichia coli: production of specific antibodies

Belloc, C.; Baird, Susan; Cosme, José; Lecoeur, Sylvaine; Gautier, Jean‐Charles; Challine, Dominique; Waziers, Isabelle de; Flinois, Jean‐Pierre; Beaune, Philippe

doi:10.1016/0300-483x(95)03178-i

Cited by 42 publications

(28 citation statements)

References 33 publications

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“…Before immunoblotting, polyclonal antibodies raised against individual human recombinant CYP isoforms (1A2, 2E1, and 3A4) 21 were immunopurified by affinity capture according to a pre-established protocol, 22 using recombinant CYP isoforms immobilized onto separate 1-mL HiTrap NHS-activated columns (Pharmacia Biotech, Saclay, France). This procedure resulted in a significant decrease of unspecific immunoreactive signal, while cross-reactivities that anti-CYP1A2 and anti-CYP3A4 antibodies exhibited with CYP1A1 and CYP3A5, respectively, persisted after immunopurification.…”

Section: Methodsmentioning

confidence: 99%

Phase I and Phase II drug-metabolizing enzymes are expressed and heterogeneously distributed in the biliary epithelium

et al. 1999

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Tissue expression of drug-metabolizing enzymes influences susceptibility to drugs and carcinogens. Because the biliary epithelium, exposed to bile-borne chemicals, may give rise to drug-induced cholangiopathies and to cholangiocarcinomas, we determined the pattern of expression of drug-metabolizing enzymes in this epithelium. We first demonstrated by blot analyses that biliary epithelial cells (BEC) isolated from human gallbladders display cytochrome P450 (CYP) 1A, 2E1, and 3A, microsomal epoxide hydrolase (mEH), ␣, , and glutathione S-transferase (GST), transcripts and proteins. We also identified CYPassociated steroid 6␤-hydroxylase activity in BEC. CYP and mEH expression was 5-to 20-fold lower in BEC than in autologous hepatocytes, and further differed by a higher ratio of CYP3A5/CYP3A4, and by CYP1A1 predominance over CYP1A2. ␣GST was highly expressed in both hepatocytes and BEC, while GST was restricted to BEC. In approximately 50% of individuals, GST was expressed in hepatocytes and at lower levels in BEC. By using the same antibodies as those used in immunoblots, we could show by immunohistochemistry that CYP2E1, CYP3A, mEH, ␣, , and GST immunoreactivities are expressed and display a heterogeneous distribution in the epithelium lining the entire biliary tract except for small intrahepatic bile ducts that were devoid of CYP3A and ␣GST immunoreactivities. In conclusion, BEC contribute to phase II, and although to a lesser extent than hepatocytes, to phase I biotransformation. The distribution of drug-metabolizing enzymes in BEC suggest that they are heterogeneous in their ability to generate and detoxicate reactive metabolites, which may contribute to specific distributions of cholangiopathies. (HEPATOLOGY 1999;30:1498-1506.)Foreign compounds including procarcinogens and drugs are widely distributed in our environment. The liver is the major organ responsible for their biotransformation into more hydrophilic products, and elimination through bile secretion. Biotransformation results from the activities of phase I and phase II metabolizing enzymes that are mainly, although not exclusively, located within hepatocytes.

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Section: Methodsmentioning

confidence: 99%

Phase I and Phase II drug-metabolizing enzymes are expressed and heterogeneously distributed in the biliary epithelium

et al. 1999

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“…The proteins were transferred onto a nitrocellulose membrane, blocked with 2% polyvinyl-pyrrolidone (PVP) and probed with a polyclonal anti-CYP2B6, 5 obtained in our lab after immunization of female New Zealand rabbits as previously described, 32 or a polyclonal antirat RED antibody (Daiichi pure chemicals Co. Ltd, Japan). After washing membranes, a secondary antibody linked to horseradish peroxidase (HRP) was used and membranes were developed using an ECL detection kit (Amersham).…”

Section: Western Blotsmentioning

confidence: 99%

A virus-directed enzyme prodrug therapy (VDEPT) strategy for lung cancer using a CYP2B6/NADPH-cytochrome P450 reductase fusion protein

Tychopoulos

Corcos

Genne³

et al. 2005

Cancer Gene Ther

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Virus-directed enzyme prodrug therapy (VDEPT) is an emerging strategy against cancer. Our approach is a P450-based VDEPT that consists of using cyclophosphamide (CPA) as a prodrug and a Cytochrome P450 2B6/NADPH cytochrome P450 reductase fusion protein (CYP2B6/RED) as a prodrug-activating enzyme. Due to the heterogenous expression of proteins in tumor cells, basal reductase activity may not be sufficient to supply CYP2B6 with electrons, the fusion protein should enable the expression of both proteins at high levels in tumor cells. CYP/RED fusion proteins have never been previously expressed in mammalian cells, to enable expression the fusion protein was cloned into an adenoviral vector and subsequently several pulmonary tumor cell lines were infected. The CYP2B6/RED fusion protein was detected by Western blot, its mRNA by Northern blot, and its heme incorporation into an active form by spectral analysis. Infection with the fusion gene increased RED activity in microsomes by a factor of 3 compared to the control. After infection and treatment with CPA, in cell lines with low endogenous RED, the fusion protein mediated significantly higher CPA-induced cytotoxicity compared to cells expressing solely CYP2B6. In conclusion, the fusion protein is functional for VDEPT by providing one protein for higher levels of CPA metabolism. Cancer Gene Therapy (2005) Keywords: VDEPT; P450-based gene therapy; cyclophosphamide; lung cancer G ene-directed enzyme prodrug therapy (GDEPT) 1,2 is an emerging strategy used to improve the selectivity of cancer chemotherapy. This is accomplished through tumor-specific activation of noncytotoxic prodrugs to active drugs by drug-metabolizing enzymes. The general scheme consists of transfection of tumor cells with an enzyme responsible for the bioactivation of a noncytotoxic prodrug and treatment with the prodrug: only cells expressing the transfected drug-metabolizing enzyme can metabolize the prodrug into cytotoxic metabolites, leading to cell death. The use of viral vectors for transgene introduction is more specifically referred to as virusdirected enzyme prodrug therapy (VDEPT). Following promising early results of a P450-based GDEPT strategy, 3 using the rat 9L gliosarcoma cell line, cyclophosphamide (CPA) as a prodrug and CYP2B1 as the prodrug activating enzyme, our approach was to improve the strategy by activating CPA with cytochrome P450 2B6 (CYP2B6) 4,5 or a CYP2B6/NADPH cytochrome P450 reductase (RED) fusion protein in order to avoid the frequent problem of low RED expression in tumor cells. This constructed protein is the fusion of two human proteins namely CYP2B6 and the human RED resulting in a single protein able to transfer electrons from NADPH right through to the heme moiety of the protein to enable metabolism of CYP2B6 substrates. In order to achieve high levels of expression in mammalian cells, the proteins were cloned into adenoviral vectors by homologous recombination.CPA belongs to the oxazaphosphorine class of molecules 6,7 and remains a widely used cytotoxic agent f...

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“…They had been raised against purified expressed CYPs, and they microsomes from four livers (HL 38,39,49,51). Incubations were performed at 37°C for 30 min in a shaker bath with recognize their specific antigens with a sensitivity of approximately 0.2 pmol but did not recognize 5 pmol of a microsomal protein content of 0.75 mg in the presence of MgCl 2 (2.5 mm), tris-HCl (50 mm, pH 7.4) and NADPH other P450 forms [24]. Bound antibodies were visualized with alkaline phosphatase conjugated anti-mouse or anti-(2 mm).…”

Section: Experiments With Specific Antibodiesmentioning

confidence: 99%

The involvement of CYP1A2 and CYP3A4 in the metabolism of clozapine

Eiermann

Engel

Johansson

et al. 1997

Brit J Clinical Pharma

194

113

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Aims Clozapine (CLZ), an atypical neuroleptic with a high risk of causing agranulocytosis, is metabolized in the liver to desmethylclozapine (DCLZ) and clozapine N-oxide (CLZ-NO). This study investigated the involvement of different CYP isoforms in the formation of these two metabolites. ; n=3). Formation of DCLZ was inhibited by fluvoxamine (53±28% at 10 mm), triacetyloleandomycin (33±15% at 10 mm), and ketoconazole (51±28% at 2 mm) and by antibodies against CYP1A2 and CYP3A4. CLZ-NO formation was inhibited by triacetyloleandomycin (34±16% at 10 mm) and ketoconazole (51±13% at 2 mm), and by antibodies against CYP3A4. There was a significant correlation between CYP3A content and DCLZ formation in microsomes from 15 human livers (r=0.67; P=0.04). A high but not significant correlation coefficient was found for CYP3A content and CLZ-NO formation (r=0.59; P=0.09). Using expression systems it was shown that CYP1A2 and CYP3A4 formed DCLZ and CLZ-NO. K m and V max values were lower in the CYP1A2 expression system compared to CYP3A4 for both metabolic reactions. Conclusions It is concluded that CYP1A2 and CYP3A4 are involved in the demethylation of CLZ and CYP3A4 in the N-oxidation of CLZ. Close monitoring of CLZ plasma levels is recommended in patients treated at the same time with other drugs affecting these two enzymes.

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Human cytochromes P450 expressed in Escherichia coli: production of specific antibodies

Cited by 42 publications

References 33 publications

Phase I and Phase II drug-metabolizing enzymes are expressed and heterogeneously distributed in the biliary epithelium

Phase I and Phase II drug-metabolizing enzymes are expressed and heterogeneously distributed in the biliary epithelium

A virus-directed enzyme prodrug therapy (VDEPT) strategy for lung cancer using a CYP2B6/NADPH-cytochrome P450 reductase fusion protein

The involvement of CYP1A2 and CYP3A4 in the metabolism of clozapine

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