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1.
Planta Med ; 67(8): 737-42, 2001 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-11731916

RESUMO

This research describes the identification of three new high molecular weight polysaccharide preparations isolated from food-grade microalgae that are potent activators of human monocytes/macrophages: "Immulina" from Spirulina platensis, "Immunon" from Aphanizomenon flos-aquae, and "Immurella" from Chlorella pyrenoidosa. These polysaccharides are structurally complex and have estimated molecular weights above ten million daltons. All three polysaccharides are highly water soluble and comprise between 0.5 % and 2.0 % of microalgal dry weight. Immunostimulatory activity was measured using a transcription factor-based bioassay for nuclear factor kappa B (NF-kappa B) activation in THP-1 human monocytes/macrophages. Using this system the EC(50) values for these microalgal polysaccharides are between 20 and 110 ng/ml (about 10pM). THP-1 activation was confirmed by measuring immune cytokine mRNA induction using reverse transcriptase-polymerase chain reaction (RT-PCR). Each polysaccharide substantially increased mRNA levels of interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha). These polysaccharides are between one hundred and one thousand times more active for in vitro monocyte activation than polysaccharide preparations that are currently used clinically for cancer immunotherapy.


Assuntos
Adjuvantes Imunológicos/isolamento & purificação , Chlorella/química , Cianobactérias/química , Preparações de Plantas/isolamento & purificação , Polissacarídeos/isolamento & purificação , Adjuvantes Imunológicos/química , Humanos , Interleucina-1/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Peso Molecular , Monócitos/efeitos dos fármacos , Monócitos/imunologia , NF-kappa B/imunologia , Preparações de Plantas/farmacologia , Polissacarídeos/química , Polissacarídeos/farmacologia , Polissacarídeos/uso terapêutico , Polissacarídeos Bacterianos , Fator de Necrose Tumoral alfa/metabolismo
2.
J Biomol Screen ; 6(2): 101-10, 2001 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-11689104

RESUMO

Cyclooxygenase-2 (COX-2) is a recognized target for cancer prevention and possibly treatment. To identify novel inhibitors of COX-2, we developed a high throughput reporter gene assay that utilizes a region of the human COX-2 promoter to drive luciferase expression. A total of 968 extracts from 266 plants were screened. Extracts from 12 plants (4.5%), including Arnebia euchroma, a medicinal plant used in the Far East to treat inflammation, inhibited the stimulation of COX-2 promoter activity. The gene promoter assay then was used to identify shikonin, a compound with known anti-inflammatory and chemopreventive properties, as an active compound in A. euchroma. To complement the gene promoter studies, we determined the effects of a mixture of shikonins on phorbol 12-myristate 13-acetate (PMA)-mediated induction of COX-2 in transformed human mammary epithelial cells. Shikonins inhibited PMA-mediated induction of COX-2 mRNA, protein, and prostaglandin E(2) synthesis. In transient transfections, PMA caused a severalfold increase in COX-2 promoter activity, an effect that was suppressed by shikonins. Shikonins also inhibited PMA-mediated stimulation of extracellular signal-regulated kinase1/2 mitogen-activated protein kinases and activator protein-1 activity. Collectively, these results demonstrate the successful development and use of a high throughput reporter gene assay for the identification of a novel inhibitor of COX-2 expression.


Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores Enzimáticos/farmacologia , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Extratos Vegetais/farmacologia , Regiões Promotoras Genéticas , Prostaglandina-Endoperóxido Sintases/genética , Transcrição Gênica , Antineoplásicos Fitogênicos/farmacologia , Northern Blotting , Western Blotting , Mama/patologia , Linhagem Celular Transformada , Ciclo-Oxigenase 2 , Dinoprostona/metabolismo , Relação Dose-Resposta a Droga , Células Epiteliais/metabolismo , Humanos , Luciferases/biossíntese , Luciferases/genética , Proteínas de Membrana , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Naftoquinonas/química , Naftoquinonas/farmacologia , Plasmídeos/metabolismo , RNA Mensageiro/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Fator de Transcrição AP-1/metabolismo , Transfecção , Células Tumorais Cultivadas
3.
J Nat Prod ; 64(10): 1282-5, 2001 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-11678651

RESUMO

Assay-guided fractionation of the ethanol extract of the twigs and leaves of Miconia myriantha yielded two new compounds, mattucinol-7-O-[4' ',6' '-O-(S)-hexahydroxydiphenoyl]-beta-D-glucopyranoside (1) and mattucinol-7-O-[4' ',6' '-di-O-galloyl]-beta-D-glucopyranoside (2), along with mattucinol-7-O-beta-D-glucopyranoside (3), ellagic acid (4), 3,3'-di-O-methyl ellagic acid-4-O-beta-D-xylopyranoside, and gallic acid. Complete (1)H and (13)C NMR assignments of compound 1, which possesses a hexahydroxydiphenoyl unit, were achieved using the HMBC technique optimized for small couplings to enhance the four-bond and two-bond H/C correlations. Compounds 1 and 4 showed inhibitory effects against Candida albicans secreted aspartic proteases, with IC(50) of 8.4 and 10.5 microM, respectively.


Assuntos
Glucosídeos/isolamento & purificação , Magnoliopsida/química , Plantas Medicinais/química , Inibidores de Proteases/isolamento & purificação , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Candida albicans/efeitos dos fármacos , Candida albicans/enzimologia , Candida albicans/metabolismo , Cromatografia em Camada Fina , Dicroísmo Circular , Ácido Elágico/química , Ácido Elágico/farmacologia , Ácido Gálico/química , Ácido Gálico/farmacologia , Glucosídeos/química , Glucosídeos/farmacologia , Concentração Inibidora 50 , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Conformação Molecular , Estrutura Molecular , Pepsina A/antagonistas & inibidores , Peru , Folhas de Planta/química , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , Espectrofotometria Infravermelho , Espectroscopia de Infravermelho com Transformada de Fourier , Relação Estrutura-Atividade
4.
J Nat Prod ; 64(8): 1001-5, 2001 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-11520214

RESUMO

Four new alkaloids, nauclealines A (1) and B (2) and naucleosides A (3) and B (4), together with six known compounds, strictosamide (5), vincosamide (6), pumiloside (7), kelampayoside A, sitosterol, and sitosteryl beta-D-glucoside, were isolated from the bark of Nauclea orientalis. The structures of 1-4 were elucidated using 1D and 2D NMR spectral methods, including COSY, DEPT, HMQC, (13)C-(1)H HMBC, and (15)N-(1)H HMBC.


Assuntos
Alcaloides/química , Alcaloides/isolamento & purificação , Indóis/química , Indóis/isolamento & purificação , Plantas Medicinais/química , Rubiaceae/química , Cromatografia em Camada Fina , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Papua Nova Guiné , Caules de Planta/química , Espectrofotometria Infravermelho , Espectrofotometria Ultravioleta , Espectroscopia de Infravermelho com Transformada de Fourier
5.
Immunopharmacol Immunotoxicol ; 23(1): 83-95, 2001 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-11322652

RESUMO

There exists a growing body of research which indicates that antimitotics such as taxol and colchicine influence cytokine gene expression. In the present study we examined the effect of podophyllotoxin and six analogs on nuclear factor kappa B (NF-kappa B) activation, and on interleukin-1 beta (IL-1beta) and tumor necrosis factor alpha (TNF-alpha) mRNA expression in human THP-1 monocytes. All compounds were inactive between 0.001microM and 10microM when tested alone. However, podophyllotoxin (0.1 microM) enhanced LPS-induced NF-kappa B activation and IL-1beta mRNA expression between 2 and 3-fold. In contrast, LPS-induced TNF-alpha mRNA expression was decreased between 3 and 6-fold. Comparable results were also observed with the three analogs acetylpodophyllotoxin, 4'-demethylpodophyllotoxin and alpha-peltatin. The remaining three analogs (podophyllotoxin-4-O-glucoside, beta-peltatin-beta-D-glucopyransoide and 1,2,3,4-dehydrodesoxypodophyllotoxin) were inactive. Clearly certain structural features such as the presence of a glycosidic group or ring aromatization results in loss of biological activity. Interestingly, the analogs that were inactive in our assays have also been previously shown to lack affinity for tubulin binding. These results suggest that during the initial hours of exposure to podophyllotoxin or specific analogs these compounds do not act as independent stimulants of human monocyte activation, but can selectively enhance or suppress LPS-induced cytokine gene expression.


Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Interleucina-1/metabolismo , Lignanas/farmacologia , Lipopolissacarídeos/farmacologia , Monócitos/efeitos dos fármacos , Fragmentos de Peptídeos/metabolismo , Podofilotoxina/análogos & derivados , Podofilotoxina/farmacologia , Fator de Necrose Tumoral alfa/metabolismo , Células Cultivadas , Eletroforese em Gel de Ágar , Humanos , Interleucina-1beta , Espectroscopia de Ressonância Magnética , Monócitos/metabolismo , NF-kappa B/metabolismo , RNA Mensageiro/metabolismo , Relação Estrutura-Atividade , Fatores de Tempo
6.
J Agric Food Chem ; 49(2): 1030-4, 2001 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-11262067

RESUMO

We have characterized a new immunostimulatory polysaccharide called Aloeride from commercial aloe vera (Aloe barbadensis) juice. Aloeride is between 4 and 7 million Da, and its glycosyl components include glucose (37.2%), galactose (23.9%), mannose (19.5%), and arabinose (10.3%). At 0.5 microg/mL Aloeride increased NF-kappa B directed luciferase expression in THP-1 human monocytic cells to levels 50% of those achieved by maximal concentrations (10 microg/mL) of LPS. Aloeride induced the expression of the mRNAs encoding IL-1beta and TNF-alpha to levels equal to those observed in cells maximally activated by LPS. Acemannan, the major carbohydrate component from aloe, used at 200 microg/mL in the macrophage assay resulted in negligible NF-kappa B activation. Analysis of acemannan and Aloeride using size-exclusion chromatography suggests that the low activity of acemannan is due to trace amounts of Aloeride. Although Aloeride comprises only 0.015% of the aloe juice dry weight, its potency for macrophage activation accounts fully for the activity of the crude juice.


Assuntos
Adjuvantes Imunológicos/química , Aloe/química , Macrófagos/efeitos dos fármacos , Plantas Medicinais , Polissacarídeos/química , Adjuvantes Imunológicos/isolamento & purificação , Adjuvantes Imunológicos/farmacologia , Animais , Linhagem Celular , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Genes Reporter , Humanos , Interleucina-1/genética , Luciferases/genética , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , NF-kappa B/genética , Extratos Vegetais/química , Polissacarídeos/isolamento & purificação , Polissacarídeos/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção , Fator de Necrose Tumoral alfa/genética
7.
Phytomedicine ; 8(6): 445-53, 2001 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-11824519

RESUMO

Aphanizomenon flos-aquae (AFA) is a fresh-water microalgae that is consumed as a nutrient-dense food source and for its health-enhancing properties. The current research characterizes the effect of a water soluble preparation from AFA on human monocyte/macrophage function and compares the effect of AFA with responses from known agents that modulate the immune system. At 0.5 pg/ml the AFA extract robustly activated nuclear factor kappa B (NF-kappa B) directed luciferase expression in THP-1 human monocytic cells to levels at 50% of those achieved by maximal concentrations (10 microg/ml) of bacterial lipopolysaccharide (LPS). In addition, the AFA extract substantially increased mRNA levels of interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha), and enhanced the DNA binding activity of NF-kappa B. The effects of AFA water soluble preparation were similar to the responses displayed by LPS, but clearly different from responses exhibited by tetradecanoyl phorbol acetate (TPA) and interferon-gamma (INF-gamma). Pretreatment of THP-1 monocytes with factors known to induce hyporesponsiveness suppressed both AFA-dependent and LPS-dependent activation. These results suggest that the macrophage-activating properties of the AFA water soluble preparation are mediated through pathways that are similar to LPS-dependent activation.


Assuntos
Extratos Celulares/farmacologia , Cianobactérias , Macrófagos/efeitos dos fármacos , Monócitos/efeitos dos fármacos , Citocinas/genética , Humanos , Interferon gama/genética , Interleucina-1/genética , Interleucina-1/metabolismo , Interleucina-10/farmacologia , Lipopolissacarídeos/farmacologia , NF-kappa B/efeitos dos fármacos , NF-kappa B/metabolismo , Polimixina B/farmacologia , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Fator de Crescimento Transformador beta/farmacologia , Fator de Necrose Tumoral alfa/metabolismo
8.
J Nat Prod ; 63(10): 1431-3, 2000 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-11076571

RESUMO

A new indanone (1) has been isolated from the filamentous marine cyanobacterium Lyngbya majuscula, and its structure determined spectroscopically. Vascular endothelial growth factor (VEGF) is an important regulator of tumor angiogenesis. Compound 1 inhibits hypoxia-induced activation of the VEGF gene promoter in Hep3B human liver tumor cells, in vitro.


Assuntos
Cianobactérias/química , Regulação da Expressão Gênica/efeitos dos fármacos , Indanos/química , Hipóxia Celular , Fatores de Crescimento Endotelial/genética , Humanos , Indanos/isolamento & purificação , Indanos/farmacologia , Linfocinas/genética , Regiões Promotoras Genéticas , Células Tumorais Cultivadas , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio Vascular
9.
Biol Pharm Bull ; 23(7): 834-7, 2000 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-10919362

RESUMO

In the course of our research on the oligoglycosidic constituents of Turkish Astragalus species, we have isolated a number of cycloartane-type triterpene glycosides. The current study examines the immunostimulatory effects of nineteen of these cycloartane-type compounds using a transcription-based bioassay for Nuclear Factor kappa B (NF-kappaB) activation in a human macrophage/monocyte cell line, THP-1. All compounds were inactive at 100 microg/ml except astragaloside I which increased NF-kappaB directed luciferase expression to levels about 65% as compared with maximal stimulation by E. coli lipopolysaccharide (LPS) at 10 microg/ml. None of the compounds were active at low dosage levels (0.1 microg/ml) in combination with 50 ng/ml LPS. Astragaloside I also increased mRNA expression of the inflammatory cytokines interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) as measured using reverse transcriptase-polymerase chain reaction (RT)-PCR. Based on these results it is clear that certain structural features are required for immunostimulation of cycloartane-type triterpene glycosides.


Assuntos
Adjuvantes Imunológicos/farmacologia , Glicosídeos/farmacologia , Macrófagos/efeitos dos fármacos , Magnoliopsida/química , Triterpenos/farmacologia , Células Cultivadas , Glicosídeos/química , Humanos , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , NF-kappa B/metabolismo , Saponinas/química , Saponinas/farmacologia , Triterpenos/química
10.
Anal Biochem ; 266(1): 48-57, 1999 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-9887212

RESUMO

Free radical-mediated oxidation of proteins results in the formation of carbonyl groups in quantities that reflect the intensity of the oxidative stress. We have developed an immunochemical technique for the quantification of carbonyl groups in protein samples prepared from small tissue samples and cell cultures. Protein samples were slot-blotted onto a polyvinylidene difluoride membrane, which was sequentially treated with 2,4-dinitrophenylhydrazine (DNPH), a primary antibody specific for the 2,4-dinitrophenol group, and a peroxidase-labeled second antibody. After the blots were developed with a chemiluminescent substrate and exposed to X-ray film, the level of immunostaining was quantitated by densitometry. Using oxidized bovine serum albumin as a standard and loading 5 microg of protein per slot, the minimum detectable carbonyl content was approximately 60 pmol carbonyl/mg protein. When necessary, nonspecific staining by noncarbonyl constituents in complex sample matrices was accounted for by using sodium borohydride-treated blanks. Results by the new method were highly correlated (r = 0.932, P < 0.0001) with those of the standard DNPH-based spectrophotometric technique. The coefficient of variation at a carbonyl level of 1.5 nmol/mg protein was 9.7%. The utility of this new method was demonstrated by measuring protein oxidation in cultured human colon cells (SW620) that were briefly exposed to H2O2.


Assuntos
Immunoblotting/métodos , Proteínas/análise , Proteínas/química , Células Cultivadas/química , Células Cultivadas/efeitos dos fármacos , Colo/citologia , DNA/química , Radicais Livres/metabolismo , Humanos , Peróxido de Hidrogênio/farmacologia , Immunoblotting/instrumentação , Immunoblotting/normas , Oxirredução , Fenil-Hidrazinas/química , Proteínas/efeitos dos fármacos , Sensibilidade e Especificidade , Soroalbumina Bovina/química , Fatores de Tempo
11.
Arch Biochem Biophys ; 358(1): 149-56, 1998 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-9750175

RESUMO

The potential involvement of vicinal dithiols in the transformation of the aryl hydrocarbon (Ah) receptor from its ligand binding to DNA binding form in Hepa-1 cells was explored through the use of diamide and phenylarsine oxide (PAO), which have been shown to specifically form a stable ring complex with vicinal sulfhydryl groups in selected proteins. Pretreatment with diamide and PAO rapidly prevented the inducer-dependent formation of the Ah receptor/xenobiotic response element complex detected by electrophoretic mobility shift assays and suppressed Ah receptor-mediated transcription. Diamide and PAO also inhibited DNA binding activity of the nuclear Ah receptor subsequent to its translocation to the nucleus but to a lesser extent than that observed with pretreatment conditions. The Ah receptor exhibited much higher sensitivity to cellular redox changes than Sp1, a transcription factor previously shown to be very sensitive to redox regulation. Diamide added to nuclear extracts inhibited Ah receptor DNA binding more than when it was added in intact cells. In contrast, Ah receptor DNA binding activity was more sensitive to PAO when it was added to intact cells than when it was added to nuclear extracts. Finally, dithiol 2,3-dimercaptopropanol was over 100 times more effective than monothiol 2-mercaptoethanol in reversing the PAO-dependent inhibition of Ah receptor DNA binding activity. This suggests that vicinal sulfhydryl residues may be involved in DNA binding of the Ah receptor.


Assuntos
DNA de Neoplasias/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Animais , Arsenicais/farmacologia , Transporte Biológico , Carcinoma Hepatocelular , Citocromo P-450 CYP1A1/biossíntese , Citocromo P-450 CYP1A1/genética , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/metabolismo , Diamida/farmacologia , Dimercaprol/farmacologia , Mercaptoetanol/farmacologia , Camundongos , Oxirredução , Receptores de Hidrocarboneto Arílico/antagonistas & inibidores , Fator de Transcrição Sp1/efeitos dos fármacos , Fator de Transcrição Sp1/metabolismo , Transcrição Gênica/efeitos dos fármacos , Células Tumorais Cultivadas , Xenobióticos/farmacologia
12.
Arch Biochem Biophys ; 356(2): 142-50, 1998 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-9705204

RESUMO

We have previously demonstrated that H2O2 downregulates CYP1A1 and CYP1A2 transcription in isolated rat hepatocytes (C. W. Barker, et al., 1994, J. Biol. Chem. 269, 3985-3990). In the present study, induction of chloramphenicol acetyltransferase (CAT) expression driven by 3.1 kb of rat CYP1A1 upstream regulatory sequences was suppressed by 56% in Hepa-1 cells treated with H2O2. Similarly, H2O2 inhibited CAT expression from vectors containing two copies of either xenobiotic-response element (XRE) 1 or XRE2. H2O2 did not inhibit basal CAT expression in cells that were not treated with the inducer beta-napthoflavone. Electrophoretic mobility shift assays demonstrated that the suppression of XRE-dependent transcription by H2O2 was not due to changes in nuclear aryl hydrocarbon (Ah) receptor DNA binding activity. Several types of experiments indicated that modulation of XRE enhancer strength by various means could modify H2O2-dependent suppression of CAT expression. Conditions that increased the transactivation potential of the Ah receptor (increase in XRE copy number or shortening of the distance between XREs and the minimal CYP1A1 promoter) attenuated the action of H2O2, while conditions that reduced XRE-mediated transactivation potential (decrease in XRE copy number, increase of the distance between the XRE and the promoter, or reduction of the number of bound Ah receptors by lowering the concentration of inducer) potentiated the inhibitory action of H2O2.


Assuntos
Citocromo P-450 CYP1A1/antagonistas & inibidores , Citocromo P-450 CYP1A1/biossíntese , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , Xenobióticos/farmacologia , Animais , Carcinoma Hepatocelular , Citocromo P-450 CYP1A1/genética , DNA/efeitos dos fármacos , DNA/metabolismo , Dosagem de Genes , Vetores Genéticos/efeitos dos fármacos , Camundongos , Ligação Proteica/efeitos dos fármacos , Ratos , Receptores de Hidrocarboneto Arílico/efeitos dos fármacos , Receptores de Hidrocarboneto Arílico/metabolismo , Sequências Reguladoras de Ácido Nucleico/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacos , Células Tumorais Cultivadas
13.
Mol Cell Biol ; 14(9): 5653-60, 1994 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-8065302

RESUMO

Aryl hydrocarbons (AHs) such as 2,3,7,8-tetrachlorodibenzo-p-dioxin and benzo[a]pyrene activate the sequence-specific DNA-binding activity of the AH receptor. In the rat hepatocyte-derived cell line LCS7, DNA-binding activity peaked after 30 min and was then down-regulated, reaching negligible levels by 2 h. Down-regulation could be blocked, and DNA-binding activity maintained at maximum for many hours by inhibiting protein or RNA synthesis, implying that down-regulation is a mediated process requiring a labile or inducible protein. CYP1A1 transcription and in vivo DNA-protein interactions at xenobiotic response elements were down-regulated in parallel with DNA-binding activity in nuclear extracts, and these changes could also be blocked by inhibitors of protein synthesis. The correlation between AH receptor DNA-binding activity, intensity of in vivo footprints at xenobiotic response elements, and CYP1A1 transcription rate implies that down-regulation of AH receptor DNA-binding activity is important in regulating CYP1A1 transcription and that receptor is required continuously to maintain transcription. This correlation extends to the murine hepatoma cell line Hepa-1c1c7, in which slower kinetics of activation and down-regulation of CYP1A1 transcription paralleled slower activation and down-regulation of AH receptor DNA-binding activity. The difference in kinetics between cell lines also implies that AH receptor DNA-binding activity is modulated by a mechanism that may be influenced by cell-specific regulatory pathways. The above observations in conjunction with mixing experiments and comparisons of cytoplasmic and nuclear extracts indicate that down-regulation of AH receptor DNA-binding activity is probably due either to degradation or to conversion of the receptor to form that is inactive in both DNA binding and transactivation.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Ativação Transcricional , Animais , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Núcleo Celular/metabolismo , Regulação para Baixo , Regulação Enzimológica da Expressão Gênica , Técnicas In Vitro , Camundongos , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Oligodesoxirribonucleotídeos/química , Ratos , Transcrição Gênica
14.
Nucleic Acids Res ; 22(9): 1741-9, 1994 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-8202380

RESUMO

In vivo footprinting experiments, augmented with gel shift and transfection analyses suggest that activation of the CYP1A1 gene by aryl hydrocarbons may be a multicomponent process. During the first 30 minutes of exposure to aryl hydrocarbon carcinogens and environmental contaminants, in vivo footprints appear at nine distinct sites within a 281 bp region centered 950 bp upstream of the CYP1A1 transcription start site. Six of these sites are unrelated in sequence to the three xenobiotic response elements (XREs) within this region, at which the aryl hydrocarbon (AH) receptor is known to bind. These six display a variety of footprint patterns, are diverse in sequence and range in G-C content from 60 to 75%. This diversity suggests that multiple nuclear factors may be responsible for these six in vivo footprints. These observations are consistent with competition gel shift experiments showing that the nuclear factors binding at two of these sites are different from each other, as well as from the AH receptor. Gel shifts also indicate that the sequence-specific factors binding at these sites are expressed constitutively. This is consistent with a model in which in vivo footprints are induced at these six sites, not through direct activation or de novo synthesis of DNA-binding factors, but through a two phase mechanism in which binding of the nuclear AH receptor complex to XREs facilitates the binding of constitutive factors at these sites. This facilitation could be mediated either through specific protein-protein interactions or through alterations in chromatin structure that make these sites accessible to constitutive nuclear factors. A function for the sequences at which aryl hydrocarbons induce in vivo footprints is suggested by transfection experiments showing that one of these sequences cooperates with a weak XRE to confer on a reporter gene responsiveness to aryl hydrocarbons.


Assuntos
Sistema Enzimático do Citocromo P-450/genética , Oxirredutases/genética , Receptores de Hidrocarboneto Arílico/metabolismo , Sequências Reguladoras de Ácido Nucleico , Ativação Transcricional , Animais , Sequência de Bases , Linhagem Celular , Citocromo P-450 CYP1A1 , DNA/metabolismo , Dados de Sequência Molecular , Ratos
15.
J Biol Chem ; 269(6): 3985-90, 1994 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-8307954

RESUMO

We have previously shown that the inflammatory mediator interleukin-1 suppressed transcription of CYP1A1 and CYP1A2 mRNAs (Barker, C.W., Fagan, J.B., and Pasco, D.S. (1992) J. Biol. Chem. 267, 8050-8055). Since many of the actions of inflammatory mediators are mimicked by oxidative stress, we treated isolated hepatocytes with 0.25-1.0 mM H2O2 to determine whether expression of these genes is also modulated by oxidative stress. Inducer-dependent accumulation of CYP1A1 and CYP1A2 mRNAs were maximally reduced approximately 50 and 70%, respectively, by 1.0 mM H2O2. Run-on transcription analysis suggested that the effect of H2O2 was mediated transcriptionally. The reduction in CYP1A mRNA levels was not due to a reduction in the levels of all mRNAs due to some general toxic effect since H2O2 did not reduce glyceraldehyde-3-phosphate dehydrogenase, alpha-tubulin, beta-fibrinogen, or albumin mRNA levels, and did not increase lactate dehydrogenase released into the medium. Insulin-mimicked H2O2 action, reducing the expression of both mRNAs, and N-acetylcysteine, which increases intracellular glutathione levels, completely reversed the insulin effect on both mRNAs and the H2O2 effect on CYP1A1 mRNA, but only partially reversed the H2O2 effect on CYP1A2 mRNA. This study indicates that the CYP1A1 and CYP1A2 genes are responsive to oxidative stress and that the majority of this responsiveness can be modified by cellular redox potential.


Assuntos
Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Fígado/metabolismo , Estresse Fisiológico/enzimologia , Animais , Benzoflavonas/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Expressão Gênica , Genes jun , Glutationa/metabolismo , Peróxido de Hidrogênio/farmacologia , Insulina/farmacologia , Masculino , Oxirredução , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Transcrição Gênica/efeitos dos fármacos , beta-Naftoflavona
16.
J Biol Chem ; 268(2): 1053-7, 1993 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-8419313

RESUMO

In vitro nuclear run-on transcription analysis using probes directed against different regions of CYP1A2 revealed that the 70-100-fold induction of CYP1A2 mRNA by polycyclic aromatic compounds is associated with a corresponding increase in the transcriptional activation of this gene in rat liver. Probes from regions of the 1st, 2nd, and 4th introns detected approximately 50-100-fold higher CYP1A2 run-on transcription in liver nuclei from inducer-treated animals than in nuclei from untreated animals. The run-on signals from untreated rats were 3-5-fold above background signals. Additional experiments using single-stranded DNA probes and a probe from a region 5' to the CYP1A2 transcription start site revealed that the inducer-dependent transcripts were colinear with the CYP1A2 mRNA and that they did not result from read through of an initiation event 5' to CYP1A2. Run-on transcription analyses were also carried out with nuclei from isolated hepatocytes using the same series of probes spanning CYP1A2. These analyses indicated that the inducer-dependent accumulation of CYP1A2 mRNA in hepatocytes is associated with at least a 20-fold increase in CYP1A2 transcription. In contrast to liver and hepatocytes, these probes failed to detect run-on transcripts from kidney nuclei, indicating that the lack of CYP1A2 mRNA in this tissue is due to the lack of transcriptional activation of this gene by polycyclic aromatic compounds.


Assuntos
Sistema Enzimático do Citocromo P-450/genética , Regulação Enzimológica da Expressão Gênica , Fígado/enzimologia , Transcrição Gênica , Animais , Benzoflavonas/farmacologia , Núcleo Celular/metabolismo , Células Cultivadas , Sistema Enzimático do Citocromo P-450/biossíntese , Sondas de DNA , Íntrons , Rim/metabolismo , Fígado/efeitos dos fármacos , Dibenzodioxinas Policloradas/farmacologia , Ratos , beta-Naftoflavona
17.
J Biol Chem ; 267(12): 8050-5, 1992 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-1569064

RESUMO

Animals subjected to immunostimulatory conditions exhibit reduced tissue levels of total cytochrome P450 and P450-dependent drug metabolism. We have investigated the possibility that depressed levels of two carcinogen-metabolizing cytochrome P450s may be due to decreased levels of the mRNAs encoding these enzymes by studying the effect of monocyte-derived cytokines on the induction of CYP1A1 and CYP1A2 mRNAs in isolated rat hepatocytes. Medium conditioned by activated human peripheral blood monocytes or by the U937 monocyte cell line suppressed the induction of both mRNAs by 2,3,7,8-tetrachlorodibenzo-p-dioxin, whereas beta-fibrinogen mRNA levels increased 30-40-fold. CYP1A2 mRNA induction was maximally inhibited more than CYP1A1 mRNA (approximately 95 and 65%, respectively), and lower concentrations of conditioned medium suppressed CYP1A2 mRNA induction (half-maximal at 1.9 and 3.1%, respectively). Low concentrations of recombinant interleukin-1 suppressed the inducer-dependent accumulation of both CYP1A1 and CYP1A2 mRNAs in a dose-dependent fashion (half-maximal at 2 and 0.5 units/ml, respectively), while two other monocyte-derived cytokines, interleukin-6 and transforming growth factor-beta, did not. Run-on transcription analysis demonstrated that conditioned medium and interleukin-1 rapidly suppressed the transcription rate of CYP1A1 and CYP1A2 in inducer-treated hepatocytes. The close correspondence between the reductions in CYP1A1 and CYP1A2 transcription rates and mRNA levels suggest that conditioned medium and interleukin-1 suppress the induction of these mRNAs principally through a transcriptional mechanism.


Assuntos
Sistema Enzimático do Citocromo P-450/genética , Interleucina-1/farmacologia , Isoenzimas/genética , Fígado/enzimologia , RNA Mensageiro/biossíntese , Animais , Northern Blotting , Humanos , Fígado/citologia , Fígado/efeitos dos fármacos , Masculino , Hibridização de Ácido Nucleico , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/efeitos dos fármacos , Ratos , Ratos Endogâmicos , Transcrição Gênica
18.
Mol Cell Biol ; 10(12): 6408-16, 1990 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-2174107

RESUMO

Three nuclear factors, the Ah receptor, XF1, and XF2, bind sequence specifically to the Ah response elements or xenobiotic response elements (XREs) of the cytochrome P450IA1 (P450c) gene. The interactions of these factors with the Ah response element XRE1 were compared by three independent methods, methylation interference footprinting, orthophenanthroline-Cu+ footprinting, and mobility shift competition experiments, using a series of synthetic oligonucleotides with systematic alterations in the XRE core sequence. These studies established the following (i) all three factors interact sequence specifically with the core sequence of XRE1; (ii) the pattern of contacts made with this sequence by the Ah receptor are different from those made by XF1 and XF2; and (iii) although XF1 and XF2 can be distinguished by the mobility shift assay, the sequence specificities of their interactions with XRE1 are indistinguishable. Further characterization revealed the following additional differences among these three factors: (i) XF1 and XF2 could be extracted from nuclei under conditions quite different from those required for extraction of the Ah receptor; (ii) XF1 and XF2 were present in the nuclei of untreated cells and did not respond to polycyclic compounds, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and beta-napthoflavone, while nuclear Ah receptor was undetectable in untreated cells and rapidly increased in response to TCDD; (iii) inhibition of protein synthesis did not affect the TCDD-induced appearance of the Ah receptor but substantially decreased the constitutive activities of XF1 and XF2, suggesting that the Ah receptor must be present in untreated cells in an inactive form that can be rapidly activated by polycyclic compounds, while the constitutive expression of XF1 and XF2 depends on the continued synthesis of a relatively unstable protein; (iv) the receptor-deficient and nuclear translocation-defective mutants of the hepatoma cell line Hepa1, which are known to lack nuclear Ah receptor, expressed normal levels of XF1 and XF2, suggesting that the former factor is genetically distinct from the latter two; and (v) a divalent metal ion, probably Zn2+, is known to be an essential cofactor for the Ah receptor but was not required for the DNA-binding activities of XF1 and XF2. Together, these findings indicate that the Ah receptor is distinct from XF1 and XF2, while the latter two activities may be related. Because the DNA-binding domains of these three factors overlap substantially, their binding to XREs is probably mutually exclusive, which suggests that the interplay of these factors at Ah response elements may be important to the regulation of CYP1A1 gene transcription. The results of preliminary transfection experiments with constructs harboring XREs upstream of the chloramphenicol acetyltransferase gene driven by a minimal simian virus 40 promoter are presented that are consistent with this hypothesis.


Assuntos
Sistema Enzimático do Citocromo P-450/genética , Proteínas de Ligação a DNA/metabolismo , Genes , Compostos Policíclicos/farmacologia , Receptores de Droga/metabolismo , Transcrição Gênica/efeitos dos fármacos , Sequência de Bases , Carcinoma Hepatocelular , Linhagem Celular , Núcleo Celular/metabolismo , Cobre , Humanos , Neoplasias Hepáticas , Dados de Sequência Molecular , Mapeamento de Nucleotídeos , Sondas de Oligonucleotídeos , Fenantrolinas , Plasmídeos , Dibenzodioxinas Policloradas/metabolismo , Receptores de Hidrocarboneto Arílico , Receptores de Droga/efeitos dos fármacos , Transfecção
19.
J Biol Chem ; 265(16): 9251-8, 1990 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-2160969

RESUMO

The aryl hydrocarbon (Ah) receptor, also called the xenobiotic or TCDD receptor, mediates transcriptional activation of the cytochrome P-450c (CYP1A1) gene by interacting with Ah or xenobiotic response elements. This paper presents evidence that a metal ion, probably Zn2+, is an essential cofactor for the Ah receptor. This paper also maps in detail the interactions between the Ah response element XRE1 and the Ah receptor from the rat hepatocyte-derived cell line LCS7. Interactions were mapped by three methods, 1) methylation interference footprinting, 2) mobility shift competition experiments, using a series of synthetic oligonucleotides with systematic alterations in the Ah response element core sequence, and 3) orthophenanthroline/Cu+ footprinting. These findings suggest the following consensus sequence for DNA recognition by the Ah receptor: CNA/TNA/TCACGCA/TA/T. The chelators 1,10-phenanthroline and oxalic acid inhibited the sequence-specific DNA-binding activity of the AH receptor in a concentration dependent manner, suggesting that the DNA-binding activity of the receptor requires divalent metal ions. Inhibition was due to metal-chelation, since: 1) inhibition was almost completely prevented by the presence of Zn2+, or other divalent metal ions having high affinity for the chelators used, while metal ions with low affinity did not protect; 2) the DNA-binding activity of the receptor could be restored by dialysis to remove 1,10-phenanthroline, but only in the presence of Zn2+, while dialysis in the absence of metal ions reversed inhibition by the nonchelating isomer 4,7-phenanthroline. The involvement of a divalent cation in receptor function, possibly bound via sulfhydryls, was also suggested by the finding that Cd2+ and Co2+ inhibited DNA-binding activity. Once bound to the XRE1 DNA sequence, the receptor could not be inhibited by 1,10-phenanthroline, suggesting that the essential metal ion must become inaccessible to chelation when the receptor binds DNA. The Zn2+ requirement of the Ah receptor is similar to that of the estrogen and the glucocorticoid receptors and is consistent with the hypothesis that the Ah receptor is a member of the steroid and thyroid hormone receptor superfamily.


Assuntos
DNA/metabolismo , Receptores de Droga/metabolismo , Zinco/farmacologia , Animais , Sequência de Bases , Sítios de Ligação , Ligação Competitiva , Cátions Bivalentes , Linhagem Celular , Núcleo Celular/metabolismo , Fígado/análise , Fígado/metabolismo , Metilação , Dados de Sequência Molecular , Mapeamento de Nucleotídeos , Oligonucleotídeos/metabolismo , Oxalatos/farmacologia , Ácido Oxálico , Fenantrolinas/farmacologia , Ratos , Receptores de Hidrocarboneto Arílico , Receptores de Droga/efeitos dos fármacos
20.
DNA ; 8(7): 535-41, 1989 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-2504573

RESUMO

An efficient, simple, and reproducible DNA-mediated gene transfer procedure has been developed for primary cultures of adult rat hepatocytes. Calcium phosphate-DNA precipitate is formed in complete culture medium during 5 hr incubation with cells. Unabsorbed precipitate is then washed out, and 40 hr later gene expression is measured. Under optimal conditions, up to 20-25% of cells in cultures transfected with the beta-galactosidase (lacZ) gene stain positively for this activity, and cells transfected with the chloramphenicol acetyl transferase (CAT) gene, fused to a strong promoter, express CAT activities of 10-14 nmoles/min per mg protein. Five conditions were optimized based on transfection efficiency, CAT expression, and cell viability. (i) Medium composition: the presence of protein, such as fetal bovine serum or bovine serum albumin, in the medium was essential. (ii) Cell substratum: tissue culture plastic was superior to calf skin collagen and Matrigel. (iii) Cell density: 0.5-1.0 X 10(6) cells/60-mm dish were superior to higher densities. (iv) Duration of exposure to calcium phosphate-DNA: 5-8 hr was better than shorter or longer times. (v) Length of time hepatocytes were maintained in culture before initiating transfection: 2-3 days was superior to earlier times. This procedure was successful with reporter genes linked to three different eukaryotic promoters. These included a chimeric promoter containing the polycyclic aromatic hydrocarbon-responsive enhancer of the cytochrome P450c gene (CYP1A1), which was shown to confer upon the CAT gene responsiveness to polycyclic aromatic hydrocarbons comparable to that of the native P450c gene. This transfection procedure should be of considerable use for the study of liver-specific gene expression in primary hepatocyte cultures.


Assuntos
Fosfatos de Cálcio/metabolismo , DNA/metabolismo , Fígado/metabolismo , Transfecção , Animais , Células Cultivadas , Cloranfenicol O-Acetiltransferase/genética , Técnicas de Cultura/métodos , Escherichia coli/genética , Genes , Genes Bacterianos , Masculino , Plasmídeos , Ratos , Ratos Endogâmicos , beta-Galactosidase/genética
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