RESUMEN
Mercaptopurines have been used as anticancer agents for more than 40 years, and most acute lymphoblastic leukemias are treated with 6-mercaptopurine (6MP) or 6-thioguanine (TG). Overexpression of the two related multidrug resistance proteins MRP4 and MRP5 has been shown to confer some resistance against mercaptopurines, which has been attributed to extrusion of mercaptopurine metabolites by these transporters. We have analyzed the mercaptopurine metabolites formed in human embryonic kidney cells and determined which metabolites are extruded by MRP4 and MRP5. Incubation with 6MP led to the formation of thioinosine and thioxanthosine metabolites and we found that thio-IMP was transported by both MRP4 and MRP5; MRP5 showed the highest transport rate. In contrast, only MRP5 transported thioxanthosine monophosphate (tXMP). During incubation with TG, the monophosphorylated form of thioguanosine was transported by both MRP4 and MRP5; the highest transport rate was for MRP4. Similarly, only 6-methyl-thio-IMP was formed during incubation with 6-methyl mercaptopurine riboside. This compound was a substrate for both MRP4 and MRP5; MRP4 showed the highest transport rate. Our results show that all major thiopurine monophosphates important in the efficacy of mercaptopurine treatment are transported by MRP4 and MRP5, although the substrate specificity of the two transporters differs in detail.
Asunto(s)
Mercaptopurina/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Proteínas Ribosómicas/metabolismo , Tioguanina/metabolismo , Transporte Biológico , Células Cultivadas , Cromatografía Líquida de Alta Presión , Interacciones Farmacológicas , Humanos , Riñón/citología , Riñón/embriología , Cinética , Mercaptopurina/farmacología , Metiltioinosina/farmacología , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/biosíntesis , Proteínas Ribosómicas/biosíntesis , Tioguanina/farmacología , TransfecciónRESUMEN
Despite accumulating evidence that multidrug resistance transporter proteins play a part in drug resistance in some clinical cancers, it remains unclear whether the relatively low levels of multidrug resistance transporter expression found in most untreated tumors could substantially affect their basal sensitivity to antineoplastic drugs. To shed light on this problem, the drug sensitivities of wild-type mouse cell lines were compared with those of lines in which the Mdr1a and Mdr1b genes encoding P-glycoprotein (P-gp) were inactivated and lines in which the Mrp1 gene was inactivated in addition to Mdr1a and Mdr1b. These models permit a clean dissection of the contribution of each transporter to drug resistance at expression levels similar to those in normal tissues and avoid complications that might arise from previous exposure of cell lines to drug selection. For substrate drugs, we found that these contributions can indeed be very substantial. Lines lacking functional P-gp were, on average, markedly more sensitive to paclitaxel (16-fold), anthracyclines (4-fold) and Vinca alkaloids (3-fold). Lines lacking both P-gp and Mrp1 were (compared with wild-type lines) hypersensitive to an even broader array of drugs, including epipodophyllotoxins (4-7-fold), anthracyclines (6-7-fold), camptothecins (3-fold), arsenite (4-fold) and Vinca alkaloids, especially vincristine (28-fold). Thus, even very low levels of P-gp and Mrp1 expression that may be difficult to detect in tumors could significantly affect their innate sensitivity to a wide range of clinically important substrate drugs. An implication is that the use of resistance reversal agents to sensitize drug-naive tumors may be appropriate in more cases than is presently appreciated.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/fisiología , Transportadoras de Casetes de Unión a ATP/fisiología , Resistencia a Múltiples Medicamentos/fisiología , Resistencia a Antineoplásicos/fisiología , Células 3T3/efectos de los fármacos , Células 3T3/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/biosíntesis , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/biosíntesis , Transportadoras de Casetes de Unión a ATP/genética , Animales , Antibióticos Antineoplásicos/farmacología , Antineoplásicos Fitogénicos/farmacología , Cruzamientos Genéticos , Resistencia a Múltiples Medicamentos/genética , Resistencia a Antineoplásicos/genética , Ensayos de Selección de Medicamentos Antitumorales , Silenciador del Gen , Genotipo , Ratones , Ratones Noqueados , Proteínas Asociadas a Resistencia a Múltiples Medicamentos , Paclitaxel/farmacologíaRESUMEN
The human multidrug transporter MDR1 P-glycoprotein and the multidrug resistance proteins MRP1 and MRP2 transport a range of cytotoxic drugs, resulting in multidrug resistance in tumour cells. To overcome this form of drug resistance in patients, several inhibitors (reversal agents) of these transporters have been isolated. Using polarized cell lines stably expressing human MDR1, MRP1 or MRP2cDNA, and 2008 ovarian carcinoma cells stably expressing MRP1 cDNA, we have investigated in this study the specificity of the reversal agents V-104 (a pipecolinate derivative), GF120918 (an acridone carboxamide derivative also known as GG918), and Pluronic L61 (a (poly)oxypropethylene and (poly)oxypropylene block copolymer). Transport experiments with cytotoxic drugs with polarized cell lines indicate that all three compounds efficiently inhibit MDR1 Pgp. Furthermore, V-104 partially inhibits daunorubicin transport by MRP1 but not vinblastine transport by MRP2. V-104 reverses etoposide resistance of 2008/MRP1 cells, whereas GF120918 does not reverse resistance due to MRP1. V-104 partially inhibits the export of the organic anion dinitrophenyl S-glutathione by MDCKII-MRP1 but not by MDCKII-MRP2 cells. Unexpectedly, export of the organic anion calcein by MDCKII-MRP1 and MDCKII-MRP2 cells is stimulated by Pluronic L61, probably because it relieves the block on entry of calcein AM into the cell by endogenous MDR1 Pgp.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Acridinas/farmacología , Resistencia a Múltiples Medicamentos , Resistencia a Antineoplásicos , Isoquinolinas/farmacología , Metanfetamina/análogos & derivados , Poloxámero/farmacología , Tetrahidroisoquinolinas , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/efectos de los fármacos , Proteínas de Transporte de Anión , Antibióticos Antineoplásicos/farmacocinética , Antineoplásicos Fitogénicos/farmacocinética , Transporte Biológico Activo/efectos de los fármacos , Proteínas Portadoras/farmacología , Daunorrubicina/farmacocinética , Etopósido/farmacocinética , Fluoresceínas/farmacología , Humanos , Metanfetamina/farmacología , Factores de Tiempo , Vinblastina/farmacocinéticaRESUMEN
Two prominent members of the ATP-binding cassette superfamily of transmembrane proteins, multidrug resistance 1 (MDR1) P-glycoprotein and multidrug resistance protein 1 (MRP1), can mediate the cellular extrusion of xenobiotics and (anticancer) drugs from normal and tumor cells. The MRP subfamily consists of at least six members, and here we report the functional characterization of human MRP5. We found resistance against the thiopurine anticancer drugs, 6-mercaptopurine (6-MP) and thioguanine, and the anti-HIV drug 9-(2-phosphonylmethoxyethyl)adenine (PMEA) in MRP5-transfected cells. This resistance is due to an increased extrusion of PMEA and 6-thioinosine monophosphate from the cells that overproduce MRP5. In polarized Madin-Darby canine kidney II (MDCKII) cells transfected with an MRP5 cDNA construct, MRP5 is routed to the basolateral membrane and these cells transport S-(2,4-dinitrophenyl)glutathione and glutathione preferentially toward the basal compartment. Inhibitors of organic anion transport inhibit transport mediated by MRP5. We speculate that MRP5 might play a role in some cases of unexplained resistance to thiopurines in acute lymphoblastic leukemia and/or to antiretroviral nucleoside analogs in HIV-infected patients.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Resistencia a Múltiples Medicamentos , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Línea Celular , Clonación Molecular , Perros , Humanos , Transporte Iónico , Datos de Secuencia Molecular , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Nucleótidos/metabolismoRESUMEN
Studies with knockout mice lacking mdr1a P-glycoprotein (P-gp) have previously shown that blood-brain barrier P-gp is important in preventing the accumulation of several drugs in the brain. Asimadoline (EMD 61753) is a peripherally selective kappa-opioid receptor agonist which is under development as a therapeutic analgaesic. From the structural characteristics of this drug and its peripheral selectivity, we hypothesized that it is transported by P-gp. Using a pig-kidney polarized epithelial cell line transfected with mdr cDNAs, we demonstrate that asimadoline is transported by the mouse mdr1a P-gp and the human MDR1 P-gp. Furthermore, we show that in mdr1a/1b double knockout mice, the absence of P-gp leads to a 9 fold increased accumulation of asimadoline in the brain. In line with this accumulation difference, mdr1a/1b (-/-) mice are at least 8 fold more sensitive to the sedative effect of asimadoline than wild-type mice. Interestingly, the oral uptake of asimadoline was not substantially altered in mdr1a/1b (-/-) mice. Our results demonstrate that for some drugs, P-gp in the blood-brain barrier can have a therapeutically beneficial effect by limiting brain penetration, whereas at the same time intestinal P-gp is not a significant impediment to oral uptake of the drug.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/fisiología , Acetamidas/metabolismo , Analgésicos Opioides/metabolismo , Barrera Hematoencefálica/fisiología , Encéfalo/metabolismo , Pirrolidinas/metabolismo , Fases del Sueño/efectos de los fármacos , Subfamilia B de Transportador de Casetes de Unión a ATP/deficiencia , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/deficiencia , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Acetamidas/farmacocinética , Acetamidas/toxicidad , Analgésicos Opioides/farmacocinética , Analgésicos Opioides/toxicidad , Animales , Disponibilidad Biológica , Encéfalo/efectos de los fármacos , Línea Celular , Humanos , Masculino , Ratones , Ratones Noqueados , Pirrolidinas/farmacocinética , Pirrolidinas/toxicidad , Porcinos , Distribución Tisular , Miembro 4 de la Subfamilia B de Casete de Unión a ATPRESUMEN
The human multidrug resistance protein (MRP1) causes drug resistance by extruding drugs from tumor cells. In addition to an MDR-like core, MRP1 contains an N-terminal membrane-bound region (TMD0) connected to the core by a cytoplasmic linker (L0). We have studied truncated MRP1 versions containing either the MDR-like core alone or the core plus linker L0, produced in the baculovirus-insect (Sf9) cell system. Their function was examined in isolated membrane vesicles. Full-length MRP1 showed ATP-dependent, vanadate-sensitive accumulation of leukotriene C4 and N-ethylmaleimide glutathione. In addition, leukotriene C4-stimulated, vanadate-dependent nucleotide occlusion was detected. The MDR-like core was virtually inactive. Co-expression of the core with the N-terminal region including L0 fully restored MRP1 function. Unexpectedly, a truncated MRP1 mutant lacking the entire TMD0 region but still containing L0 behaved like wild-type MRP1 in vesicle uptake and nucleotide trapping experiments. We also expressed the MRP1 constructs in polarized canine kidney derived MDCKII cells. Like wild-type MRP1, the MRP1 protein without the TMD0 region was routed to the lateral plasma membrane and transported dinitrophenyl glutathione and daunorubicin. The TMD0L0 and the MRP1 minus TMD0L0 remained in an intracellular compartment. Taken together, these experiments strongly suggest that the TMD0 region is neither required for the transport function of MRP1 nor for its proper routing to the plasma membrane.
Asunto(s)
Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos , Estructura Secundaria de Proteína , Animales , Baculoviridae , Disparidad de Par Base , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Clonación Molecular , Perros , Glutatión/análogos & derivados , Glutatión/farmacocinética , Humanos , Cinética , Leucotrieno C4/farmacocinética , Maleimidas/farmacocinética , Modelos Moleculares , Proteína 3 Homóloga de MutS , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Eliminación de Secuencia , Spodoptera , Transfección , Vanadatos/farmacologíaRESUMEN
The canalicular (apical) membrane of the hepatocyte contains an ATP-dependent transport system for organic anions, known as the multispecific organic anion transporter (cMOAT). The deduced amino acid sequence of cMOAT is 49% identical to that of the human multidrug resistance- associated protein (MRP) MRP1, and cMOAT and MRP1 are members of the same sub-family of adenine nucleotide binding cassette transporters. In contrast to MRP1, cMOAT was predominantly found intracellularly in nonpolarized cells, suggesting that cMOAT requires a polarized cell for plasma membrane routing. Therefore, we expressed cMOAT cDNA in polarized kidney epithelial MDCK cell lines. When these cells are grown in a monolayer, cMOAT localizes to the apical plasma membrane. We demonstrate that cMOAT causes transport of the organic anions S-(2,4-dinitrophenyl)-glutathione, the glutathione conjugate of ethacrynic acid, and S-(PGA1)-glutathione, a substrate not shown to be transported by organic anion transporters previously. Transport is inhibited only inefficiently by compounds known to block MRP1. We also show that cMOAT causes transport of the anticancer drug vinblastine to the apical side of a cell monolayer. We conclude that cMOAT is a 5'-adenosine triphosphate binding cassette transporter that potentially might be involved in drug resistance in mammalian cells.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas Portadoras/metabolismo , Células Epiteliales/metabolismo , Proteínas de Transporte de Anión , Transporte Biológico Activo , Compartimento Celular , Línea Celular , Membrana Celular/metabolismo , Polaridad Celular , Técnica del Anticuerpo Fluorescente Indirecta , Glutatión/análogos & derivados , Glutatión/metabolismo , Humanos , Inmunohistoquímica , Riñón/citología , Riñón/metabolismo , Transfección , Vinblastina/metabolismoRESUMEN
The mdr1-type P-glycoproteins (P-gps) confer multidrug resistance to cancer cells by active extrusion of a wide range of drugs from the cell. To study their physiological roles, we have generated mice genetically deficient in the mdr1b gene [mdr1b (-/-) mice] and in both the mdr1a and mdr1b genes [mdr1a/1b (-/-) mice]. In spite of the host of functions speculatively attributed to the mdrl-type P-gps, we found no physiological abnormalities in either strain. Viability, fertility, and a range of histological, hematological, serum-chemical, and immunological parameters were not abnormal in mdr1a/1b (-/-) mice. The high level of mdrlb P-gp normally present in the pregnant uterus did not protect fetuses from a drug (digoxin) in the bloodstream of the mother, although the protein did reduce drug accumulation in the adrenal gland and ovaries. Pharmacologically, mdr1a/1b (-/-) mice behaved similarly to the previously analyzed mdr1a (-/-) mice, displaying, for instance, increased brain penetration and reduced elimination of digoxin. However, both mdr1a and mdr1b P-gps contributed to the extrusion of rhodamine from hematopoietic progenitor cells, suggesting a potential role for the endogenous mdr1-type P-gps in protection of bone marrow against cytotoxic anticancer drugs. This, and the normal viability of mdr1a/1b (-/-) mice, has implications for the use of P-gp-blocking agents in cancer and other chemotherapy. mdr1a/1b (-/-) mice should provide a useful model system to further test the pharmacological roles of the drug-transporting P-gps and to analyze the specificity and effectivity of P-gp-blocking drugs.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/fisiología , Resistencia a Múltiples Medicamentos/genética , Ratones Noqueados/fisiología , Animales , Digoxina/farmacocinética , Desarrollo Embrionario y Fetal/genética , Inhibidores Enzimáticos/farmacocinética , Femenino , Ratones , Ratones Noqueados/embriología , EmbarazoRESUMEN
The human multidrug resistance protein MRP1 mediates transport of organic substrates conjugated to glutathione, glucuronide, or sulfate. The naturally occurring prostaglandins A1 and A2 can form two diastereomeric glutathione S-conjugates, and it has been speculated that these might be substrates for MRP1. Here we present evidence that polarized MDCKII cells expressing MRP1 cDNA transport PGA1-GS to the basolateral side of a cell monolayer, in accordance with the lateral localization of human MRP1 in these cells. Furthermore, we show that vesicles made from yeast cells expressing MRP1 cDNA and from mouse erythrocytes (known to contain mrpl) actively accumulate both diastereomers of PGA2-GS with a similar efficiency. Recently, we generated mice with a homozygous mutant mrp1 allele. Uptake of PGA2-GS in vesicles made from erythrocytes of these mice was 3.2 times lower than in wild-type vesicles, but was still significantly above background. This residual transport activity was partly inhibited by methotrexate and cAMP, whereas mrp1-mediated activity was unaffected by these compounds. We conclude that mouse erythrocytes contain at least two transport systems for PGA2-GS. One of these is mrp1; the other one has not been identified yet, but can be inhibited by methotrexate and cAMP.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/fisiología , Glutatión/análogos & derivados , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Adenosina Trifosfato/fisiología , Animales , Transporte Biológico , Línea Celular , Polaridad Celular , AMP Cíclico/farmacología , Perros , Eritrocitos , Ácido Etacrínico/metabolismo , Glutatión/metabolismo , Humanos , Riñón/citología , Metotrexato/farmacología , Ratones , Ratones Mutantes , Microsomas/metabolismo , Prostaglandinas A Sintéticas/metabolismo , EstereoisomerismoRESUMEN
The mouse mdr1a (also called mdr3) P-GP is abundant in the blood-brain barrier, and its absence in mdr1a (-/-) mice leads to highly increased levels of the drugs ivermectin, vinblastine, digoxin, and cyclosporin A in the brain. We show here that the drugs loperamide, domperidone, and ondansetron are transported substrates for the mouse mdr1a P-GP and its human homologue MDR1. Phenytoin is a relatively weaker substrate for each, and the drugs haloperidol, clozapine, and flunitrazepam are transported hardly or not at all. Tissue distribution studies demonstrated that the relative brain penetration of radiolabeled ondansetron and loperamide (and their metabolites) is increased four- and sevenfold, respectively, in mdr1a (-/-) mice. A pilot toxicity study with oral loperamide showed that this peripherally acting antidiarrheal agent gains potent opiatelike activity in the central nervous system of mdr1a (-/-) mice. mdr1a (-/-) mice also showed increased sensitivity to neurolepticlike side effects of oral domperidone. These results point to the possible role that the drug-transporting P-GP(s) may play in the clinical use of many drugs, especially those with potential targets in the central nervous system.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Barrera Hematoencefálica , Encéfalo/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/sangre , Animales , Transporte Biológico , Encéfalo/efectos de los fármacos , Línea Celular , Clozapina/farmacocinética , Difusión , Domperidona/farmacocinética , Domperidona/farmacología , Epitelio/metabolismo , Flunitrazepam/farmacocinética , Haloperidol/farmacocinética , Humanos , Riñón , Cinética , Loperamida/farmacocinética , Masculino , Ratones , Ratones Endogámicos , Ondansetrón/farmacocinética , Fenitoína/farmacocinética , Proteínas Recombinantes/metabolismo , Relación Estructura-Actividad , Porcinos , Distribución Tisular , TransfecciónRESUMEN
The human multidrug resistance-associated protein MRP confers resistance to various cytotoxic drugs by lowering the intracellular drug concentration. Recent evidence indicates that MRP can also transport glutathione S-conjugates across membranes. To study the transport properties of MRP in intact cells, we have expressed human MRP cDNA in the polarized pig kidney epithelial cell line LLC-PK1. MRP mainly localized to the basolateral plasma membrane of these cells, and not to the apical membrane, as determined by immunocytochemistry using confocal laser scanning and electron microscopy. In accordance with this localization, MRP caused increased transport of the glutathione S-conjugate S-(2, 4-dinitrophenyl)-glutathione and of the anticancer drug daunorubicin to the basal side of the epithelial cell layer. Sulfinpyrazone and probenecid, known inhibitors of multispecific organic anion transport, inhibited this basolateral transport, but not the apical transport of daunorubicin mediated by the apically localized human MDR1 P-glycoprotein in MDR1-transfected LLC-PK1 cells. Probenecid and sulfinpyrazone may therefore be useful lead compounds for the development of clinical reversal agents specific for MRP-mediated drug resistance.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/análisis , Resistencia a Múltiples Medicamentos , Riñón/química , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Transporte Biológico , Línea Celular , Daunorrubicina/farmacocinética , Glutatión/análogos & derivados , Glutatión/metabolismo , Humanos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos , PorcinosRESUMEN
We have previously shown that absence of the mouse mdr1a (also called mdr3) P-glycoprotein in mdr1a (-/-) "knockout" mice has a profound effect on the tissue distribution and elimination of vinblastine and ivermectin, and hence on the toxicity of these compounds. We show here that the mouse mdr1a and the human MDR1 P-glycoprotein actively transport ivermectin, dexamethasone, digoxin, and cyclosporin A and, to a lesser extent, morphine across a polarized kidney epithelial cell layer in vitro. Injection of these radio-labeled drugs in mdr1a (-/-) and wild-type mice resulted in markedly (20- to 50-fold) higher levels of radioactivity in mdr1a (-/-) brain for digoxin and cyclosporin A, with more moderate effects for dexamethasone (2- to 3-fold) and morphine (1.7-fold). Digoxin and cyclosporin A were also more slowly eliminated from mdr1a (-/-) mice. Our findings show that P-glycoprotein can be a major determinant for the pharmacology of several medically important drugs other than anti-cancer agents, especially in the blood-brain barrier. These results may explain a range of pharmacological interactions observed between various drugs in patients.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/fisiología , Cardiotónicos/farmacocinética , Ciclosporina/farmacocinética , Dexametasona/farmacocinética , Digoxina/farmacocinética , Glucocorticoides/farmacocinética , Inmunosupresores/farmacocinética , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/análisis , Animales , Transporte Biológico , Humanos , Masculino , Ratones , Morfina/farmacocinética , Porcinos , Distribución TisularRESUMEN
Drug resistance, be it intrinsic or acquired, is a major problem in cancer chemotherapy. In vitro, one well characterised form of resistance against many different cytotoxic drugs is caused by the MDR1 P-glycoprotein, a large plasma membrane protein that protects the cell by actively pumping substrate drugs out. Available evidence suggests that this protein may cause drug resistance in at least some clinical tumours. Drugs inhibiting the MDR1 P-glycoprotein activity are, therefore, co-administered during chemotherapy of these tumours. To predict the biological and pharmacological effects of the blocking of this protein, we have generated mice with a genetic disruption of the drug-transporting mdr1a P-glycoprotein. These mice are overall healthy, but they accumulate much higher levels of substrate drugs in the brain, and have markedly slower elimination of these drugs from the circulation. For some drugs, this leads to dramatically increased toxicity, indicating that P-glycoprotein inhibitors should be used with caution in patients.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/deficiencia , Resistencia a Múltiples Medicamentos , Resistencia a Antineoplásicos , Ratones Noqueados/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/antagonistas & inhibidores , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Animales , Ratones , Ratones Noqueados/genética , FenotipoRESUMEN
The human MDR3 (or MDR2) P-glycoprotein is probably involved in the transport of phospholipids from liver hepatocytes into bile (Smit et al. (1993) Cell 75, 451-462). In accordance with this function, MDR3 is highly expressed in human liver, but lower mRNA levels were also found in adrenal, heart, muscle and cells of the B-cell compartment. We have cloned and analyzed the MDR3 promoter region. It is GC-rich, and contains neither a TATA nor a CAAT box, but it does contain multiple putative SP1 binding sites, features also found in so-called housekeeping genes. RNase protection and primer extension analyses indicate that the MDR3 gene has multiple transcription start sites in a GC-rich region with considerable homology to the putative mouse mdr2 promoter. A 3 kb genomic fragment containing the MDR3 start sites directs transcription of a chloramphenicol acetyltransferase (CAT) reporter gene upon transient transfection in the human hepatoma cell line HepG2. This transcription is orientation dependent, and stimulated by a SV40 enhancer, indicating that the 3 kb insert contains the core promoter elements of the MDR3 gene. The promoter region contains several consensus sequences where known or putative liver-specific (C/EBP, HNF5) or lymphoid specific (Pu.1, ets-1) transcription factors may bind.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Genes , Regiones Promotoras Genéticas , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/biosíntesis , Animales , Secuencia de Bases , Carcinoma Hepatocelular/patología , Clonación Molecular , Secuencia de Consenso , ADN Complementario/genética , Elementos de Facilitación Genéticos , Exones/genética , Regulación de la Expresión Génica , Genes Reporteros , Humanos , Neoplasias Hepáticas/patología , Ratones/genética , Datos de Secuencia Molecular , Empalme del ARN , Proteínas Recombinantes de Fusión/biosíntesis , Alineación de Secuencia , Homología de Secuencia de Ácido Nucleico , Transcripción Genética , Células Tumorales CultivadasRESUMEN
We have generated mice homozygous for a disruption of the mdr1a (also called mdr3) gene, encoding a drug-transporting P-glycoprotein. The mice were viable and fertile and appeared phenotypically normal, but they displayed an increased sensitivity to the centrally neurotoxic pesticide ivermectin (100-fold) and to the carcinostatic drug vinblastine (3-fold). By comparison of mdr1a (+/+) and (-/-) mice, we found that the mdr1a P-glycoprotein is the major P-glycoprotein in the blood-brain barrier and that its absence results in elevated drug levels in many tissues (especially in brain) and in decreased drug elimination. Our findings explain some of the side effects in patients treated with a combination of carcinostatics and P-glycoprotein inhibitors and indicate that these inhibitors might be useful in selectively enhancing the access of a range of drugs to the brain.
Asunto(s)
Barrera Hematoencefálica/fisiología , Proteínas Portadoras/genética , Ivermectina/toxicidad , Glicoproteínas de Membrana/genética , Vinblastina/farmacocinética , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP , Animales , Barrera Hematoencefálica/efectos de los fármacos , Capilares/química , Proteínas Portadoras/análisis , Proteínas Portadoras/fisiología , Resistencia a Medicamentos/genética , Células Epiteliales , Femenino , Intestino Delgado/química , Ivermectina/sangre , Ivermectina/farmacocinética , Masculino , Glicoproteínas de Membrana/análisis , Glicoproteínas de Membrana/fisiología , Ratones , Ratones Noqueados/genética , Mutagénesis Insercional , ARN Mensajero/análisis , Distribución Tisular , Vinblastina/toxicidadRESUMEN
Two types of P-glycoprotein have been found in mammals: the drug-transporting P-glycoproteins and a second type, unable to transport hydrophobic anticancer drugs. The latter is encoded by the human MDR3 (also called MDR2) and the mouse mdr2 genes, and its tissue distribution (bile canalicular membrane of hepatocytes, B cells, heart, and muscle) suggests a specialized metabolic function. We have generated mice homozygous for a disruption of the mdr2 gene. These mice develop a liver disease that appears to be caused by the complete inability of the liver to secrete phospholipid into the bile. Mice heterozygous for the disrupted allele had no detectable liver pathology, but half the level of phospholipid in bile. We conclude that the mdr2 P-glycoprotein has an essential role in the secretion of phosphatidylcholine into bile and hypothesize that it may be a phospholipid transport protein or phospholipid flippase.
Asunto(s)
Bilis/metabolismo , Proteínas Portadoras/genética , Hepatopatías/genética , Glicoproteínas de Membrana/genética , Fosfolípidos/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP , Alelos , Animales , Secuencia de Bases , Bilirrubina/sangre , Proteínas Portadoras/análisis , Enzimas/sangre , Homocigoto , Hígado/fisiopatología , Glicoproteínas de Membrana/análisis , Ratones , Ratones Mutantes , Datos de Secuencia Molecular , Mutagénesis , ARN Mensajero/análisis , Recombinación GenéticaRESUMEN
In this paper, we review recent work on multidrug resistance (MDR) in Amsterdam. We have generated mice homozygous for a disruption of one of their P-glycoprotein (Pgp) genes. The mutations do not interfere with viability or fertility, showing that these Pgps have no indispensable role in early development or metabolism. Mice homozygous for a disruption of their mdr2 gene, however, develop liver disease and this appears to be due to their complete inability to secrete phospholipids into bile. This suggests that the mdr2 Pgp (and, by inference, its human MDR3 homologue) is essential for translocating phospholipids through the hepatocyte canalicular membrane in which this Pgp is located. These and other results show the importance of the genetic approach for studying drug metabolism. MDR is not only caused by increased activity of Pgps. When the human non-small cell lung carcinoma cell line SW-1573 is selected in vitro for low level doxorubicin resistance, the resistant variants are nearly always multidrug resistant, but this is not due to increased Pgp activity. Only when resistance is pushed to higher levels does activation of the MDR1 Pgp gene occur. This suggests that clinically relevant levels of drug resistance in some cells may be caused predominantly by non-Pgp-mediated drug resistance mechanisms. The protein responsible for MDR in the SW-1573 cells has not yet been identified and experiments are in progress to find the gene encoding it.
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Proteínas Portadoras/genética , Proteínas Portadoras/fisiología , Resistencia a Medicamentos/genética , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/fisiología , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP , Animales , Carcinoma de Células Pequeñas/tratamiento farmacológico , Doxorrubicina/uso terapéutico , Humanos , Hepatopatías/genética , Ratones , Esteroides/metabolismo , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
Amplification and overexpression of the neu gene have been found in several human adenocarcinomas. We have obtained monoclonal antibodies to the human neu protein by immunizing a Balb/c mouse with a Balb/c cell line expressing the human neu gene by transfection. The monoclonal antibodies reacted with neu protein on intact cells by immunofluorescence and immunoprecipitated neu in metabolically labeled cells, also in the presence of tunicamycin. We tested possible down-modulating effects of these monoclonal antibodies on SKBR-3 mammary tumor cells, which express high levels of wild-type human neu protein. We also used NIH3T3 cells transfected with either a normal or a mutated human neu gene, encoding a protein with a valine to glutamic acid substitution in the transmembrane domain. Down-modulation of the normal cell-surface neu protein was inefficient. In contrast, the antibodies induced 50-65% down-modulation in NIH3T3 cells expressing the mutated human neu protein and could inhibit these cells to form colonies in soft agar. We propose that these differences are due to changed aggregation properties of the point-mutated protein.
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Anticuerpos Monoclonales/inmunología , Regulación Neoplásica de la Expresión Génica , Mutación , Proteínas Proto-Oncogénicas/genética , Proto-Oncogenes , Animales , Adhesión Celular , División Celular , Línea Celular , Células Cultivadas , Células Clonales , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Humanos , Neoplasias Mamarias Experimentales , Ratones , Ratones Endogámicos BALB C/inmunología , Plásmidos , Proteínas Tirosina Quinasas , Proteínas Proto-Oncogénicas/inmunología , Receptor ErbB-2RESUMEN
In mouse embryos, the int-1 proto-oncogene is transiently expressed in areas of the developing neural system. Retinoic acid-treated P19 embryonal carcinoma cells have often been used as an in vitro model for the molecular basis of neural development. We shown here that int-1 is transiently expressed in differentiated P19 cells. The time course and retinoic acid dose dependence of int-1 expression suggest that the gene is specifically expressed during early neural differentiation. P19 cells may be a useful model to assist in the study, at the cellular level, of the role of int-1 in neural development.
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Proto-Oncogenes , Teratoma/genética , Proteínas de Pez Cebra , Animales , Diferenciación Celular/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/patología , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas/genética , Proto-Oncogenes/efectos de los fármacos , Teratoma/metabolismo , Teratoma/patología , Tretinoina/farmacología , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/metabolismo , Células Tumorales Cultivadas/patología , Proteínas Wnt , Proteína Wnt1RESUMEN
The int-1 gene is often activated by proviral insertion in mouse mammary tumors. Direct evidence for the normal function of this gene and its role in tumorigenesis has therefore been lacking. To examine possible biological effects of int-1 activation in in vitro cell systems, we have constructed recombinant molecules of genomic int-1 DNA, transcriptionally activated by retroviral promoters. Transfection of these constructs into cuboidal RAC311C mammary cells leads to morphological transformation of the cells and rapid tumorigenicity.