Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.2.1.23 (beta-galactosidase)
 14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Fusion proteins constructed between beta-galactosidase and six different segments of either cytochrome P450IIB1 or cytochrome P450IIB2 (ranging from 18 to 33 amino acids in length) were expressed in Escherichia coli. Rabbit antibodies raised against these fusion proteins were first adsorbed through a beta-galactosidase column and then immunopurified on a second column containing the corresponding fusion protein. With the exception of the antibodies directed against the hydrophobic amino-terminal segment of cytochrome P450IIB1, all the antipeptide antibodies recognized the major phenobarbital-inducible cytochromes P450IIB1 and -IIB2 on immunoblots of liver microsomal proteins. Two of the antibodies were raised against regions where cytochromes P450IIB1 and -IIB2 differ in primary structure, and were differentially reactive toward these two highly homologous cytochromes. Several of the antipeptide antibodies were also reactive with a third phenobarbital-inducible microsomal protein expressed in livers of some individual Sprague-Dawley rats which was shown to be more highly related to P450IIB1 than P450IIB2. This P450IIB1-related P450, designated P450IIB1*, was purified to apparent homogeneity and shown to hydroxylate the steroid hormones testosterone and androstenedione with the well-defined regiospecificity and high catalytic activity characteristic of P450IIB1. A fourth microsomal protein detected using the antipeptide antibodies appeared to be more highly related to P450IIB2. Because the segments on the P450 molecules recognized by these antipeptide antibodies are known, it is possible to predict where P450IIB1* and the P450IIB2-related protein differ from cytochromes P450IIB2 and -IIB1, respectively. These studies demonstrate the utility of site-specific anti-P450 antibodies raised to fusion peptides for studies on the expression of structurally related P450s and polymorphic variants within the cytochrome P450 gene superfamily.
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PMID:Antibodies targeted against hypervariable and constant regions of cytochromes P450IIB1 and P450IIB2. 253 47

Cytochromes P450 are inserted into and anchored to the endoplasmic reticulum (ER) membrane by a hydrophobic signal sequence at the NH2 terminus. To determine whether the NH2-terminal sequence might also have an ER retention function, the NH2-terminal 29 amino acids of cytochrome P450 2C1, with and without an additional 29 amino acids containing an N-glycosylation site, were fused either to a soluble cytoplasmic protein, Escherichia coli beta-galactosidase, or to a secreted protein, E. coli alkaline phosphatase, and the hybrid proteins were expressed in COS1 cells. Subcellular fractionation indicated that both the beta-galactosidase and alkaline phosphatase hybrid proteins cosedimented with marker enzymes for ER membranes, and localization by immunofluorescent staining was consistent with an ER location. Hybrid proteins with the NH2-terminal glycosylation site were glycosylated in COS1 cells, and the carbohydrate moiety was sensitive to endoglycosidase H digestion, providing further evidence that the proteins were retained in the ER. In vitro studies of membrane insertion of the alkaline phosphatase hybrid indicated that fusion to alkaline phosphatase hybrid indicated that fusion to alkaline phosphatase did not alter the topological properties of the cytochrome P450 NH2-terminal sequence. In addition, alkaline phosphatase fused to the extracellular and transmembrane domains of epidermal growth factor receptor was transported to the plasma membrane in COS1 cells, which establishes that alkaline phosphatase as a cytoplasmic domain does not prevent transport from the ER. These observations indicate that the large cytoplasmic domain of cytochrome P450 is not required for retention in the ER and suggest that a specific sequence or structure within the NH2-terminal 29 amino acids functions as an ER retention signal.
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PMID:The amino-terminal 29 amino acids of cytochrome P450 2C1 are sufficient for retention in the endoplasmic reticulum. 836 Jan 66

The cancer chemotherapeutic agent cyclophosphamide (CPA) and its isomer ifosfamide (IFA) are alkylating agent prodrugs that require metabolism by liver cytochrome P450 (P450) enzymes for antitumor activity. The therapeutic effectiveness of these oxazaphosphorines is limited by the hematopoietic, renal, and cardiac toxicity that accompanies the systemic distribution of liver-derived activated drug metabolites. Transfer of a liver cytochrome P450 gene, CYP2B1, into human breast MCF-7 cancer cells is presently shown to greatly sensitize these cells to oxazaphosphorine toxicity as a consequence of the acquired capacity for intratumoral CPA and IFA activation. Thus, CPA and IFA were highly cytotoxic to MCF-7 cells following stable transfection of CYP2B1 but exhibited no toxicity to parental tumor cells or to a beta-galactosidase-expressing MCF-7 transfectant. This cytotoxicity could be appreciably blocked by the CYP2B1 inhibitor metyrapone. Cell cycle analysis revealed that CPA arrested the CYP2B1-expressing cells, but not CYP2B1-negative cells, at G(2)-M phase. A strong bystander cytotoxicity effect that does not require direct cell-cell contact was mediated by CYP2B1-expressing MCF-7 cells on non-CYP2B1 cells. Intratumoral CYP2B1 expression conferred a distinct therapeutic advantage when treating MCF-7 tumors grown in nude mice with CPA, as revealed by a 15-20-fold greater in vivo cytotoxicity, determined by tumor excision/colony formation assay, and by the substantially enhanced antitumor activity, monitored by tumor growth delay, for CYP2B1-e xpressing MCF-7 tumors as compared to CYP2B1-negative control tumors. These enhanced therapeutic effects were obtained without any apparent increase in host toxicity. To evaluate the extent to which a CPA/P450 gene therapy strategy may be generally applicable to other tumor cell types, a replication-defective recombinant adenovirus carrying the CYP2B1 gene driven by the cytomegalovirus (CMV) promotor ad.CMV-2B1 was constructed and used to infect a panel of human tumor cell lines. Ad.CMV-2B1 infection rendered each of the cell lines highly sensitive to CPA and IFA cytotoxicity, with substantial chemosensitization seen at multiplicities of infection as low as 10. The CPA/P450 prodrug activation system may thus serve as a useful paradigm for further development of novel cancer gene therapy strategies that utilize drug susceptibility genes to significantly potentiate the antitumor activity of conventional cancer chemotherapeutic agents.
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PMID:Sensitization of human breast cancer cells to cyclophosphamide and ifosfamide by transfer of a liver cytochrome P450 gene. 864 Aug 22

Using the mRNA differential display technique and Western blot analysis, the present study demonstrates that induction of KAR2 occurs when misfolded membrane-bound cytochrome P450, mutated in its cytosolically exposed domain, is expressed in Saccharomyces cerevisiae. Using various KAR2 promoter constructs in front of the Escherichia coli beta-galactosidase reporter gene, we found a fast and strong induction through the heat shock element (HSE), which was enhanced several fold by its adjacent GC-rich region. Additionally, a less pronounced induction was detected for the UPR element (UPRE). As expected, this response was absent in the ire1 disruptant strain. However, the HSE-mediated induction was enhanced upon disruption of IRE1 suggesting that the HSE pathway can compensate for the lack of a functional UPR pathway. Western blotting confirmed that Kar2p levels were increased to the same extent in the ire1 disruptant and in the non-disruptant strain. Removal of the P450 membrane-spanning region also abolished the UPRE-mediated induction of KAR2 transcription, but the HSE-mediated response remained. The data show for the first time that the transcription of KAR2 is significantly induced in response to a misfolded membrane-bound endoplasmic reticulum protein, and identifies the HSE and UPRE regions as KAR2 promoter elements responding to the misfolded cytosolic P450 domain and to the membrane-integrated mutant P450, respectively.
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PMID:Misfolded membrane-bound cytochrome P450 activates KAR2 induction through two distinct mechanisms. 1057 60

In vitro and in vivo effects of naringin on microsomal monooxygenase were studied to evaluate the drug interaction of this flavonoid. In vitro addition of naringin up to 500 microM had no effects on benzo(a)pyrene hydroxylase (AHH) activity of mouse liver microsomes. In contrast, the aglycone naringenin at 300 to 500 microM decreased AHH activity by 50% to 60%. Analysis of Lineweaver-Burk and Dixon plots indicated that naringenin competitively inhibited AHH activity with an estimated Ki of 39 microM. Naringenin at 100 microM also reduced metabolic activation of benzo(a)pyrene to genotoxic products as monitored by umuC gene expression response in Salmonella typhimurium TA1535/pSK1002. In the presence of equimolar naringenin and benzo(a)pyrene, umuC gene expression presented as beta-galactosidase activity was reduced to a level similar to the control value. Administration of a liquid diet containing 10 mg/ml naringin for 7 days caused 38% and 49% decreases of AHH and 7-methoxyresorufin O-demethylase activities, respectively. In contrast, the administration had no effects on cytochrome P450 (P450)-catalyzed oxidations of 7-ethoxyresorufin, 7-ethoxycoumarin, N-nitrosodimethylamine, nifedipine, erythromycin and testosterone. Microsomal P450 and cytochrome b5 contents and NADPH-P450 reductase activity were not affected. Immunoblot analysis using MAb 1-7-1, which immunoreacted with both P450 1A1 and 1A2, revealed that the level of P450 1A2 protein was decreased by 38%. These results demonstrate that naringenin is a potent inhibitor of AHH activity in vitro and naringin reduces the P450 1A2 protein level in vivo. These effects may indicate a chemopreventive role of naringin against protoxicants activated by P450 1A2.
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PMID:In vitro and in vivo effects of naringin on cytochrome P450-dependent monooxygenase in mouse liver. 1061 67

The cytochrome P450 mono-oxygenase system represents a major defence against chemical challenge from the environment, constituting part of an adaptive response mounted by an organism following exposure to harmful agents. Cytochrome P450s are also able to catalyse the activation of compounds to toxic products, and participate in a variety of essential 'housekeeping' functions, such as biosynthesis of steroid hormones and fatty acid oxidation. It is clear that the modulation of expression of these enzymes can have a significant effect on chemical toxicity, carcinogenicity and mutagenicity. The concept of cancer chemoprevention, i.e. the administration of a (non-toxic) chemical or dietary component in order to prevent neoplastic disease or to inhibit its progression, is an attractive one. Despite this, relatively little work has been done to characterize the ability of putative chemopreventive agents to modulate P450 expression, or to understand the interaction between P450s and chemopreventive agents. Before chemopreventive treatment can become a reality, it is essential that this complex issue is addressed; for instance, it is likely that any single chemopreventive agent will induce more than one P450 isoenzyme, and while altered expression of a particular P450 may attenuate the effects of one toxic agent, the effects of others might well be potentiated. Our laboratory has created a transgenic mouse line in which the rat CYP1A1 promoter drives expression of the beta-galactosidase gene. These mice can be used to define which compounds act via the Ah receptor, in which tissues, and at which stage of development. We are currently developing another mouse line in which beta1-galactosidase expression is controlled by the mouse GstA1 promoter, allowing us to define the role of the antioxidant responsive element in the action of chemopreventive agents. Finally, using cre-loxP transgenic technology, we have generated a mouse line in which P450 reductase can be deleted in a conditional, i.e. tissue-specific, manner, permitting us to investigate the role of P450s in chemoprevention in a more defined manner.
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PMID:Cytochrome P450s and chemoprevention. 1081 96

Vinca alkaloids are important chemotherapeutic agents, and their pharmacokinetic properties display significant interindividual variations, possibly due to CYP3A4-mediated metabolism. We have evaluated the relevance of this metabolism for the chemotherapeutic and the toxicological properties of these drugs. Analysis was performed using Chinese hamster ovary cell lines that expressed either CYP2D6 or CYP3A4. The latter cells metabolized vinblastine with a turnover number of 0.4 min(-1), resulting in a decreased cytotoxicity of this compound. Whereas vincristine and vinblastine at a concentration of 100 nM killed more than 90% of the parental cells, more than 50 and 35%, respectively, of cells that coexpressed CYP3A4 and cytochrome P450 (P450) reductase survived these treatments. No additional increase in cytotoxicity was noted above 100 nM. Similarly, preincubation of vinblastine with bacterial membranes that contained recombinant CYP3A4 and P450 reductase decreased the cytotoxicity of vinblastine for parental Chinese hamster ovary cells. We also demonstrate that the presence of vinblastine in a coculture of cells that expressed beta-galactosidase together with cells that expressed CYP3A4 strongly selected for the latter cells, resulting in an increased level of CYP3A4 in the surviving cell population. Similarly, treatment of the human colon adenocarcinoma cell line LS174T with vinblastine selected for a cell population with higher levels of endogenous CYP3A4 as revealed by immunohistochemistry without simultaneous increase of multidrug resistance protein 1 (MDR1). This is the first evidence that tumor P450s have the potential to contribute to the development of drug resistance during chemotherapy.
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PMID:Detoxication of vinca alkaloids by human P450 CYP3A4-mediated metabolism: implications for the development of drug resistance. 1087 37

Recent work suggesting that cellular oxidative stress exerts an inhibitory effect on aromatic hydrocarbon receptor (AHR)-dependent gene expression led us to test the hypothesis that pro-oxidant environmental pollutants might alter the induction of detoxification genes by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), an AHR ligand. We found that, in mouse hepatoma Hepa-1 cells, TCDD-inducible cytochrome P450, Cyp1a1, and nicotinamide adenine dinucleotide phosphate-quinone oxidoreductase (Nqo1) mRNA accumulation were differentially affected by cadmium (Cd(2+)), chromium (Cr(6+)), and arsenic (As(3+)). Cadmium or arsenic did not change Cyp1a1 mRNA levels but did enhance TCDD-inducible levels of Nqo1 mRNA, an effect that paralleled the ability of these metals to activate a beta-galactosidase gene reporter system regulated by an electrophile response promoter element. Chromium inhibited mRNA accumulation for both Cyp1a1 and Nqo1. Manipulation of cellular thiol status did not modify the response to combined chromium-TCDD exposure, suggesting that the response was not caused by oxidative stress. Chromium did not block DNA-binding competence of the AHR and did not have an effect on mRNA stability, but it inhibited Cyp1a1 gene transcription and the expression of an AHR-dependent luciferase reporter. These data indicate that coexposure to pro-oxidant metals and AHR ligands, which is common in the environment, can disrupt the regulation of phase I and phase II detoxification genes, leading to imbalances in gene expression that may have important consequences for the toxicity of complex mixtures.
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PMID:Disruption of dioxin-inducible phase I and phase II gene expression patterns by cadmium, chromium, and arsenic. 1097 92

The major adrenal steroid dehydroepiandrosterone (DHEA) enhances memory and immune function but has no known dedicated receptor; local metabolism may govern its activity. We described a cytochrome P450 expressed in brain and other tissues, CYP7B, that catalyzes the 7alpha-hydroxylation of oxysterols and 3beta-hydroxysteroids including DHEA. We report here that CYP7B mRNA and 7alpha-hydroxylation activity are widespread in rat tissues. However, steroids related to DHEA are reported to be modified at positions other than 7alpha, exemplified by prominent 6alpha-hydroxylation of 5alpha-androstane-3beta,17beta-diol (A/anediol) in some rodent tissues including brain. To determine whether CYP7B is responsible for these and other activities we disrupted the mouse Cyp7b gene by targeted insertion of an IRES-lacZ reporter cassette, placing reporter enzyme activity (beta-galactosidase) under Cyp7b promoter control. In heterozygous mouse brain, chromogenic detection of reporter activity was strikingly restricted to the dentate gyrus. Staining did not exactly reproduce the in situ hybridization expression pattern; post-transcriptional control is inferred. Lower level staining was detected in cerebellum, liver, and kidney, and which largely paralleled mRNA distribution. Liver and kidney expression was sexually dimorphic. Mice homozygous for the insertion are viable and superficially normal, but ex vivo metabolism of DHEA to 7alpha-hydroxy-DHEA was abolished in brain, spleen, thymus, heart, lung, prostate, uterus, and mammary gland; lower abundance metabolites were also eliminated. 7alpha-Hydroxylation of 25-hydroxycholesterol and related substrates was also abolished, as was presumed 6alpha-hydroxylation of A/anediol. These different enzyme activities therefore derive from the Cyp7b gene. CYP7B is thus a major extrahepatic steroid and oxysterol hydroxylase and provides the predominant route for local metabolism of DHEA and related molecules in brain and other tissues.
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PMID:Neurosteroid hydroxylase CYP7B: vivid reporter activity in dentate gyrus of gene-targeted mice and abolition of a widespread pathway of steroid and oxysterol hydroxylation. 1129 Jul 41

The importance of environmental and dietary arylamines, and heterocyclic amines in the etiology of human cancer is of growing interest. These pre-carcinogens are known to undergo bioactivation by cytochrome P450 (CYP)-directed oxidation, which then become substrates for the UDP-glucuronosyltransferases (UGTs). Thus, glucuronidation may contribute to the elimination of CYP-mediated reactive intermediate metabolites, preventing a toxic event. In this study, human UGTs were analyzed for their ability to modulate the mutagenic actions of N-hydroxy-arylamines formed by CYP1A2. Studies with recombinant human UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B4, UGT2B7 and UGT2B15 expressed in heterologous cell culture confirmed that UGT1A9 glucuronidated the mutagenic arylamines N-hydroxy-2-acetylaminofluorene (N-hydroxy-2AAF) and 2-hydroxyamino-1-methyl-6-phenylimidazo(4,5-b)pyridine (N-hydroxy-PhIP). To examine the mutagenic potential of these agents, a genotoxicity assay was employed using Salmonella typhimurium NM2009, a bacterial strain expressing the umuC SOS response gene fused to a beta-galactosidase reporter lacZ gene. DNA modification results in the induction of the umuC gene and subsequent enhancement of beta-galactosidase activity. Both N-hydroxy-2AAF and N-hydroxy-PhIP stimulated a dose-dependent increase in bacterial beta-galactosidase activity. In addition, the procarcinogens 2AAF and PhIP were efficiently bioactivated to bacterial mutagens when incubated with Escherichia coli membranes expressing CYP1A2 and NADPH reductase. CYP1A2 generated 2AAF- and PhIP-mediated DNA damage, but only the action of N-hydroxy-2AAF was blocked by expressed UGT1A9. These results indicate that UGT1A9 can control the outcome of a genotoxic response. The results also indicate that while a potential toxicant such as N-hydroxy-PhIP can serve as substrate for glucuronidation, its biological actions can exceed the capacity of the detoxification pathway to prevent the mutagenic episode.
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PMID:The contribution of UDP-glucuronosyltransferase 1A9 on CYP1A2-mediated genotoxicity by aromatic and heterocyclic amines. 1137 3


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