RESUMEN
For analysis of multidrug resistance, a major barrier to effective cancer chemotherapy, we profiled mRNA expression of the 48 known human ABC transporters in 60 diverse cancer cell lines (the NCI-60) used by the National Cancer Institute to screen for anticancer activity. The use of real-time RT-PCR avoided artifacts commonly encountered with microarray technologies. By correlating the results with the growth inhibitory profiles of 1,429 candidate anticancer drugs tested against the cells, we identified which transporters are more likely than others to confer resistance to which agents. Unexpectedly, we also found and validated compounds whose activity is potentiated, rather than antagonized, by the MDR1 multidrug transporter. Such compounds may serve as leads for development.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Resistencia a Múltiples Medicamentos , Resistencia a Antineoplásicos , Perfilación de la Expresión Génica , Neoplasias/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/metabolismo , Estadística como AsuntoRESUMEN
Methotrexate (MTX) is a substrate for numerous human ATP-binding cassette (ABC) efflux transporters, yet the impact of these transporters on MTX pharmacokinetics (PK) over a large dose range has not been examined. To investigate the effects of two transporters-ABC subfamily C member 2 (Abcc2; multidrug resistance protein 2) and ABC subfamily C member 3 (Abcc3; multidrug resistance protein 3)-involved in MTX hepatobiliary disposition in vivo, MTX plasma, urine, and feces concentrations were analyzed after 10, 50, and 200 mg/kg i.v. doses to groups of wild type (WT), Abcc2(-/-), and Abcc3(-/-) mice. The absence of Abcc2 caused a decrease in total clearance of MTX relative to WT mice at all dose levels yet was accompanied by compensatory increases in renal excretion and metabolism to 7-hydroxymethotrexate (7OH-MTX). In Abcc3(-/-) mice, total clearance was elevated at the two lower dose levels and was attributed to stimulation of biliary excretion and confirmed by elevated fecal excretion; however, at the high 200 mg/kg dose, clearance was severely retarded and could be attributed to hepatotoxicity because conversion to 7OH-MTX was diminished. The findings confirmed that both Abcc2 and Abcc3 significantly influenced the PK properties of MTX, and depending on the MTX dose and strain, alternate elimination pathways were elicited and saturable.
Asunto(s)
Metotrexato/farmacocinética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/deficiencia , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Sistema Biliar/metabolismo , Heces/química , Hígado/metabolismo , Masculino , Metotrexato/análogos & derivados , Metotrexato/sangre , Metotrexato/metabolismo , Metotrexato/orina , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismoRESUMEN
The apical sodium-dependent bile acid transporter (Asbt) is responsible for transport across the intestinal brush border membrane; however, the carrier(s) responsible for basolateral bile acid export into the portal circulation remains to be determined. Although the heteromeric organic solute transporter Ostalpha-Ostbeta exhibits many properties predicted for a candidate intestinal basolateral bile acid transporter, the in vivo functions of Ostalpha-Ostbeta have not been investigated. To determine the role of Ostalpha-Ostbeta in intestinal bile acid absorption, the Ostalpha gene was disrupted by homologous recombination in mice. Ostalpha(-/-) mice were physically indistinguishable from wild-type mice. In everted gut sac experiments, transileal transport of taurocholate was reduced by >80% in Ostalpha(-/-) vs. wild-type mice; the residual taurocholate transport was further reduced to near-background levels in gut sacs prepared from Ostalpha(-/-)Mrp3(-/-) mice. The bile acid pool size was significantly reduced (>65%) in Ostalpha(-/-) mice, but fecal bile acid excretion was not elevated. The decreased pool size in Ostalpha(-/-) mice resulted from reduced hepatic Cyp7a1 expression that was inversely correlated with ileal expression of fibroblast growth factor 15 (FGF15). These data indicate that Ostalpha-Ostbeta is essential for intestinal bile acid transport in mice. Unlike a block in intestinal apical bile acid uptake, genetic ablation of basolateral bile acid export disrupts the classical homeostatic control of hepatic bile acid biosynthesis.
Asunto(s)
Ácidos y Sales Biliares/metabolismo , Homeostasis , Mucosa Intestinal/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Animales , Transporte Biológico/efectos de los fármacos , Colesterol 7-alfa-Hidroxilasa/metabolismo , Ácido Cólico/administración & dosificación , Ácido Cólico/farmacología , Heces/química , Regulación de la Expresión Génica/efectos de los fármacos , Marcación de Gen , Homeostasis/efectos de los fármacos , Absorción Intestinal/efectos de los fármacos , Mucosa Intestinal/efectos de los fármacos , Intestino Delgado/efectos de los fármacos , Intestino Delgado/metabolismo , Intestinos/efectos de los fármacos , Lípidos/aislamiento & purificación , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Proteínas de Transporte de Membrana/deficiencia , Ratones , Ratones Noqueados , Modelos Biológicos , Fenotipo , Membrana Serosa/efectos de los fármacos , Membrana Serosa/metabolismoRESUMEN
PURPOSE: Successful treatment of acute myeloid leukemia (AML) remains a therapeutic challenge, with a high percentage of patients suffering from persistent or relapsed disease. Resistance to drug therapy can develop from increased drug export and/or altered intracellular signaling. Both mechanisms are mediated by the efflux transporters ABCC4 (MRP4), ABCC5 (MRP5), and ABCC11 (MRP8), which are involved in cellular efflux of endogenous signaling molecules (e.g., cyclic adenosine 3', 5'-monophosphate and cyclic guanosine 3',5'-monophosphate) and nucleoside analogues. The nucleoside analogue cytosine arabinoside (AraC) is administered to all patients with AML. EXPERIMENTAL DESIGN: Expression of ABCC transporters MRP4, MRP5, and MRP8 in blast samples from 50 AML patients was investigated by real-time reverse transcription-PCR analysis and correlated with clinical outcome measures. Accumulation of radiolabeled AraC, transport of AraC metabolites, and AraC cytotoxicity were analyzed in MRP8-transfected LLC-PK1 cells. RESULTS: Regression analysis revealed that high expression of MRP8 is associated with a low probability of overall survival assessed over 4 years (P<0.03). MRP8-transfected LLC-PK1 cells accumulated reduced intracellular levels of AraC (63% of the parental vector-transfected LLC-PK1 control cells) as well as AraC metabolites. Furthermore, AraC monophosphate was transported by MRP8-enriched membrane vesicles (116+/-6 versus 65+/-13 pmol/mg/10 minutes by control vesicles), and MRP8-transfected cells were resistant to AraC. CONCLUSION: These data suggest that MRP8 is differentially expressed in AML blasts, that expression of MRP8 serves as a predictive marker for treatment outcome in AML, and that efflux of AraC metabolites by MRP8 is a mechanism that contributes to resistance of AML blasts.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Crisis Blástica , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Adulto , Anciano , Membrana Celular/metabolismo , Citarabina/metabolismo , Resistencia a Antineoplásicos , Femenino , Humanos , Células LLC-PK1 , Leucemia Mieloide Aguda/mortalidad , Masculino , Persona de Mediana Edad , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Pronóstico , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Madre/metabolismo , Células Madre/patología , Tasa de SupervivenciaRESUMEN
Nucleoside-based analogues are mainstays in the treatment of cancer, viral infections, and inflammatory diseases. Recent studies showing that the ATP-binding cassette transporter, multidrug resistance protein 4, is able to efflux nucleoside and nucleotide analogues from transfected cells suggests that the pump may affect the efficacy of this class of agents. However, the in vivo pharmacologic functions of the pump are largely unexplored. Here, using Mrp4(-/-) mice as a model system, and the nucleotide analogue, 9'-(2'-phosphonylmethoxyethyl)-adenine (PMEA) as a probe, we investigate the ability of Mrp4 to function in vivo as an endogenous resistance factor. In the absence of alterations in plasma PMEA levels, Mrp4-null mice treated with PMEA exhibit increased lethality associated with marked toxicity in several tissues. Affected tissues include the bone marrow, spleen, thymus, and gastrointestinal tract. In addition, PMEA penetration into the brain is increased in Mrp4(-/-) mice. These findings indicate that Mrp4 is an endogenous resistance factor, and that the pump may be a component of the blood-brain barrier for nucleoside-based analogues. This is the first demonstration that an ATP-binding cassette transporter can affect in vivo tissue sensitivity towards this class of agents.
Asunto(s)
Adenina/análogos & derivados , Hipersensibilidad a las Drogas/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Organofosfonatos/farmacología , Adenina/sangre , Adenina/farmacocinética , Adenina/farmacología , Animales , Médula Ósea/efectos de los fármacos , Médula Ósea/metabolismo , Encéfalo/metabolismo , Hipersensibilidad a las Drogas/etiología , Hipersensibilidad a las Drogas/genética , Mucosa Intestinal/metabolismo , Intestinos/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/deficiencia , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Organofosfonatos/sangre , Organofosfonatos/farmacocinética , Bazo/efectos de los fármacos , Bazo/metabolismo , Timo/efectos de los fármacos , Timo/metabolismoRESUMEN
ABCC11 (Multidrug resistance protein 8; MRP8), a plasma membrane ATP-binding cassette transporter, has been implicated in drug resistance of breast cancer by virtue of its ability to confer resistance to fluoropyrimidines and to efflux methotrexate, and by its expression in this tumor. Expression of ABCC11 in breast, a hormonally regulated tissue, as well as the pump's ability to transport estrogen conjugates, suggest the possibility that expression of ABCC11 may be susceptible to regulation by estrogen. However, nothing is currently known about regulation of this gene. In this study, estradiol (E(2)) treatment reduced expression of ABCC11 mRNA in estrogen receptor (ER)-alpha-positive MCF7 cells, and E(2) antagonists such as ICI 182 780 and tamoxifen (TAM) abrogated E(2)-mediated downregulation. ABCC11 expression was positively correlated with ER-alpha expression in both breast cell lines, and two independent series of tumors from postmenopausal patients. In addition, expression of ABCC11 was upregulated in MCF7 cells exposed to TAM for 72 h, and was overexpressed in TAM-resistant cell lines. Drug sensitivity analysis of the TAM-resistant cells indicated that they were also resistant to 5-fluorouracil (5-FU), consistent with the reported ability of ABCC11 to confer resistance to this agent. These studies indicate that ABCC11 expression is negatively regulated by E(2), but that ABCC11 expression is high in high-expressing ER-alpha breast cancers. Our findings support the notion that expression of ABCC11 in ER-alpha-positive breast cancers may contribute to decreased sensitivity to chemotherapy combinations that include 5-FU. ABCC11 may be a potential predictive tool in the choice of anticancer therapies in ER-positive breast cancers resistant to TAM.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Estradiol/farmacología , Receptor alfa de Estrógeno/genética , Posmenopausia , Tamoxifeno/farmacología , Transportadoras de Casetes de Unión a ATP/metabolismo , Anciano , Anciano de 80 o más Años , Western Blotting , Mama/metabolismo , Mama/patología , Neoplasias de la Mama/patología , Carcinoma Ductal de Mama/tratamiento farmacológico , Carcinoma Ductal de Mama/metabolismo , Carcinoma Ductal de Mama/secundario , Receptor alfa de Estrógeno/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Persona de Mediana Edad , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales CultivadasRESUMEN
To investigate the mechanism of cellular resistance to 6-MP, we established a 6-MP resistant cell line (CEM-MP5) by stepwise selection of the human T-lymphoblastic leukemia cell line (CEM). CEM-MP5 cells were about 100-fold resistant to 6-MP compared with parental CEM cells. Western blot analysis demonstrated that multidrug resistant protein 4 (MRP4) was increased in CEM-MP5 cells, whereas the levels of the nucleoside transporters hENT1, hCNT2 and hCNT3 were decreased compared with those of parental CEM cells. Consistent with the operation of an efflux pump, accumulation of [14C]6-MP and/or its metabolites was reduced, and ATP-dependent efflux was increased in CEM-MP5 cells. Taken together these results showed that up-regulation of MRP4 and down-regulation of influx transporters played a major role in 6-MP resistance of CEM-MP5 cells.
Asunto(s)
Antimetabolitos Antineoplásicos/farmacología , Leucemia/tratamiento farmacológico , Mercaptopurina/farmacología , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/fisiología , Proteínas de Transporte de Nucleósidos/fisiología , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/análisis , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Transportadoras de Casetes de Unión a ATP/análisis , Línea Celular , Resistencia a Antineoplásicos , Tranportador Equilibrativo 1 de Nucleósido/antagonistas & inhibidores , Transportador Equilibrativo 2 de Nucleósido/antagonistas & inhibidores , Humanos , Hipoxantina Fosforribosiltransferasa/metabolismo , Leucemia/metabolismo , Proteínas de Transporte de Membrana/análisis , Mercaptopurina/farmacocinética , Metiltransferasas/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/análisis , Proteínas de Neoplasias/análisis , Proteínas de Transporte de Nucleósidos/análisis , Regulación hacia ArribaRESUMEN
The disposition of fexofenadine, a commonly used antihistamine drug, is governed primarily by active transport. Biliary excretion of the parent compound is the major route of systemic clearance. Previous studies demonstrated that fexofenadine hepatic uptake is mediated by organic anion transporting polypeptides. Recently, we showed that in mice fexofenadine is excreted into bile primarily by multidrug resistance-associated protein (Mrp) 2 (Abcc2). In the present study, the roles of Mrp3 (Abcc3) and Mrp4 (Abcc4) in the hepatobiliary disposition of fexofenadine were examined in knockout mice using in situ liver perfusion. Compared with that in wild-type mice, basolateral excretion of fexofenadine was impaired, resulting in a approximately 50% decrease in perfusate recovery in Abcc3(-/-) mice; in contrast, fexofenadine hepatobiliary disposition was unaltered in Abcc4(-/-) mice. As expected, in Abcc2(-/-) mice, fexofenadine was redirected from the canalicular to the basolateral membrane for excretion. In Abcc2(-/-)/Abcc3(-/-) double-knockout mice, fexofenadine biliary excretion was impaired, but perfusate recovery was similar to that in wild-type mice and more than 2-fold higher than that in Abcc3(-/-) mice, presumably due to compensatory basolateral transport mechanism(s). These results demonstrate that multiple transport proteins are involved in the hepatobiliary disposition of fexofenadine. In addition to Mrp2 and Mrp3, other transport proteins play an important role in the biliary and hepatic basolateral excretion of this zwitterionic drug.
Asunto(s)
Antagonistas de los Receptores Histamínicos H1 no Sedantes/metabolismo , Hígado/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Terfenadina/análogos & derivados , Animales , Bilis/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Terfenadina/metabolismoRESUMEN
PURPOSE: Knowledge of drug concentrations in tumors is critical for understanding the determinants of drug accumulation in tumors. Because significant obstacles prevent making these measurements in humans, development of a predictive pharmacokinetic model would be of great value to the translation of preclinical data to the clinic. Our goal was to show how the latter could be achieved for temozolomide, an agent used in the treatment of brain tumors, using an orthotopic brain tumor model in rats. EXPERIMENTAL DESIGN: Rats bearing i.c. tumors received 20 mg/kg i.v. of temozolomide followed by the subsequent measurement of serial plasma, cerebrospinal fluid (CSF), normal brain, and brain tumor temozolomide concentrations. The resultant data provided the framework to develop a hybrid physiologically based pharmacokinetic model for temozolomide in brain. The preclinical pharmacokinetic model was scaled to predict temozolomide concentrations in human CSF, normal brain, and brain tumor, and through a series of Monte Carlo simulations, the accumulation of temozolomide in brain tumors under conditions of altered blood-brain barrier permeability, fractional blood volume, and clinical dosing schedules was evaluated. RESULTS: The developed physiologically based pharmacokinetic model afforded a mechanistic and accurate prediction of temozolomide brain disposition in rats, which through model scale-up procedures accurately predicted the CSF/plasma area under the drug concentration-time curve ratios of 0.2 reported in patients. Through a series of model simulations, it was shown that the brain tumor accumulation of temozolomide varied substantially based on changes in blood-brain barrier permeability and fractional tumor blood volume but minimally based on clinical dosing regimens. CONCLUSIONS: A physiologically based pharmacokinetic modeling approach offers a means to translate preclinical to clinical characteristics of drug disposition in target tissues and, thus, a means to select appropriate drug dosing regimens for achieving optimal target tissue drug concentrations.
Asunto(s)
Antineoplásicos Alquilantes/farmacocinética , Neoplasias Encefálicas/patología , Encéfalo/metabolismo , Dacarbazina/análogos & derivados , Animales , Antineoplásicos Alquilantes/líquido cefalorraquídeo , Antineoplásicos Alquilantes/uso terapéutico , Encéfalo/efectos de los fármacos , Neoplasias Encefálicas/tratamiento farmacológico , Dacarbazina/líquido cefalorraquídeo , Dacarbazina/farmacocinética , Dacarbazina/uso terapéutico , Humanos , Modelos Animales , Ratas , TemozolomidaRESUMEN
Methotrexate (MTX) is an anticancer agent that is widely used in a variety of human cancers including primary central nervous system lymphoma (PCNSL). Important pharmacological properties that directly bear on the use of MTX in PCNSL, such as mechanisms that govern its uptake into brain tumors, are poorly defined, but are amenable to investigation in mouse models. In order to pursue such preclinical pharmacological studies, a rapid and sensitive liquid chromatography-tandem mass spectrometry (LC/MS/MS) method for the determination of MTX and its metabolite, 7-hydroxymethotrexate (7-OH MTX) in plasma and microdialysate samples from brain tumors and cerebrospinal fluid (CSF) is needed. The plasma assay was based on 10 microl samples and following a protein precipitation procedure enabled direct injection onto a LC/MS/MS system using positive electrospray ionization. A column switching technique was employed for desalting and the clean-up of microdialysate samples from brain tissues. The methods were validated for MTX and 7-OH MTX in both plasma and microdialysate samples from brain tumor and CSF, and produced lower limits of quantification (LLOQ) in plasma of 3.7 ng/ml for MTX and 7.4 ng/ml for 7-OH MTX, and in microdialysate samples of 0.7 ng/ml for both MTX and 7-OH MTX. The utility of the method was demonstrated by estimation of pharmacokinetic (PK) and brain distribution properties of MTX and 7-OH MTX in conscious mice. The method has the advantages of low sample volume, rapid clean-up, and the simultaneous measurement of MTX and 7-OH MTX in plasma and brain tissues allowing detailed PK studies to be completed in individual mice.
Asunto(s)
Antimetabolitos Antineoplásicos/análisis , Cromatografía Liquida/métodos , Metotrexato/análogos & derivados , Metotrexato/análisis , Metotrexato/metabolismo , Espectrometría de Masas en Tándem/métodos , Animales , Antimetabolitos Antineoplásicos/sangre , Encéfalo/metabolismo , Neoplasias Encefálicas/metabolismo , Metotrexato/sangre , Metotrexato/química , Metotrexato/farmacocinética , Ratones , Ratones Endogámicos C57BL , Microdiálisis , Estructura Molecular , Estándares de Referencia , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Espectrometría de Masa por Ionización de Electrospray/métodosRESUMEN
Human MRP4 (ABCC4, MOAT-B) is a lipophilic anion transporter that is able to confer resistance to nucleotide analogues and methotrexate (MTX). We previously investigated the implications of the ability of MRP4 to confer resistance to nucleotide analogues and determined that the pump is competent in the MgATP-energized transport of cyclic nucleotides and estradiol 17beta-D-glucuronide. Here we examine the potential role of MRP4 in conferring resistance to MTX and related processes by determining the selectivity of the transporter for MTX, MTX polyglutamates, and physiological folates. In so doing, it is shown that MRP4 is active in the transport of MTX as well as the physiological folates folic acid (FA) and N(5)-formyltetrahydrofolic acid (leucovorin). MTX, FA, and leucovorin are subject to high capacity [V(max(MTX)), 0.24 +/- 0.05 nmol/mg/min; V(max (FA)), 0.68 +/- 0.14 nmol/mg/min; V(max(leucovorin)), 1.95 +/- 0.18 nmol/mg/min], low affinity [K(m(MTX)), 0.22 +/- 0.01 mM; K(m(FA)), 0.17 +/- 0.02 mM; K(m (leucovorin)), 0.64 +/- 0.23 mM] transport by MRP4. In addition, as would be expected were MRP4 a component of the MTX efflux system, its capacity to transport this agent is abrogated by the addition of a single glutamyl residue. It is also shown that glutamylation similarly affects the ability of MRP2 to transport MTX. On the basis of these transport properties, it is concluded that the efflux system for MTX includes MRP2 and MRP4, in addition to MRP1 and MRP3, and that MRP4 represents a common efflux system for both MTX and certain nucleotide analogues.
Asunto(s)
Antimetabolitos Antineoplásicos/farmacocinética , Proteínas Portadoras/metabolismo , Ácido Fólico/farmacocinética , Metotrexato/farmacocinética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Receptores de Superficie Celular , Animales , Baculoviridae/genética , Línea Celular , Receptores de Folato Anclados a GPI , Insectos/citología , Insectos/metabolismo , Insectos/virología , Células LLC-PK1/metabolismo , Liposomas , Metotrexato/análogos & derivados , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Ósmosis , Ácido Poliglutámico/análogos & derivados , Ácido Poliglutámico/farmacocinética , Porcinos , TransfecciónRESUMEN
Mutations in human multidrug resistance protein 6 (MRP6, ABCC6), a member of the MRP family of drug efflux pumps, are the genetic basis of Pseudoxanthoma elasticum, a disease that affects elastin fibers in the skin, retina, and blood vessels. However, little is known about the functional characteristics of the protein, including its potential activity as a resistance factor for anticancer agents. Here, we report the results of investigations of the in vitro transport properties and drug resistance activity of MRP6. Using membrane vesicles prepared from Chinese hamster ovary cells transfected with MRP6 expression vector, it is shown that expression of MRP6 is specifically associated with the MgATP-dependent transport of the glutathione S-conjugates leukotriene C(4) and S-(2, 4-dinitrophenyl)glutathione and the cyclopentapeptide BQ123 but not glucuronate conjugates such as 17beta-estradiol 17-(beta-D-glucuronide). Analysis of the drug sensitivity of MRP6-transfected cells revealed low levels of resistance to several natural product agents, including etoposide, teniposide, doxorubicin, and daunorubicin. These results indicate that MRP6 is a glutathione conjugate pump that is able to confer low levels of resistance to certain anticancer agents.
Asunto(s)
Resistencia a Múltiples Medicamentos/fisiología , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Animales , Antineoplásicos/farmacocinética , Antineoplásicos/farmacología , Transporte Biológico Activo , Células CHO , Cricetinae , Resistencia a Antineoplásicos , Etopósido/farmacocinética , Etopósido/farmacología , Leucotrieno C4/farmacocinética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/biosíntesis , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Tenipósido/farmacocinética , Tenipósido/farmacología , TransfecciónRESUMEN
The multidrug resistance protein (MRP) family consists of nine members that can be categorized according to whether or not a third (NH(2)-terminal) membrane-spanning domain is present. Three (MRP1, MRP2, and MRP3) of the four members that have this structural feature are able to confer resistance to natural product anticancer agents. We previously established that MRP7, the remaining family member that has three membrane-spanning domains, possesses the cardinal biochemical activity of MRPs in that it is able to transport amphipathic anions such as 17beta-estradiol 17-(beta-d-glucuronide). However, the drug resistance profile of the pump has not been determined. In this study, the drug resistance capabilities of MRP7 are evaluated by analyzing the resistance profiles of two clones of HEK293 cells in which the pump was ectopically expressed. MRP7-transfected HEK293 cells exhibited the highest levels of resistance toward docetaxel (9-13-fold). In addition, lower levels of resistance were observed for paclitaxel (3-fold), vincristine (3-fold), and vinblastine (3-4-fold). Consistent with the operation of an ATP-dependent efflux pump, MRP7-transfected cells exhibited reduced accumulation of radiolabeled paclitaxel compared with HEK293 cells transfected with parental plasmid. These results indicate that MRP7, unlike other MRPs, is a resistance factor for taxanes.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Taxoides/farmacología , Antineoplásicos Fitogénicos/farmacocinética , Línea Celular , Docetaxel , Resistencia a Antineoplásicos , Humanos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/biosíntesis , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Paclitaxel/farmacocinética , Paclitaxel/farmacología , Taxoides/farmacocinética , TransfecciónRESUMEN
ABCG2 is a plasma membrane efflux pump that is able to confer resistance to several anticancer agents, including mitoxantrone, camptothecins, anthracyclines, and flavopiridol. The antimetabolite methotrexate (MTX) was inferred recently to be an additional substrate of the pump based on the analysis of ABCG2-overexpressing cell lines. However, the transport characteristics of the pump with regard to this agent have not been determined. In addition, physiological substrates of ABCG2 have not been identified. Here we examine the in vitro transport properties of the pump using membrane vesicles prepared from HEK293 cells transfected with ABCG2 expression vector. In so doing it is shown that MTX is a high capacity low affinity substrate of the pump, with K(m) and V(max) values of 1.34 +/- 0.18 mM and 687 +/- 87 pmol/mg/min, respectively. Unlike previously characterized multidrug resistance protein family members, ABCG2 is also able to transport MTX diglutamate and MTX triglutamate. However, addition of even one more glutamyl residue is sufficient to completely abrogate ABCG2-mediated transport. By contrast with the wild-type protein (ABCG2-R482), two ABCG2 variants that have been identified in drug selected cell lines, R482T and R482G, were unable to transport MTX to any extent. Similarly, folic acid was subject to efflux by the wild-type protein but not by the two mutants. However, transport of the reduced folate leucovorin was not detected for either the wild-type or the mutant proteins. Finally, it is shown that ABCG2 is capable of transporting E(2)17betaG with K(m) and V(max) values of 44.2 +/- 4.3 micro M and 103 +/- 17 pmol/mg/min, respectively. These results indicate that ABCG2 is a component of the energy-dependent efflux system for certain folates and antifolates, but that its transport characteristics with respect to polyglutamates and reduced folates are not identical to those of multidrug resistance protein family members. In addition, it is demonstrated that R482 mutations observed in drug-resistant cell lines have profound effects on the in vitro transport properties of the pump.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Estradiol/análogos & derivados , Estradiol/farmacocinética , Metotrexato/análogos & derivados , Metotrexato/farmacocinética , Mutación , Proteínas de Neoplasias , Ácido Poliglutámico/análogos & derivados , Ácido Poliglutámico/farmacocinética , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Transportadoras de Casetes de Unión a ATP/genética , Transporte Biológico/efectos de los fármacos , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Humanos , Indoles/farmacología , TransfecciónRESUMEN
The MRP family is comprised of nine related ABC transporters that are able to transport structurally diverse lipophilic anions and function as drug efflux pumps. Investigations of this family have provided insights not only into cellular resistance mechanisms associated with natural product chemotherapeutic agents, antifolates and nucleotide analogs, but also into factors that influence drug distribution in the body, membrane systems that are involved in the extrusion of reduced folates, cysteinyl leukotrienes and bile acids, and the molecular basis of two hereditary conditions in humans. The review will describe the biochemical properties, drug resistance activities and potential in vivo functions of these unusual pumps.
Asunto(s)
Resistencia a Antineoplásicos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Animales , Transporte Biológico , Humanos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/química , Estructura Terciaria de ProteínaRESUMEN
ETV6/ARG, a novel fusion gene composed of the ETV6 HLH oligomerization domain and most of sequences of the ARG protein tyrosine, was recently identified in human leukemia cells. The presence of the ETV6/ARG translocation raises the possibility that the resulting fusion protein functions as an oncogene. However, the transforming activity of the ETV6/ARG protein has not been determined and its contribution to leukemogenesis is therefore unknown. Here we address this question by analysing the oncogenic activity of ETV6/ARG in hematopoietic and fibroblast cells. It is demonstrated that expression of ETV6/ARG confers IL3-independent growth to Ba/F3 cells and anchorage independent growth to Rat-1 fibroblasts. It is also shown that multiple signaling molecules, including PI3K, SHC, ras-GAP and CRK-L, are tyrosine phosphorylated in Ba/F3 cells that express ETV6/ARG. Analysis of four different types of ETV6/ARG transcripts previously identified in the AML-M3 leukemia cell line HT93A suggest that ETV6 HLH domain is required for oncogenic activity. Based upon these results it is concluded that ARG can be activated as an oncogene in human malignancy and that the ETV6/ARG oncoprotein triggers some of the same signaling pathways associated with activated ABL oncogenes.
Asunto(s)
Transformación Celular Neoplásica/metabolismo , Proteínas de Unión al ADN/fisiología , Leucemia Mieloide/genética , Proteínas Oncogénicas/metabolismo , Proteínas Tirosina Quinasas/fisiología , Proteínas Represoras/fisiología , Animales , Adhesión Celular , División Celular/fisiología , Transformación Celular Neoplásica/genética , Ensayo de Unidades Formadoras de Colonias , Fibroblastos/metabolismo , Proteínas de Fusión bcr-abl/química , Proteínas de Fusión bcr-abl/genética , Genes abl/fisiología , Humanos , Interleucina-3/genética , Interleucina-3/metabolismo , Leucemia Mieloide/metabolismo , Proteínas Oncogénicas/genética , Fosforilación , Fosfotirosina/metabolismo , Plásmidos , Proteínas Proto-Oncogénicas c-ets , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Células Tumorales Cultivadas , Tirosina/metabolismo , Proteína ETS de Variante de Translocación 6RESUMEN
UNLABELLED: Overexpression of multidrug resistance (MDR1) P-glycoprotein (Pgp) remains an important barrier to successful chemotherapy in cancer patients and impacts the pharmacokinetics of many important drugs, thus evoking a need to noninvasively interrogate Pgp transport activity in vivo. METHODS: Cell tracer transport experiments as well as mouse biodistribution and microPET imaging studies were performed to characterize a nonmetabolized gallium(III) complex, gallium(III)-(bis(3-ethoxy-2-hydroxy-benzylidene)-N,N'-bis(2,2-dimethyl-3-amino-propyl)ethylenediamine) (Ga-[3-ethoxy-ENBDMPI])(+), as a candidate SPECT ((67)Ga) and generator-produced PET ((68)Ga) radiopharmaceutical recognized by MDR1 Pgp. RESULTS: The (67)Ga-complex showed high membrane potential-dependent accumulation in drug-sensitive KB3-1 cells and modulator-reversible low accumulation in MDR KB8-5 cells. In KB8-5 cells, the median effective concentrations (EC(50)) of MDR modulators LY335979, PSC 833, and cyclosporin A were 69 nmol/L, 1 micromol/L, and 3 micromol/L, respectively. Using a variety of cells stably expressing MDR1 Pgp, multidrug resistance-associated proteins (MRP1-MRP6), or the breast cancer resistance protein (BCRP/MXR), the (67)Ga-complex was shown to be readily transported by MDR1 Pgp and, to a much lesser extent, by MRP1, but not MRP2-MRP6 or BCRP/MXR. In a nude mouse xenograft tumor model, the (67)Ga-complex produced a readily detected 3-fold difference between Pgp-expressing tumors and drug-sensitive tumors in the opposite flank. In mdr1a/1b(-/-) gene-deleted mice, the (67)Ga-complex showed 17-fold greater brain uptake and retention compared with wild-type mice with no net difference in blood pharmacokinetics, consistent with transport in vivo by Pgp expressed at the capillary blood-brain barrier. This could be readily observed with microPET using the (68)Ga-complex. Incidentally, wild-type mice showed heart-to-blood ratios of >100 by 1 h after injection and heart-to-liver ratios of 2.2 by 120 min. CONCLUSION: Molecular imaging of the functional transport activity of MDR1 Pgp with ((67/68)Ga-[3-ethoxy-ENBDMPI])(+) may enable noninvasive SPECT/PET monitoring of the blood-brain barrier, chemotherapeutic regimens, and MDR1 gene therapy protocols in vivo. These Pgp-directed properties of the radiopharmaceutical may also translate favorably to myocardial perfusion imaging.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Barrera Hematoencefálica/diagnóstico por imagen , Barrera Hematoencefálica/metabolismo , Carcinoma de Células Escamosas/diagnóstico por imagen , Carcinoma de Células Escamosas/metabolismo , Radioisótopos de Galio/farmacocinética , Animales , Línea Celular Tumoral , Humanos , Masculino , Tasa de Depuración Metabólica , Ratones , Ratones Noqueados , Ratones Desnudos , Especificidad de Órganos , Tomografía de Emisión de Positrones/métodos , Transporte de Proteínas , Radiofármacos/farmacocinética , Distribución Tisular , Tomografía Computarizada de Emisión de Fotón Único/métodosRESUMEN
Multidrug resistance-associated protein 1 and P-glycoprotein are major ATP-binding cassette transporters that function as efflux pumps and confer resistance to a variety of structurally unrelated anticancer agents. To evaluate the comparative importance of these transporters with respect to anticancer agents, we established and characterized SV40-immortalized [mrp1(-/-)] (KO), [mdr1a/1b(-/-)] (DKO), and combined [mrp1 (-/-), mdr1a/1b(-/-)] (TKO) deficient fibroblast lines derived from primary embryonic fibroblasts of knockout mice. Western blot analyses demonstrated that KO and DKO fibroblasts exhibited similar levels of P-glycoprotein and mrp1, respectively, to that of wild-type (WT) fibroblasts. In addition, semiquantitative reverse transcription-PCR measurements of other multidrug resistance-associated protein (mrp) family members demonstrated that TKO fibroblasts displayed expression profiles of mrps 2-7 comparable to that of WT fibroblasts. These results indicate that loss of mrp1, P-glycoprotein, or both transporters does not cause overt compensatory changes in the expression of the other determined transporters. Using cell viability and calcein accumulation assays, we demonstrated that KO and DKO fibroblasts exhibited a low to moderate increase in sensitivity to vincristine and etoposide and in calcein accumulation compared to WT fibroblasts, whereas TKO fibroblasts displayed a markedly enhanced sensitivity to these agents and further elevated calcein accumulation. Furthermore, verapamil, an inhibitor of both mrp1 and P-glycoprotein, significantly sensitized WT fibroblasts to both vincristine and etoposide while having no effect on the sensitivity of TKO cells to these agents. Collectively, these findings indicate that mrp1 and P-glycoprotein are major determinants of drug sensitivity in immortalized mouse embryonic fibroblasts. They also suggest the existence of a compensatory mechanism by which the loss of one transporter can be functionally offset by the other in the transport of common drug substrates.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/fisiología , Quimiocinas CC/fisiología , Fibroblastos/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Animales , Anticarcinógenos/farmacología , Antineoplásicos/farmacología , Antineoplásicos Fitogénicos/farmacología , Transporte Biológico , Western Blotting , Supervivencia Celular , Células Cultivadas , Relación Dosis-Respuesta a Droga , Etopósido/farmacología , Fluoresceínas/metabolismo , Fluoresceínas/farmacología , Concentración 50 Inhibidora , Ratones , Ratones Noqueados , Pruebas de Precipitina , ARN/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo , Transfección , Células Tumorales Cultivadas , Vincristina/farmacologíaRESUMEN
To investigate the mechanisms of cellular resistance to 6-mercaptopurine (6-MP) in chronic myeloid leukemia (CML), a 6-MP resistant cell line (K562-MP5) was established by stepwise selection of the CML cell line (K562). The results of the drug sensitivity analysis of the K562-MP5 cell line revealed the cells to be 339-fold more resistant to 6-MP compared with the parental K562 cells. K562-MP5 cells exhibited decreased accumulation and increased efflux of [(14)C]6-MP and its metabolites. In addition, K562-MP5 cells showed increased [(3)H]MTX transport. K562-MP5 cells over-expressed P-glycoprotein (P-gp) and up-regulated MDR1 mRNA levels. Taken together, these results suggest that the up-regulation of P-gp, which contributes to the decreased accumulation by increasing the efflux of 6-MP and its metabolites, underlies the mechanism of 6-MP resistance in K562 cells.