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1.
FEBS J ; 274(2): 439-50, 2007 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17229149

RESUMO

cGMP secretion from cells can be mediated by ATP-binding cassette (ABC) transporters ABCC4, ABCC5, and ABCC11. Indirect evidence suggests that ABCC4 and ABCC5 contribute to cGMP transport by erythrocytes. We have re-investigated the issue using erythrocytes from wild-type and transporter knockout mice. Murine wild-type erythrocyte vesicles transported cGMP with an apparent Km that was 100-fold higher than their human counterparts, the apparent Vmax being similar. Whereas cGMP transport into human vesicles was efficiently inhibited by the ABCC4-specific substrate prostaglandin E1, cGMP transport into mouse vesicles was inhibited equally by Abcg2 and Abcc4 inhibitors/substrates. Similarly, cGMP transport into vesicles from Abcc4-/- and Abcg2-/- mice was 42% and 51% of that into wild-type mouse vesicles, respectively, whereas cGMP transport into vesicles from Abcc4(-/-)/Abcg2(-/-) mice was near background. The knockout mice were used to show that Abcg2-mediated cGMP transport occurred with lower affinity but higher Vmax than Abcc4-mediated transport. Involvement of Abcg2 in cGMP transport by Abcc4-/- erythrocyte vesicles was supported by higher transport at pH 5.5 than at pH 7.4, a characteristic of Abcg2-mediated transport. The relative contribution of ABCC4/Abcc4 and ABCG2/Abcg2 in cGMP transport was confirmed with a new inhibitor of ABCC4 transport, the protease inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride.


Assuntos
GMP Cíclico/metabolismo , Eritrócitos/citologia , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/genética , Alprostadil/metabolismo , Animais , Transporte Biológico , Relação Dose-Resposta a Droga , Membrana Eritrocítica/metabolismo , Eritrócitos/metabolismo , Humanos , Camundongos , Camundongos Knockout , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Proteínas de Neoplasias/genética
2.
Mol Pharmacol ; 71(1): 240-9, 2007 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17032904

RESUMO

Some cellular uptake systems for (anti)folates function optimally at acidic pH. We have tested whether this also applies to efflux from cells by breast cancer resistance protein (BCRP; ABCG2), which has been reported to transport folic acid, methotrexate, and methotrexate di- and triglutamate at physiological pH. Using Spodoptera frugiperda-BCRP membrane vesicles, we showed that the ATP-dependent vesicular transport of 1 muM methotrexate by BCRP is 5-fold higher at pH 5.5 than at physiological pH. The transport of methotrexate was saturable at pH 5.5, with apparent Km and Vmax values of 1.3 +/- 0.2 mM and 44 +/- 2.5 nmol/mg of protein/min, respectively, but was linear with drug concentration at pH 7.3 up to 6 mM methotrexate. In contrast to recent reports, we did not detect transport of methotrexate diglutamate at physiological pH, but we did find transport at pH 5.5. We also found that 7-hydroxy-methotrexate, the major metabolite of methotrexate, is transported by BCRP both at physiological pH and (more efficiently) at low pH. The pH effect was also observed in intact BCRP-overexpressing cells: we found a 3-fold higher level of resistance to both methotrexate and the prototypical BCRP substrate mitoxantrone at pH 6.5 as at physiological pH. Furthermore, with MDCKII-BCRP monolayers, we found that resveratrol, which is a neutral compound at pH < or = 7.4, is efficiently transported by BCRP at pH 6.0, whereas we did not detect active transport at pH 7.4. We conclude that BCRP transports substrate drugs more efficiently at low pH, independent of the dissociation status of the substrate.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Antineoplásicos/farmacocinética , Neoplasias da Mama/metabolismo , Ácido Fólico/farmacocinética , Concentração de Íons de Hidrogênio , Metotrexato/análogos & derivados , Metotrexato/farmacocinética , Mitoxantrona/farmacocinética , Proteínas de Neoplasias/metabolismo , Estilbenos/farmacocinética , Topotecan/farmacocinética , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Animais , Transporte Biológico , Linhagem Celular , Linhagem Celular Tumoral , Cães , Feminino , Humanos , Cinética , Fígado/metabolismo , Coelhos , Resveratrol
3.
Arterioscler Thromb Vasc Biol ; 26(9): 2168-74, 2006 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16840717

RESUMO

OBJECTIVE: The protein C anticoagulant pathway is an essential process for attenuating thrombin generation by the membrane-bound procoagulant complexes tenase and prothrombinase. In this pathway, protein S (PS) serves as a cofactor for activated protein C. PS circulates in plasma both in a free form and in complex with complement component 4b-binding protein (C4BP). C4BP is a known acute phase reactant, thereby suggesting a relation between PS and the acute phase response. Interleukin (IL)-6 has been shown to increase both PS and C4BP gene expression. Our objective was to study the regulation of PS gene expression by IL-6 in detail. METHODS AND RESULTS: IL-6 upregulates both PS mRNA and protein levels in liver-derived HepG2 cells. The promoter of the PS gene (PROS1) was cloned upstream from a luciferase reporter gene. After transfection in HepG2 cells, the luciferase activity was shown to be stimulated by the addition of IL-6. IL-6 exerts its effect through Signal Transducer and Activator of Transcription 3 (STAT3) that interacts with the PROS1 promoter at a binding site in between nucleotides 229 to 207 upstream from the translational start. CONCLUSIONS: IL-6 induces PS expression via STAT3. A possible function for IL-6-induced PS expression in cell survival is discussed.


Assuntos
Interleucina-6/fisiologia , Proteína S/biossíntese , Fator de Transcrição STAT3/fisiologia , Linhagem Celular Tumoral , Regulação da Expressão Gênica , Humanos , Interleucina-6/farmacologia , Fosforilação , Regiões Promotoras Genéticas , Proteína S/genética , Proteína S/metabolismo , RNA Mensageiro/biossíntese , Elementos de Resposta , Fator de Transcrição STAT3/metabolismo
4.
J Biol Chem ; 281(26): 17635-43, 2006 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-16672217

RESUMO

Protein S (PS) is a vitamin K-dependent plasma protein that inhibits blood coagulation by serving as a nonenzymatic cofactor for activated protein C in the protein C anticoagulant pathway. Low PS levels are a risk factor for the development of deep venous thrombosis. The regulation of PS levels through transcriptional regulation of the PS gene was investigated in this report. A minimal PS gene promoter 370 bp upstream from the translational initiation codon was sufficient for maximal promoter activity in transient transfections regardless of the cell type. A pivotal role for Sp1 in the constitutive expression of the PS gene was demonstrated through electrophoretic mobility shift assay experiments, transient expression of mutant PS promoter-reporter gene constructs, and chromatin immunoprecipitations in HepG2 cells. At least four Sp-binding sites were identified. The two sites most proximal to the translational start codon were found to be indispensable for PS promoter activity, whereas mutation of the two most distal Sp-binding sites had a negligible influence on basal promoter activity. In addition, all other major promoter-binding proteins that were found by electrophoretic mobility shift assay could be positively identified in supershift assays. We identified binding sites for the hepatocyte-specific forkhead transcription factor FOXA2, nuclear factor Y, and the cAMP-response element-binding protein/activating transcription factor family of transcription factors. Their relevance was investigated using site-directed mutagenesis.


Assuntos
Regiões Promotoras Genéticas/fisiologia , Proteína S/genética , Proteína S/metabolismo , Fator de Transcrição Sp1/metabolismo , Fator de Transcrição Sp3/metabolismo , Sequência de Bases , Sítios de Ligação/fisiologia , Fator de Ligação a CCAAT/metabolismo , Carcinoma Hepatocelular , Cromatina/fisiologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Endotélio Vascular/citologia , Regulação da Expressão Gênica/fisiologia , Células HeLa , Fator 3-beta Nuclear de Hepatócito/metabolismo , Humanos , Neoplasias Hepáticas , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteína S/química , Fatores de Transcrição/metabolismo , Transfecção , Veias Umbilicais/citologia
5.
Chem Res Toxicol ; 15(5): 614-22, 2002 May.
Artigo em Inglês | MEDLINE | ID: mdl-12018981

RESUMO

Tamoxifen is a major drug used for adjuvant chemotherapy of breast cancer; however, its use has been associated with a small but significant increase in risk of endometrial cancer. In rats, tamoxifen is a hepatocarcinogen, and DNA adducts have been observed in both rat and human tissues. Tamoxifen has been shown previously to be metabolized to reactive products that have the potential to form protein and DNA adducts. Previous studies have suggested a role for P450 3A4 in protein adduct formation in human liver microsomes, via a catechol intermediate; however, no clear correlation was seen between P450 3A4 content of human liver microsomes and adduct formation. In the present study, we investigated the P450 forms responsible for covalent drug-protein adduct formation and the possibility that covalent adduct formation might occur via alternative pathways to catechol formation. Recombinant P450 3A4 catalyzed adduct formation, and this correlated with the level of uncoupling in the P450 incubation, consistent with a role of reactive oxygen species in potentiating adduct formation after enzymatic formation of the catechol metabolite. Whereas P450s 1A1, 2D6, and 3A5 generated catechol metabolite, no covalent adduct formation was observed with these forms. By contrast, P450 2B6, 2C19, and rat liver microsomes catalyzed drug-protein adduct formation but not catechol formation. Drug protein adducts formed specifically with P450 3A4 in incubations using membranes isolated from bacteria expressing P450 3A4 and reductase, as well as in reconstitutions of purified 3A4, suggesting that the electrophilic species reacted preferentially with the P450 enzymes concerned.


Assuntos
Anticarcinógenos/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Microssomos Hepáticos/metabolismo , Tamoxifeno/metabolismo , Animais , Anticarcinógenos/efeitos adversos , Catecóis/metabolismo , Cromatografia Líquida de Alta Pressão , Sistema Enzimático do Citocromo P-450/fisiologia , Adutos de DNA/biossíntese , Humanos , Masculino , Microssomos Hepáticos/enzimologia , Ratos , Ratos Wistar , Tamoxifeno/efeitos adversos
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