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1.
Int J Mol Sci ; 20(9)2019 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-31083300

RESUMO

TCDD-inducible poly-ADP-ribose polymerase (TIPARP) is an aryl hydrocarbon receptor (AHR) target gene that functions as part of a negative feedback loop to repress AHR activity. Tiparp-/- mice exhibit increased sensitivity to the toxicological effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), including lethal wasting syndrome. However, it is not known whether Tiparp-/- mice also exhibit increased sensitivity to other AHR ligands. In this study, we treated male Tiparp-/- or wild type (WT) mice with a single injection of 100 mg/kg 3-methylcholanthrene (3MC). Consistent with TIPARP's role as a repressor of AHR signaling, 3MC-treated Tiparp-/- mice exhibited increased hepatic Cyp1a1 and Cyp1b1 levels compared with WT mice. No 3MC-treated Tiparp-/- mice survived beyond day 16 and the mice exhibited chylous ascites characterized by an accumulation of fluid in the peritoneal cavity. All WT mice survived the 30-day treatment and showed no signs of fluid accumulation. Treated Tiparp-/- mice also exhibited a transient and mild hepatotoxicity with inflammation. 3MC-treated WT, but not Tiparp-/- mice, developed mild hepatic steatosis. Lipid deposits accumulated on the surface of the liver and other abdominal organs in the 3MC-Tiparp-/- mice. Our study reveals that Tiparp-/- mice have increased sensitivity to 3MC-induced liver toxicity, but unlike with TCDD, lethality is due to chylous ascites rather than wasting syndrome.


Assuntos
Ascite Quilosa/induzido quimicamente , Ascite Quilosa/enzimologia , Metilcolantreno/toxicidade , Poli(ADP-Ribose) Polimerases/metabolismo , Dibenzodioxinas Policloradas/toxicidade , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/patologia , Animais , Compostos Azo/farmacologia , Ascite Quilosa/patologia , Citocinas/metabolismo , Fígado Gorduroso/enzimologia , Fígado Gorduroso/patologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Inflamação/patologia , Mediadores da Inflamação/metabolismo , Fígado/efeitos dos fármacos , Fígado/enzimologia , Fígado/patologia , Masculino , Camundongos Knockout , Poli(ADP-Ribose) Polimerases/genética , Pirazóis/farmacologia , Receptores de Hidrocarboneto Arílico/metabolismo , Transdução de Sinais , Análise de Sobrevida
2.
Biochem J ; 2018 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-30373764

RESUMO

Here we report the biochemical characterization of the mono-ADP-ribosyltransferase 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) poly-ADP-ribose-polymerase (TIPARP/ARTD14/PARP7), which is known to repress aryl hydrocarbon receptor (AHR)-dependent transcription. We found that the nuclear localization of TIPARP was dependent on a short N-terminal sequence and its zinc finger domain. Deletion and in vitro ADP-ribosylation studies identified amino acids 400-657 as the minimum catalytically active region, which retained its ability to mono-ADP-ribosylate AHR. However, the ability of TIPARP to ADP-ribosylate and repress AHR in cells was dependent on both its catalytic activity and zinc-finger domain. The catalytic activity of TIPARP was resistant to meta-iodobenzylguanidine but sensitive to iodoacetamide and hydroxylamine, implicating cysteines and acidic side chains as ADP-ribosylated target residues. Mass spectrometry identified multiple ADP-ribosylated peptides in TIPARP and AHR. Electron transfer dissociation analysis of the TIPARP peptide 33ITPLKTCFK41 revealed cysteine 39 as a site for mono-ADP-ribosylation. Mutation of cysteine 39 to alanine resulted in a small, but significant, reduction in TIPARP auto-ribosylation activity, suggesting that additional amino acid residues are modified, but loss of cysteine 39 did not prevent its ability to repress AHR. Our findings characterize the subcellular localization and mono-ADP-ribosyltransferase activity of TIPARP, identify cysteine as a mono-ADP-ribosylated residue targeted by this enzyme, and confirm the TIPARP-dependent mono-ADP-ribosylation of other protein targets, such as AHR.

3.
Toxicol Sci ; 165(2): 347-360, 2018 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-29873790

RESUMO

The aryl hydrocarbon receptor (AHR) mediates the toxic effects of dioxin (2, 3, 7, 8-tetrachlorodibenzo-p-dioxin; TCDD), which includes thymic atrophy, steatohepatitis, and a lethal wasting syndrome in laboratory rodents. Although the mechanisms of dioxin toxicity remain unknown, AHR signaling in hepatocytes is necessary for dioxin-induced liver toxicity. We previously reported that loss of TCDD-inducible poly(adenosine diphosphate [ADP]-ribose) polymerase (TIPARP/PARP7/ARTD14), an AHR target gene and mono-ADP-ribosyltransferase, increases the sensitivity of mice to dioxin-induced toxicities. To test the hypothesis that TIPARP is a negative regulator of AHR signaling in hepatocytes, we generated Tiparpfl/fl mice in which exon 3 of Tiparp is flanked by loxP sites, followed by Cre-lox technology to create hepatocyte-specific (Tiparpfl/flCreAlb) and whole-body (Tiparpfl/flCreCMV; TiparpEx3-/-) Tiparp null mice. Tiparpfl/flCreAlb and TiparpEx3-/- mice given a single injection of 10 µg/kg dioxin did not survive beyond days 7 and 9, respectively, while all Tiparp+/+ mice survived the 30-day treatment. Dioxin-exposed Tiparpfl/flCreAlb and TiparpEx3-/- mice had increased steatohepatitis and hepatotoxicity as indicated by greater staining of neutral lipids and serum alanine aminotransferase activity than similarly treated wild-type mice. Tiparpfl/flCreAlb and TiparpEx3-/- mice exhibited augmented AHR signaling, denoted by increased dioxin-induced gene expression. Metabolomic studies revealed alterations in lipid and amino acid metabolism in liver extracts from Tiparpfl/flCreAlb mice compared with wild-type mice. Taken together, these data illustrate that TIPARP is an important negative regulator of AHR activity, and that its specific loss in hepatocytes is sufficient to increase sensitivity to dioxin-induced steatohepatitis and lethality.

4.
FEBS Lett ; 591(14): 2147-2154, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28649707

RESUMO

The third step of protein splicing is cyclization of Asn coupled to peptide bond cleavage. In two related cyanobacterial inteins, this step is facilitated by Asn or Gln. For a Synechococcus sp. PCC7002 intein, the isolated third step of protein splicing is more efficient with its native Asn than with substitution to Gln. For a Trichodesmium erythraeum intein, its native Gln facilitates the third step as efficiently as with Asn. Despite these differences, the yield of splicing is not affected, suggesting that the third step is influenced by mechanism-linked conformational changes. A conserved catalytic His and the penultimate residue also play roles in promoting side-chain cyclization.


Assuntos
Inteínas/genética , Processamento de Proteína , Synechococcus/genética , Trichodesmium/genética , Mutação
5.
Biochemistry ; 56(8): 1042-1050, 2017 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-28165720

RESUMO

Protein splicing is a post-translational reaction facilitated by an intein, or intervening protein, which involves the removal of the intein and the ligation of the flanking polypeptides, or exteins. A DNA polymerase II intein from Pyrococcus abyssi (Pab PolII intein) can promote protein splicing in vitro on incubation at high temperature. Mutation of active site residues Cys1, Gln185, and Cys+1 to Ala results in an inactive intein precursor, which cannot promote the steps of splicing, including cleavage of the peptide bond linking the N-extein and intein (N-terminal cleavage). Surprisingly, coupling the inactivating mutations to a change of the residue at the C-terminus of the N-extein (N-1 residue) from the native Asn to Asp reactivates N-terminal cleavage at pH 5. Similar "aspartic acid effects" have been observed in other proteins and peptides but usually only occur at lower pH values. In this case, however, the unusual N-terminal cleavage is abolished by mutations to catalytic active site residues and unfolding of the intein, indicating that this cleavage effect is mediated by the intein active site and the intein fold. We show via mass spectrometry that the reaction proceeds through cyclization of Asp resulting in anhydride formation coupled to peptide bond cleavage. Our results add to the richness of the understanding of the mechanism of protein splicing and provide insight into the stability of proteins at moderately low pH. The results also explain, and may help practitioners avoid, a side reaction that may complicate intein applications in biotechnology.


Assuntos
Ácido Aspártico/metabolismo , DNA Polimerase II/química , Inteínas , Ácido Aspártico/química , Domínio Catalítico , Ciclização , DNA Polimerase II/genética , DNA Polimerase II/metabolismo , Temperatura Alta , Concentração de Íons de Hidrogênio , Mutação , Processamento de Proteína Pós-Traducional , Pyrococcus abyssi/enzimologia
6.
Sci Rep ; 7: 40539, 2017 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-28079158

RESUMO

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor historically known for its toxic responses to man-made pollutants such as dioxin. More recently, the AhR has emerged as a suppressor of inflammation, oxidative stress and apoptosis from cigarette smoke by mechanisms that may involve the regulation of microRNA. However, little is known about the AhR regulation of miRNA expression in the lung in response to inhaled toxicants. Therefore, we exposed Ahr-/- and Ahr+/- mice to cigarette smoke for 4 weeks and evaluated lung miRNA expression by PCR array. There was a dramatic regulation of lung miRNA by the AhR in the absence of exogenous ligand. In response to cigarette smoke, there were more up-regulated miRNA in Ahr-/- mice compared to Ahr+/- mice, including the cancer-associated miRNA miR-96. There was no significant change in the expression of the AhR regulated proteins HuR and cyclooxygenase-2 (COX-2). There were significant increases in the anti-oxidant gene sulfiredoxin 1 (Srxn1) and FOXO3a- predicted targets of miR-96. Collectively, these data support a prominent role for the AhR in regulating lung miRNA expression. Further studies to elucidate a role for these miRNA may further uncover novel biological function for the AhR in respiratory health and disease.


Assuntos
Regulação da Expressão Gênica , Pulmão/metabolismo , MicroRNAs/genética , Receptores de Hidrocarboneto Arílico/metabolismo , Fumar/efeitos adversos , Fumar/genética , Animais , Quimiotaxia/genética , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Citocinas/metabolismo , Proteína Semelhante a ELAV 1/genética , Proteína Semelhante a ELAV 1/metabolismo , Proteína Forkhead Box O3/genética , Proteína Forkhead Box O3/metabolismo , Deleção de Genes , Cinética , Pulmão/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , MicroRNAs/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/genética , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , Receptores de Hidrocarboneto Arílico/deficiência
7.
Free Radic Biol Med ; 89: 342-57, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26408075

RESUMO

The aryl hydrocarbon receptor (AhR) is a ubiquitously expressed receptor/transcription factor that mediates toxicological responses of environmental contaminants such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Emerging evidence indicates that the AhR suppresses apoptosis and proliferation independent of exogenous ligands, including suppression of apoptosis by cigarette smoke, a key risk factor for chronic obstructive pulmonary disease (COPD). As cigarette smoke is a potent inducer of oxidative stress, a feature that may contribute to the development of COPD, we hypothesized that the AhR prevents smoke-induced apoptosis by regulating oxidative stress. Utilizing primary lung fibroblasts derived from AhR(+/+) and AhR(-/-) mice as well as A549 human lung adenocarcinoma cells deficient in AhR expression (A549-AhR(ko)), we first show that AhR(-/-) fibroblasts and A549-AhR(ko) epithelial cells have a significant increase in cigarette smoke extract (CSE)-induced oxidative stress compared to wild type. CSE induced a significant increase in the mRNA expression of key antioxidant genes, including Nqo1 and Srxn1, predominantly in AhR(+/+) fibroblasts, with significantly less induction in AhR(-/-) cells. The induction of Srxn1, but not Nqo1, was independent of dioxin-response element (DRE) binding as AhR(DBD/DBD) lung fibroblasts, which express an AhR incapable of binding the DRE, increased Srxn1 to a degree similar to wild-type cells in response to CSE. There was no difference in Nrf2 expression or activation based on AhR expression. Lung fibroblasts derived from COPD subjects have significantly less AhR protein expression compared with both never-smokers (Normal) and smokers (At Risk). Consequently, COPD-derived fibroblasts were less robust in their induction of both Nqo1 and Srxn1 mRNA after exposure to CSE, which also failed to activate the AhR in the COPD fibroblasts. Taken together, these results support a new role for the AhR in regulating antioxidant defense in lung structural cells, such that low AhR expression may facilitate the development or progression of COPD.


Assuntos
Estresse Oxidativo/efeitos dos fármacos , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , Dibenzodioxinas Policloradas/metabolismo , Doença Pulmonar Obstrutiva Crônica/metabolismo , Receptores de Hidrocarboneto Arílico/fisiologia , Elementos de Resposta/genética , Fumaça/efeitos adversos , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Estudos de Casos e Controles , Células Cultivadas , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Técnicas Imunoenzimáticas , Pulmão/citologia , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/genética , Doença Pulmonar Obstrutiva Crônica/etiologia , Doença Pulmonar Obstrutiva Crônica/patologia , RNA Mensageiro/genética , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa
8.
J Biol Chem ; 290(27): 16824-40, 2015 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-25975270

RESUMO

The aryl hydrocarbon receptor (AHR) mediates the toxic effects of the environmental contaminant dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin; TCDD). Dioxin causes a range of toxic responses, including hepatic damage, steatohepatitis, and a lethal wasting syndrome; however, the mechanisms are still unknown. Here, we show that the loss of TCDD-inducible poly(ADP-ribose) polymerase (Tiparp), an ADP-ribosyltransferase and AHR repressor, increases sensitivity to dioxin-induced toxicity, steatohepatitis, and lethality. Tiparp(-/-) mice given a single injection of 100 µg/kg dioxin did not survive beyond day 5; all Tiparp(+/+) mice survived the 30-day treatment. Dioxin-treated Tiparp(-/-) mice exhibited increased liver steatosis and hepatotoxicity. Tiparp ADP-ribosylated AHR but not its dimerization partner, the AHR nuclear translocator, and the repressive effects of TIPARP on AHR were reversed by the macrodomain containing mono-ADP-ribosylase MACROD1 but not MACROD2. These results reveal previously unidentified roles for Tiparp, MacroD1, and ADP-ribosylation in AHR-mediated steatohepatitis and lethality in response to dioxin.


Assuntos
Dioxinas/toxicidade , Fígado Gorduroso/enzimologia , Fígado Gorduroso/mortalidade , Poli(ADP-Ribose) Polimerases/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fígado Gorduroso/induzido quimicamente , Fígado Gorduroso/genética , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Poli(ADP-Ribose) Polimerases/genética , Receptores de Hidrocarboneto Arílico/genética , Receptores de Hidrocarboneto Arílico/metabolismo
9.
Toxicol Appl Pharmacol ; 280(3): 511-25, 2014 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-25178717

RESUMO

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor implicated in the regulation of apoptosis and proliferation. Although activation of the AhR by xenobiotics such as dioxin inhibits the cell cycle and control apoptosis, paradoxically, AhR expression also promotes cell proliferation and survival independent of exogenous ligands. The microRNA (miRNA) miR-196a has also emerged as a regulator of proliferation and apoptosis but a relationship between the AhR and miR-196a is not known. Therefore, we hypothesized that AhR-dependent regulation of endogenous miR-196a expression would promote cell survival and proliferation. Utilizing lung fibroblasts from AhR deficient (AhR(-/-)) and wild-type (AhR(+/+)) mice, we show that there is ligand-independent regulation of miRNA, including low miR-196a in AhR(-/-) cells. Validation by qRT-PCR revealed a significant decrease in basal expression of miR-196a in AhR(-/-) compared to AhR(+/+) cells. Exposure to AhR agonists benzo[a]pyrene (B[a]P) and FICZ as well as AhR antagonist CH-223191 decreased miR-196a expression in AhR(+/+) fibroblasts concomitant with decreased AhR protein levels. There was increased proliferation only in AhR(+/+) lung fibroblasts in response to serum, corresponding to a decrease in p27(KIP1) protein, a cyclin-dependent kinase inhibitor. Increasing the cellular levels of miR-196a had no effect on proliferation or expression of p27(KIP1) in AhR(-/-) fibroblasts but attenuated cigarette smoke-induced apoptosis. This study provides the first evidence that AhR expression is essential for the physiological regulation of cellular miRNA levels- including miR-196a. Future experiments designed to elucidate the functional relationship between the AhR and miR-196a may delineate additional novel ligand-independent roles for the AhR.


Assuntos
Apoptose/fisiologia , Fibroblastos/metabolismo , Regulação da Expressão Gênica/fisiologia , Pulmão/metabolismo , MicroRNAs/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Animais , Compostos Azo/farmacologia , Benzo(a)pireno/farmacologia , Western Blotting , Carbazóis/farmacologia , Proliferação de Células/fisiologia , Inibidor de Quinase Dependente de Ciclina p27/genética , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Imuno-Histoquímica , Pulmão/citologia , Camundongos Knockout , MicroRNAs/genética , Pirazóis/farmacologia , RNA/química , RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa
10.
Cell ; 156(5): 907-19, 2014 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-24581492

RESUMO

Recent studies recognize a vast diversity of noncoding RNAs with largely unknown functions, but few have examined interspersed repeat sequences, which constitute almost half our genome. RNA hybridization in situ using C0T-1 (highly repeated) DNA probes detects surprisingly abundant euchromatin-associated RNA comprised predominantly of repeat sequences (C0T-1 RNA), including LINE-1. C0T-1-hybridizing RNA strictly localizes to the interphase chromosome territory in cis and remains stably associated with the chromosome territory following prolonged transcriptional inhibition. The C0T-1 RNA territory resists mechanical disruption and fractionates with the nonchromatin scaffold but can be experimentally released. Loss of repeat-rich, stable nuclear RNAs from euchromatin corresponds to aberrant chromatin distribution and condensation. C0T-1 RNA has several properties similar to XIST chromosomal RNA but is excluded from chromatin condensed by XIST. These findings impact two "black boxes" of genome science: the poorly understood diversity of noncoding RNA and the unexplained abundance of repetitive elements.


Assuntos
Cromossomos de Mamíferos/química , Eucromatina/química , Interfase , RNA não Traduzido/análise , Animais , Núcleo Celular/química , Humanos , Células Híbridas , Elementos Nucleotídeos Longos e Dispersos , Camundongos , RNA não Traduzido/genética , Sequências Repetitivas de Ácido Nucleico , Transcrição Genética
11.
Mol Pharmacol ; 78(6): 1004-11, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20805301

RESUMO

Cytochrome P450 2W1 (CYP2W1) is expressed at high levels in colorectal cancer cells. Moreover, we have shown previously that a higher tumor expression is associated with less survival. In this study, we characterize post-translational modification, inverted endoplasmic reticulum (ER) topology, and catalytic activity of CYP2W1. The analysis of colorectal normal and cancer tissues and CYP2W1 overexpressing human embryonic kidney (HEK) 293 cells showed that a fraction of CYP2W1 is modified by N-glycosylation. Bioinformatic analysis identified Asn177 as the only possible glycosylation site of CYP2W1, which was supported by the inability of an N177A mutant to be glycosylated in HEK 293 cells. Analysis of the membrane topology indicated that unlike other cytochromes P450, CYP2W1 in HEK 293-transfected cells and in nontransfected Caco2TC7 and HepG2 cells is oriented toward the lumen of the ER, a topology making CYP2W1 available to the ER glycosylation machinery. Immunofluorescence microscopy and cell surface biotinylation experiments revealed approximately 8% of the CYP2W1 on the cell surface. Despite the reverse orientation of CYP2W1 in the ER membrane, apparently making functional interactions with NADPH-cytochrome P450 reductase impossible, CYP2W1 in HEK 293 cells was active in the metabolism of indoline substrates and was able to activate aflatoxin B1 into cytotoxic products. The study identifies for the first time a cytochrome P450 enzyme with a luminal ER orientation and still retaining catalytic activity. Together, these results suggest the possibility of using CYP2W1 as a drug target in the treatment of colon cancer using antibodies and/or specific CYP2W1 activated prodrugs.


Assuntos
Domínio Catalítico/fisiologia , Neoplasias Colorretais/enzimologia , Sistema Enzimático do Citocromo P-450/metabolismo , Líquido Intracelular/enzimologia , Aflatoxina B1/metabolismo , Aflatoxina B1/toxicidade , Asparagina/metabolismo , Biotransformação , Células CACO-2 , Linhagem Celular Tumoral , Neoplasias Colorretais/metabolismo , Sistema Enzimático do Citocromo P-450/biossíntese , Sistema Enzimático do Citocromo P-450/genética , Família 2 do Citocromo P450 , Flavoproteínas Transferidoras de Elétrons/metabolismo , Glicosilação , Células HEK293 , Células Hep G2 , Humanos , Indóis/metabolismo , Líquido Intracelular/metabolismo
12.
Pharmacogenomics ; 11(5): 625-7, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20415549

RESUMO

A significant number of instances of interindividual variability in drug response, where a clear phenotypic consequence is evident in the population, have not yet been associated with variations in gene sequence. Epigenetics, along with, for example, drug interactions and disease, provides answers to some of these inconsistencies. The role of epigenetics in drug response has just started to be perceived, but its impending influence on drug metabolism and disposition promises to be important. Research in this area can provide us with novel mechanisms for this variation and also with novel biomarkers that can be useful for understanding drug response, as well as for the development of new drugs and which can, in addition, constitute predictive biomarkers for today's and tomorrow's pharmacotherapy, yielding a substantial improvement in human health.


Assuntos
Preparações Farmacêuticas/metabolismo , Interações de Medicamentos , Previsões , Humanos , Inativação Metabólica/genética
13.
Hum Genet ; 127(1): 1-17, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19823875

RESUMO

The cytochromes P450 (CYPs) are very efficient catalysts of foreign compound metabolism and are responsible for the major part of metabolism of clinically important drugs. The enzymes are important in cancer since they (a) activate dietary and environmental components to ultimate carcinogens, (b) activate or inactivate drugs used for cancer treatment, and (c) are potential targets for anticancer therapy. The genes encoding the CYP enzymes active in drug metabolism are highly polymorphic, whereas those encoding metabolism of precarcinogens are relatively conserved. A vast amount of literature is present where investigators have tried to link genetic polymorphism in CYPs to cancer susceptibility, although not much conclusive data have hitherto been obtained, with exception of CYP2A6 polymorphism and tobacco induced cancer, to a great extent because of lack of important functional polymorphisms in the genes studied. With respect to anticancer treatment, the genetic CYP polymorphism is of greater importance, where treatment with tamoxifen, but also with cyclophosphamide and maybe thalidomide is influenced by CYP genetic variants. In the present review we present updates on CYP genetics, cancer risk and treatment and also epigenetic aspects of interindividual variability in CYP expression and the use of these enzymes as targets for cancer therapy. We conclude that the CYP polymorphism does not predict cancer susceptibility to any large extent but that this polymorphism might be an important factor for optimal cancer therapy using selected anticancer agents.


Assuntos
Antineoplásicos/uso terapêutico , Sistema Enzimático do Citocromo P-450/genética , Epigênese Genética , Neoplasias/tratamento farmacológico , Neoplasias/genética , Animais , Antineoplásicos/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Predisposição Genética para Doença , Humanos , Família Multigênica , Polimorfismo Genético , Fatores de Risco
14.
Extremophiles ; 13(6): 905-15, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19763742

RESUMO

Strains of hyperthermophilic anaerobic hydrothermal vent archaea maintained in the culture collection assembled by Holger Jannasch at the Woods Hole Oceanographic Institution between 1984 and 1998 were identified and partially characterized by Denaturing Gradient Gel Electrophoresis, 16S rRNA gene sequencing, and by growth tests at different temperatures and on different organic carbon and nitrogen sources. All strains were members of the genera Thermococcus and Pyrococcus. The greatest phylogenetic diversity was found in strains from a single Guaymas Basin core isolated by serial dilution from four different depth horizons of heated sediment incubated at the corresponding in situ temperatures. In contrast, geographically distinct vent locations and sample materials yielded a lower diversity of isolates when enriched under uniform temperature regimes and without prior dilution of the source material.


Assuntos
Sedimentos Geológicos/microbiologia , Fontes Termais/microbiologia , Pyrococcus/isolamento & purificação , Thermococcus/isolamento & purificação , Técnicas Bacteriológicas , Meios de Cultura , DNA Bacteriano/genética , Biologia Marinha , México , Dados de Sequência Molecular , Filogenia , Pyrococcus/classificação , Pyrococcus/genética , Pyrococcus/crescimento & desenvolvimento , Pyrococcus/metabolismo , RNA Bacteriano/genética , RNA Ribossômico 16S/genética , Ribotipagem , Especificidade da Espécie , Temperatura Ambiente , Thermococcus/classificação , Thermococcus/genética , Thermococcus/crescimento & desenvolvimento , Thermococcus/metabolismo
15.
Pharmacogenomics ; 10(7): 1067-76, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19604079

RESUMO

Although pharmacogenetics has been instrumental in describing interindividual variations in drug metabolism, epigenetic factors offer another blanket of information that could give a more vivid picture and help in developing a more personalized therapy. The dynamic aspect of epigenetics could likewise provide more definite answers to the role of changing environmental factors in drug response: the bridge that connects the environment to the genome. In this review we discuss known epigenetic and microRNA-dependent regulation of the human drug-metabolizing cytochromes P450 to help explain the unknown factors of variable drug response.


Assuntos
Sistema Enzimático do Citocromo P-450/genética , DNA/genética , Epigênese Genética/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , MicroRNAs/fisiologia , Biossíntese de Proteínas/genética , Animais , Sistema Enzimático do Citocromo P-450/biossíntese , DNA/biossíntese , Humanos , MicroRNAs/biossíntese , MicroRNAs/genética
16.
Pharmacogenomics J ; 9(3): 208-17, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19274061

RESUMO

The basis for interindividual variation in the CYP1A2 gene expression is not fully understood and the known genetic polymorphisms in the gene provide no explanation. We investigated whether the CYP1A2 gene expression is regulated by DNA methylation and displays allele-specific expression (ASE) using 65 human livers. Forty-eight percent of the livers displayed ASE not associated to the CYP1A2 mRNA levels. The extent of DNA methylation of a CpG island including 17 CpG sites, close to the translation start site, inversely correlated with hepatic CYP1A2 mRNA levels (P=0.018). The methylation of two separate core CpG sites was strongly associated with the CYP1A2 mRNA levels (P=0.005) and ASE phenotype (P=0.01), respectively. The CYP1A2 expression in hepatoma B16A2 cells was strongly induced by treatment with 5-aza-2'-deoxycytidine. In conclusion, the CYP1A2 gene expression is influenced by the extent of DNA methylation and displays ASE, mechanisms contributing to the large interindividual differences in CYP1A2 gene expression.


Assuntos
Alelos , Citocromo P-450 CYP1A2/genética , Metilação de DNA , Regulação Enzimológica da Expressão Gênica , Fígado/enzimologia , RNA Mensageiro/metabolismo , Sequência de Bases , DNA , Humanos , Dados de Sequência Molecular , Fenótipo , Reação em Cadeia da Polimerase , RNA Mensageiro/genética
17.
Expert Rev Clin Pharmacol ; 2(1): 55-65, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24422771

RESUMO

Our knowledge of epigenetics has increased in recent times and its role in various aspects of cellular physiology and disease cannot be overemphasized, even though many issues still need to be clarified. The role of epigenetics in drug therapy is one aspect that necessitates more work. Although a few epigenetic drugs are already being used clinically and others are being developed for such use, other aspects of drug therapy that are affected by epigenetic alterations need to be considered. We want to emphasize the role of environment as an important factor that modifies the epigenome, creating variation among individuals and that can ultimately affect how they respond to drug therapy. The numerous gene products that are being utilized as drug targets can likewise be affected epigenetically and would thus require special attention.

18.
Pharmacogenomics ; 8(10): 1315-25, 2007 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17979506

RESUMO

INTRODUCTION: CYP2W1 is a novel enzyme shown to be selectively expressed in rat fetal colon and in human colon cancer and has previously been suggested as a potential drug target for cancer therapy. Here, the expression and gene methylation of CYP2W1 were analyzed in human colon carcinoma cell lines, colon tumors and in corresponding normal colon tissue. METHODS: CYP2W1 mRNA and protein expression in HepG2 and Caco-2TC7 cells and normal colon and colon tumor tissue samples were analyzed using real-time PCR and Western blotting. CYP2W1 gene methylation status in the same samples was analyzed using the sodium bisulfite sequencing method. RESULTS & DISCUSSION: CYP2W1 mRNA was detected in all (n = 39) tumor samples analyzed. Moreover, in 60% (12/20) of the colon tumors, CYP2W1 mRNA levels were substantially higher than in corresponding normal tissues. CYP2W1 protein was detected in most of the colon tumor samples analyzed (n = 16), which appeared to be of two apparent phenotypes: those with five- to ten-fold induced CYP2W1 (approximately 50% of the tumors), and those with low expression, harboring similar or only slightly higher amounts of CYP2W1 as compared with surrounding control tissue. Methylation analysis of the CpG island in the exon 1-intron 1 junction of the CYP2W1 gene from both cell lines, tumors and normal tissues revealed that demethylated CpG dinucleotides appeared as a requirement for high CYP2W1 gene expression. CONCLUSION: The expression of CYP2W1 is colon tumor-specific and is associated with methylation status of the CYP2W1 gene, suggesting a potential causal link between the gene hypomethylation and its enhanced expression.


Assuntos
Neoplasias do Colo/genética , Sistema Enzimático do Citocromo P-450/genética , Metilação de DNA , Regulação Neoplásica da Expressão Gênica , Oxigenases de Função Mista/genética , Carcinoma Hepatocelular/enzimologia , Carcinoma Hepatocelular/genética , Linhagem Celular Tumoral , Colo/enzimologia , Neoplasias do Colo/enzimologia , Família 2 do Citocromo P450 , DNA de Neoplasias/genética , DNA de Neoplasias/isolamento & purificação , Humanos , Neoplasias Hepáticas/enzimologia , Neoplasias Hepáticas/genética , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , Valores de Referência
19.
Pharmacol Ther ; 116(3): 496-526, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18001838

RESUMO

The polymorphic nature of the cytochrome P450 (CYP) genes affects individual drug response and adverse reactions to a great extent. This variation includes copy number variants (CNV), missense mutations, insertions and deletions, and mutations affecting gene expression and activity of mainly CYP2A6, CYP2B6, CYP2C9, CYP2C19 and CYP2D6, which have been extensively studied and well characterized. CYP1A2 and CYP3A4 expression varies significantly, and the cause has been suggested to be mainly of genetic origin but the exact molecular basis remains unknown. We present a review of the major polymorphic CYP alleles and conclude that this variability is of greatest importance for treatment with several antidepressants, antipsychotics, antiulcer drugs, anti-HIV drugs, anticoagulants, antidiabetics and the anticancer drug tamoxifen. We also present tables illustrating the relative importance of specific common CYP alleles for the extent of enzyme functionality. The field of pharmacoepigenetics has just opened, and we present recent examples wherein gene methylation influences the expression of CYP. In addition microRNA (miRNA) regulation of P450 has been described. Furthermore, this review updates the field with respect to regulatory initiatives and experience of predictive pharmacogenetic investigations in the clinics. It is concluded that the pharmacogenetic knowledge regarding CYP polymorphism now developed to a stage where it can be implemented in drug development and in clinical routine for specific drug treatments, thereby improving the drug response and reducing costs for drug treatment.


Assuntos
Sistema Enzimático do Citocromo P-450/genética , Epigênese Genética , Farmacogenética , Polimorfismo Genético , Animais , Doenças Cardiovasculares/tratamento farmacológico , Doenças Cardiovasculares/genética , Mapeamento Cromossômico , Metilação de DNA , Depressão/tratamento farmacológico , Depressão/genética , Gastroenteropatias/tratamento farmacológico , Gastroenteropatias/genética , Dosagem de Genes , Humanos , MicroRNAs/fisiologia , Neoplasias/tratamento farmacológico , Neoplasias/genética , Personalidade/genética , Polimorfismo de Nucleotídeo Único
20.
Biochem Biophys Res Commun ; 341(2): 451-8, 2006 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-16426568

RESUMO

A novel human cytochrome P450, CYP2W1, was cloned and expressed heterologously. No or very low CYP2W1 mRNA levels were detected in fetal and adult human tissues, expression was however seen in 54% of human tumor samples investigated (n=37), in particular colon and adrenal tumors. Western blotting also revealed high expression of CYP2W1 in some human colon tumors. In rat tissues, CYP2W1 mRNA was expressed preferentially in fetal but also in adult colon. The CYP2W1 gene was shown to encompass one functional CpG island in the exon 1-intron 1 region which was methylated in cell lines lacking CYP2W1 expression, but unmethylated in cells expressing CYP2W1. Re-expression of CYP2W1 was seen following demethylation by 5-Aza-2'-deoxycytidine. Transfection of HEK293 cells with CYP2W1 caused the formation of a properly folded enzyme, which was catalytically active with arachidonic acid as a substrate. It is concluded that CYP2W1 represents a tumor-specific P450 isoform with potential importance as a drug target in cancer therapy.


Assuntos
Colo/metabolismo , Sistema Enzimático do Citocromo P-450/biossíntese , Regulação Neoplásica da Expressão Gênica , Regulação da Expressão Gênica , Oxigenases de Função Mista/biossíntese , Neoplasias das Glândulas Suprarrenais/metabolismo , Animais , Ácido Araquidônico/metabolismo , Azacitidina/análogos & derivados , Azacitidina/metabolismo , Western Blotting , Catálise , Linhagem Celular , Linhagem Celular Tumoral , Clonagem Molecular , Colo/embriologia , Neoplasias do Colo/metabolismo , Ilhas de CpG , Sistema Enzimático do Citocromo P-450/genética , Família 2 do Citocromo P450 , Metilação de DNA , Decitabina , Éxons , Humanos , Íntrons , Dobramento de Proteína , Isoformas de Proteínas , RNA Mensageiro/metabolismo , Ratos , Transfecção
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