RESUMEN
Multidrug resistance-associated protein (MRP) 3/ABCC3 and MRP4/ABCC4 are ATP-binding cassette (ABC) transporters expressed in the sinusoidal membrane of hepatocytes. The purpose of the present study was to establish organic anion-transporting polypeptide (OATP) 1B1/MRP2/MRP3 and OATP1B1/MRP2/MRP4 triple transfectants as in vitro model of the hepatobiliary transport of anionic drugs. To find in vivo relevant Mrp3 probes, wild-type and Mrp3(-/-) mice were given gemfibrozil, 6-hydroxy-5,7-dimethyl-2-methylamino-4-(3-pyridymethyl)benzothiazole (E3040), troglitazone, bisphenol A, and 4-methylumbelliferone orally. Plasma concentrations of the glucuronide conjugates were significantly lower in Mrp3(-/-) mice than in wild-type mice. The systemic exposure of gemfibrozil, E3040, and troglitazone were similar in wild-type and Mrp3(-/-) mice. 4-Methylumbelliferone and bisphenol A were undetectable in the plasma. In MRP3-expressing membrane vesicles, ATP-dependent uptakes of the glucuronide conjugates of estradiol, gemfibrozil, E3040, and troglitazone were markedly greater than those in controls, whereas MRP4-expressing membrane vesicles exhibited significant ATP-dependent uptake of gemfibrozil glucuronide and estradiol glucuronide. MRP3 or MRP4 was expressed in the OATP1B1/MRP2 double transfectants using adenovirus. The expression levels of OATP1B1 and MRP2 proteins were maintained both in the OATP1B1/MRP2/MRP3 and OATP1B1/MRP2/MRP4 triple transfectants, whereas MRP3 and MRP4 were localized in the basal membrane. Significant reductions in the basal-to-apical flux of the glucuronide conjugates of estradiol, gemfibrozil, E3040, and troglitazone were observed in the OATP1B1/MRP2/MRP3 triple transfectants compared with those in the double transfectants, whereas significant reduction was observed only for gemfibrozil glucuronide and estradiol glucuronide in the OATP1B1/MRP2/MRP4 triple transfectants. These results suggest that MRP3- or MRP4-triple transfectants provide a simple and useful in vitro system for evaluating their importance in the hepatobiliary transport of drugs.
Asunto(s)
Proteínas Asociadas a Resistencia a Múltiples Medicamentos/fisiología , Transportadores de Anión Orgánico Sodio-Independiente/genética , Transportadores de Anión Orgánico/genética , Transfección , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Células Cultivadas , Hepatocitos , Himecromona/farmacología , Masculino , Ratones , Ratones Noqueados , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Transportadores de Anión Orgánico/fisiología , Transportadores de Anión Orgánico Sodio-Independiente/fisiología , PorcinosRESUMEN
Multidrug resistance-associated protein ABCC2 (MRP2) is widely expressed in mammalian tissues including intestine, liver and kidney, and it has been shown to be located exclusively on the apical membrane of polarized cells. Recently, several reports suggest that apical membrane localization of ABCC2 (Mrp2) was regulated by radixin in rodent liver. To investigate the mechanism underlying this apical membrane targeting of MRP2 in human intestine, we chose Caco-2 cells as a model to examine the unique roles of ezrin and radixin. Following immunostaining, radixin and ezrin were found to be concentrated at the apical membrane of Caco-2 cells. Using the RNAi method, radixin and ezrin stable knockdown Caco-2 cells were constructed. A cell surface biotinylation experiment with radixin or ezrin stable knockdown Caco-2 cells showed that radixin or ezrin deficiency caused the loss of ABCC2 (MRP2) from the cell surface. An immunoprecipitation assay showed that radixin and ezrin were associated with ABCC2 (MRP2). These findings indicate that both ezrin and radixin are independently required for the apical membrane localization of ABCC2 (MRP2) in Caco-2 cells. Radixin and ezrin play similar roles in the apical membrane localization of ABCC2 (MRP2) and their expression level and subcellular distribution are important factors in the regulation of ABCC2 (MRP2) at the post-transcriptional level.
Asunto(s)
Membrana Celular/metabolismo , Polaridad Celular , Proteínas del Citoesqueleto/metabolismo , Células Epiteliales/citología , Intestinos/citología , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Actinas/metabolismo , Células CACO-2 , Técnica del Anticuerpo Fluorescente , Humanos , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Fosfoproteínas/metabolismo , Unión Proteica , Transporte de Proteínas , ARN Interferente Pequeño/metabolismo , Proteína de la Zonula Occludens-1RESUMEN
Phosphorylated double-stranded RNA-dependent protein kinase (PKR) is thought to play an important role during ER stress induced cell death, but its molecular mechanism of action has not yet been clarified completely. To resolve this issue, we employed a PKR inhibitor together with ER stress inducers (tunicamycin, thapsigargin, and 2-deoxyglucose) and found that this treatment applied to SK-N-SH and HepG2 cells suppressed the expressional induction of 94kDa glucose regulated protein (GRP94) but not GRP78 proteins at both protein and mRNA levels. Although GRP94 mRNA increased, no significant difference was observed in the mRNA level of spliced X box binding protein 1 (XBP1) and reporter gene assay using GRP78 and GRP94 promoter with an ER stress response element (ERSE) showed that PKR inhibitor did not affect their activity. These results suggest that a novel mechanism other than ERSE-dependent mRNA transcription is required for the induction of GRP94 and phosphorylation of PKR contributes to the induction of GRP94 under ER stress.
Asunto(s)
Retículo Endoplásmico/metabolismo , Regulación de la Expresión Génica/fisiología , Glicoproteínas de Membrana/biosíntesis , eIF-2 Quinasa/metabolismo , Línea Celular , Desoxiglucosa/farmacología , Retículo Endoplásmico/efectos de los fármacos , Chaperón BiP del Retículo Endoplásmico , Inhibidores Enzimáticos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Glicoproteínas de Membrana/genética , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Fosforilación , ARN Mensajero/biosíntesis , Elementos de Respuesta , eIF-2 Quinasa/antagonistas & inhibidoresRESUMEN
Vectorial transport of bile acids across hepatocytes is a major driving force for bile flow, and bile acid retention in the liver causes hepatotoxicity. The basolateral and apical transporters for bile acids are thought to be targets of drugs that induce cholestasis. Previously, we constructed polarized LLC-PK1 cells that express both a major bile acid uptake transporter human Na+/taurocholate cotransporting polypeptide (SLC10A1) (NTCP) and the bile acid efflux transporter human bile salt export pump (ABCB 11) (BSEP) and showed that monolayers of such cells can be used to characterize vectorial transcellular transport of bile acids. In the present study, we investigated whether cholestasis-inducing drugs could inhibit bile acid transport in such cells. Because fluorescent substrates allow the development of a high-throughput screening method, we examined the transport by NTCP and BSEP of fluorescent bile acids as well as taurocholate. The aminofluorescein-tagged bile acids, chenodeoxycholylglycylamidofluorescein and cholylglycylamidofluorescein, were substrates of both NTCP and BSEP, and their basal-to-apical transport rates across coexpressing cell monolayers were 4.3 to 4.5 times those of the vector control, although smaller than for taurocholate. The well known cholestatic drugs, rifampicin, rifamycin SV, glibenclamide, and cyclosporin A, reduced the basal-to-apical transport and the apical efflux clearance of taurocholate across NTCP- and BSEP-coexpressing cell monolayers. Further analysis indicated that the drugs inhibited both NTCP and BSEP. Our study suggests that such coexpressing cells can provide a useful system for the identification of inhibitors of these two transport systems, including potential drug candidates.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , Ácidos y Sales Biliares/metabolismo , Ciclosporina/toxicidad , Transportadores de Anión Orgánico Sodio-Dependiente/antagonistas & inhibidores , Rifampin/toxicidad , Rifamicinas/toxicidad , Simportadores/antagonistas & inhibidores , Miembro 11 de la Subfamilia B de Transportador de Casetes de Unión al ATP , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Transporte Biológico/efectos de los fármacos , Colestasis/etiología , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos/métodos , Fluoresceínas/metabolismo , Cinética , Células LLC-PK1 , Transportadores de Anión Orgánico Sodio-Dependiente/genética , Transportadores de Anión Orgánico Sodio-Dependiente/metabolismo , Oxadiazoles/metabolismo , Porcinos , Simportadores/genética , Simportadores/metabolismo , Ácido Taurocólico/análogos & derivados , Ácido Taurocólico/metabolismo , TransfecciónRESUMEN
Na(+)-taurocholate-cotransporting peptide (NTCP)/SLC10A1 and bile salt export pump (BSEP)/ABCB11 synergistically play an important role in the transport of bile salts by the hepatocyte. In this study, we transfected human NTCP and BSEP or rat Ntcp and Bsep into LLC-PK1 cells, a cell line devoid of bile salts transporters. Transport by these cells was characterized with a focus on substrate specificity between rats and humans. The basal to apical flux of taurocholate across NTCP- and BSEP-expressing LLC-PK1 monolayers was 10 times higher than that in the opposite direction, whereas the flux across the monolayer of control and NTCP or BSEP single-expressing cells did not show any vectorial transport. The basal to apical flux of taurocholate was saturated with a K(m) value of 20 microM. Vectorial transcellular transport was also observed for cholate, chenodeoxycholate, ursodeoxycholate, their taurine and glycine conjugates, and taurodeoxycholate and glycodeoxycholate, whereas no transport of lithocholate was detected. To evaluate the respective functions of NTCP and BSEP and to compare them with those of rat Ntcp and Bsep, we calculated the clearance by each transporter in this system. A good correlation in the clearance of the examined bile salts (cholate, chenodeoxycholate, ursodeoxycholate, and their taurine or glycine conjugates) was observed between transport by human and that of rat transporters in terms of their rank order: for NTCP, taurine conjugates > glycine conjugates > unconjugated bile salts, and for BSEP, unconjugated bile salts and glycine conjugates > taurine conjugates. In conclusion, the substrate specificity of human and rat NTCP and BSEP appear to be very similar at least for monovalent bile salts under physiological conditions.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/fisiología , Ácidos y Sales Biliares/farmacocinética , Células LLC-PK1/metabolismo , Proteínas de Transporte de Membrana/fisiología , Transportadores de Anión Orgánico Sodio-Dependiente/fisiología , Simportadores/fisiología , Miembro 11 de la Subfamilia B de Transportador de Casetes de Unión al ATP , Animales , Técnicas de Cocultivo , Humanos , Ratas , Especificidad por Sustrato , Porcinos , TransfecciónRESUMEN
The bile salt export pump (BSEP) of hepatocyte secretes conjugated bile salts across the canalicular membrane in an ATP-dependent manner. The biliary bile salts of human differ from those of rat in containing a greater proportion of glycine conjugates and taurolithocholate 3-sulfate (TLC-S). In the present study, the transport properties of hBSEP and rBsep were investigated using membrane vesicles from HEK293 cells infected with recombinant adenoviruses containing hBSEP or rBsep cDNA. ATP-dependent uptake of radiolabeled glycine-, taurine-conjugated bile salts, and [(3)H]cholate was observed when hBSEP or rBsep was expressed. Comparison of initial uptake rates indicated that for both transporters, taurine-conjugated bile salts were transported more rapidly than glycine-conjugated bile salts, however, hBSEP transported glycine conjugates to an extent that was approximately 2-fold greater than rBsep. In addition, [(3)H]TLC-S was significantly transported by hBSEP, and hardly transported by rBsep. The mean K(m) value for the uptake of [(3)H]TLC-S by hBSEP was 9.5+/-1.5 microM, a value similar to that for hMRP2 (8.2+/-1.3 microM). In conclusion, both hBSEP and rBsep transport taurine-conjugated bile salts better than glycine-conjugated bile salts, but hBSEP transports glycine conjugates to a greater extent as compared to rBsep. TLC-S, which is present in human bile but not rodent bile, is more avidly transported by hBSEP compared with rBsep.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Ácidos y Sales Biliares/metabolismo , Ácido Taurolitocólico/análogos & derivados , Vesículas Transportadoras/metabolismo , Miembro 11 de la Subfamilia B de Transportador de Casetes de Unión al ATP , Animales , Ácidos y Sales Biliares/química , Transporte Biológico , Membrana Celular/metabolismo , Células Cultivadas , Glicina/química , Glicina/metabolismo , Humanos , Cinética , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Ratas , Especificidad de la Especie , Taurina/química , Taurina/metabolismo , Ácido Taurocólico/farmacocinética , Ácido Taurolitocólico/metabolismoRESUMEN
PURPOSE: The efflux transporter, P-glycoprotein (P-gp), located in the brush-border membrane of intestinal absorptive cells, reduces the bioavailability of a wide range of orally administered drugs. Using P-gp inhibitors in transport experiments in Caco-2 cell monolayers is widely accepted as an efficient way to estimate the contribution of P-gp to the intestinal absorption of drugs. However, there still remain some arguments that the inhibitors might affect the function of other proteins. Multidrug resistance 1 gene (MDR1) specifically inhibited Caco-2 cells were constructed, therefore, as a better in vitro evaluation system of intestinal drug absorption. METHODS: The effective sites of RNAi were selected using siRNA libraries and single siRNAs and MDR1 stable knockdown Caco-2 cells were constructed using a tRNA(val)-shRNA expression vector. RESULTS: In siRNA stably expressed Caco-2 cells, the expression level of MDR1 was reduced at mRNA and protein levels. Transcellular transport studies using digoxin revealed that the P-gp function was suppressed completely, similar to that in verapamil-treated cells. CONCLUSIONS: MDR1 stable knockdown Caco-2 cells were successfully constructed by RNAi technology. This will consequently allow the development of a selection system for candidate drugs with improved absorption properties.
Asunto(s)
Genes MDR/genética , Absorción Intestinal , Proteínas de Transporte de Membrana/metabolismo , Transporte Biológico Activo , Western Blotting , Células CACO-2 , Bloqueadores de los Canales de Calcio/farmacología , Biblioteca de Genes , Vectores Genéticos , Humanos , Modelos Biológicos , Organismos Modificados Genéticamente , Interferencia de ARN/fisiología , ARN Mensajero/biosíntesis , ARN Mensajero/genética , ARN Interferente Pequeño/síntesis química , ARN Interferente Pequeño/genética , ARN de Transferencia de Valina/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transfección , Verapamilo/farmacologíaRESUMEN
PURPOSE: ME3229, an ester-type prodrug of a hydrophilic glycoprotein IIb/IIIa antagonist (ME3277), failed to show improved oral absorption. Okudaira et al. (J. Pharmacol. Exp. Ther. 294. 580-587, 2000) provided a piece of evidence that this is ascribed to an efflux system, distinct from P-gp and MRP2, that extrudes ME3277 formed from ME3229 in the intestinal epithelial cells. The aim of the present study is to examine the involvement of breast cancer resistant protein (BCRP/ABCG2) as a cause of low oral absorption of ME3229. METHODS: The transport activity of ME3277 in the presence and absence of ATP was determined using a rapid filtration method with the membrane vesicles prepared from LLC-PK1 cells expressing BCRP. The plasma concentrations of ME3229 and its metabolites were compared between Bcrp1(-/-) mice and wild-type mice after a single-pass perfusion of small intestine with ME3229. RESULTS: The ATP-dependent uptake of ME3277 was greater in BCRP-expressing membrane vesicles than that in the control vesicles. Furthermore, it was found that after intestinal perfusion with ME3229 for 60 min, the plasma concentrations of ME3277 and PM-5, a metabolite of ME3229, increased 2-fold and 3-fold, respectively, in Bcrp1 knockout mice. It is possible that BCRP acts synergistically with intestinal carboxylesterases. CONCLUSION: These results suggest that Bcrp1 plays an important role in the intestinal efflux of ME3277 and, probably, PM-10 and PM-11, metabolites of ME3229, and limits its BA after oral administration of ME3229.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Amidas/metabolismo , Piperidinas/farmacocinética , Profármacos/farmacocinética , Tiofenos/farmacocinética , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Transportadoras de Casetes de Unión a ATP/genética , Amidas/sangre , Animales , Disponibilidad Biológica , Línea Celular , Femenino , Absorción Intestinal , Yeyuno/metabolismo , Ratones , Ratones Noqueados , Perfusión , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/antagonistas & inhibidores , Vesículas Transportadoras/metabolismoRESUMEN
Double-strand RNA dependent protein kinase (PKR) plays an important role in control of cell death. We previously reported that activation of PKR is associated with hippocampal neuronal loss in Alzheimer's disease (AD). Recent studies have reported that Parkinson's (PD) and Huntington's (HD) disease brains displayed progressive hippocampal neuronal loss in extrastriatal degeneration. However, association between PKR and hippocampal neuronal loss in PD and HD brains is not known. In this report, brain tissues from patients with PD and HD displayed strong induction of phosphorylated-PKR (p-PKR) in hippocampal neurons. Immunoblotting analysis also demonstrated that levels of nuclear p-PKR in the hippocampus affected by these diseases were increased compared with age-matched disease controls. These results suggest that a close association exists between PKR and extrastriatal degeneration in PD and HD pathology.
Asunto(s)
Hipocampo/enzimología , Hipocampo/patología , Enfermedad de Huntington/enzimología , Enfermedad de Huntington/patología , Degeneración Nerviosa/enzimología , Degeneración Nerviosa/patología , Enfermedad de Parkinson/enzimología , Enfermedad de Parkinson/patología , eIF-2 Quinasa/fisiología , Adulto , Anciano , Recuento de Células , Muerte Celular , Núcleo Celular/enzimología , Femenino , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , FosforilaciónRESUMEN
Various types of stress, such as disruption of calcium homeostasis, inhibition of protein glycosylation and reduction of disulfide bonds, result in accumulation of misfolded proteins in the endoplasmic reticulum (ER). The initial cellular response involves removal of such proteins by the ER, but excessive and/or long-term stress results in apoptosis. In this study, we used a randomized ribozyme library and ER stress-mediated apoptosis (tunicamycin-induced apoptosis) in SK-N-SH human neuroblastoma cells as a selective phenotype to identify factors involved in this process. We identified a double-stranded RNA-dependent protein kinase (PKR) as one of the participants in this process. The level of nuclear PKR was elevated, but the level of cytoplasmic PKR barely changed in tunicamycin-treated SK-N-SH cells. Furthermore, tunicamycin also raised levels of phosphorylated PKR in the nucleus. We also detected the accumulation of phosphorylated PKR in the nuclei of autopsied brain tissues in Alzheimer's disease. Thus, PKR might play a role in ER stress-induced apoptosis and in Alzheimer's disease.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Apoptosis , Tunicamicina/farmacología , eIF-2 Quinasa/metabolismo , Anciano , Encéfalo/metabolismo , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Retículo Endoplásmico/metabolismo , Humanos , Fosforilación , ARN Catalítico/genética , Transducción de Señal , Células Tumorales Cultivadas , eIF-2 Quinasa/genéticaRESUMEN
In normal cells, tumor necrosis factor-alpha (TNF-alpha) activates caspase 8 in both mitochondrion-dependent and mitochondrion-independent apoptotic pathways. It is believed that these two pathways converge, with resultant activation of effector caspases, such as caspase 6 and caspase 7. However, the precise mechanism of the activation of caspases 6 and 7 remains unknown. In this study, in order to focus on the mitochondrion-dependent pathway, we employed MCF7 human breast carcinoma cells, which do not have a functional mitochondrion-independent (caspase 3-dependent) pathway. We specifically targeted the transcript of Bid, a proapoptotic facilitator that is a substrate of caspase 8 in the mitochondrial pathway. In the TNF-alpha-treated MCF7 cells that expressed Bid-targeted ribozymes, the release of cytochrome c and the activation of caspase 9, but not of caspase 8, was delayed. Furthermore, the proteolysis of procaspase 7 was also delayed in Bid ribozyme-expressing cells. Because MCF7 cells are caspase 3 deficient, the direct cross-talk between caspase 8 and caspase 3 does not take place. Therefore, it became clear for the first time that caspase 9 by itself can activate caspase 7 in the absence of the caspase 3-dependent pathway in TNF-alpha-induced apoptosis by the use of specific ribozymes.
Asunto(s)
Apoptosis , Proteínas Portadoras/metabolismo , Caspasas/metabolismo , Mitocondrias/metabolismo , ARN Catalítico/metabolismo , Proteína Proapoptótica que Interacciona Mediante Dominios BH3 , Secuencia de Bases , Neoplasias de la Mama/patología , Caspasa 3 , Caspasa 7 , Caspasa 8 , Caspasa 9 , Línea Celular Tumoral , Citocromos c/metabolismo , Activación Enzimática , Precursores Enzimáticos/metabolismo , Humanos , Immunoblotting , Modelos Biológicos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Plásmidos/metabolismo , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Transfección , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Cyclin-dependent kinase 5 (Cdk5), a cdc2-related kinase expressed in postmitotic neurons, is activated by association with a brain-specific activator, p35. It has been suggested that the conversion of p35 to p25 by the protease calpain is involved in neuronal cell death. However, p35 protein is turned over rapidly via proteasomal degradation in living neurons. In this study we show that the phosphorylation of p35 by Cdk5 suppresses the cleavage to p25 by calpain, whereas phosphorylation facilitates the proteasomal degradation of p35. The phosphorylation site in p35 that might be involved in preventing calpain cleavage was distinct from the phosphorylation site involved in facilitating proteasomal degradation. A phosphorylated form of p35 that was resistant to cleavage by calpain was more prevalent in the fetal brain, whereas the unphosphorylated form of p35 occurred in the adult brain. These results suggest that the phosphorylation of p35 serves as a protective mechanism that suppresses the generation of p25 in developing brains.
Asunto(s)
Encéfalo/embriología , Encéfalo/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Factores de Edad , Animales , Encéfalo/crecimiento & desarrollo , Calpaína/metabolismo , Células Cultivadas , Quinasa 5 Dependiente de la Ciclina , Quinasas Ciclina-Dependientes/metabolismo , Cisteína Endopeptidasas/metabolismo , Complejos Multienzimáticos/metabolismo , Fosforilación , Complejo de la Endopetidasa Proteasomal , RatasRESUMEN
When cells are exposed to death-inducing molecules such as tumor necrosis factor-alpha or Fas, caspase 8 is activated and cleaves an apoptotic facilitator, Bid, that is a member of the Bcl-2 family. After additional modification, the C-terminal moiety of Bid is translocated to the mitochondria and induces the release of cytochrome c into the cytoplasm. In an attempt to directly observe the cleavage of Bid and the following events in living cells, we constructed a vector that encoded Bid fused with yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP) (YFP-Bid-CFP). On expression of YFP-Bid-CFP in mammalian cells, we were able to observe the efficient transfer of energy from excited CFP to YFP within the YFP-Bid-CFP molecule and, importantly, the fusion protein YFP-Bid-CFP was fully functional in cells. When YFP-Bid-CFP was cleaved by caspase 8, on activation by anti-Fas Abs but not by Abeta or tunicamycin, no such transfer of energy was detected. To our knowledge, this is the first report of (i) visualization of the activation of Bid by proteolytic cleavage, with direct observation of the cleavage of YFP-Bid-CFP in the cytoplasm and subsequent translocation of the cleaved Bid to mitochondria and (ii) the absence of Abeta- or tunicamycin-mediated significant activation of caspase 8 in individual living cells.
Asunto(s)
Péptidos beta-Amiloides/fisiología , Apoptosis/fisiología , Proteínas Portadoras/metabolismo , Caspasas/metabolismo , Hígado/metabolismo , Células 3T3 , Animales , Proteína Proapoptótica que Interacciona Mediante Dominios BH3 , Proteínas Bacterianas/genética , Células COS , Proteínas Portadoras/genética , Caspasa 8 , Caspasa 9 , Chlorocebus aethiops , Clonación Molecular , ADN Complementario/genética , Activación Enzimática , Biblioteca de Genes , Genes Reporteros , Proteínas Fluorescentes Verdes , Humanos , Proteínas Luminiscentes/genética , Ratones , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Eliminación de Secuencia , Espectrometría de Fluorescencia , TransfecciónRESUMEN
Now that the sequences of many genomes are available, methods are required for the rapid identification of functional genes. We describe here a simple system for the isolation of genes that function in the tumor necrosis factor-alpha (TNF-alpha)-mediated pathway of apoptosis, using RNA helicase-associated ribozyme libraries with randomized substrate-binding arms. Because target-site accessibility considerably limits the effective use of intracellular ribozymes, the effectiveness of a conventional ribozyme library has been low. To overcome this obstacle, we attached to ribozymes an RNA motif (poly(A)-tail) able to interact with endogenous RNA helicase(s) so that the resulting helicase-attached, hybrid ribozymes can more easily attack target sites regardless of their secondary or tertiary structures. When the phenotype of cells changes upon introduction of a ribozyme library, genes responsible for these changes may be identified by sequencing the active ribozyme clones. In the case of TNF-alpha-mediated apoptosis, when a ribozyme library was introduced into MCF-7 cells, surviving clones were completely or partially resistant to TNF-alpha-induced apoptosis. We identified many pro-apoptotic genes and partial sequences of previously uncharacterized genes using this method. Our gene discovery system should be generally applicable to the identification of functional genes in various systems.
Asunto(s)
Apoptosis/genética , Perfilación de la Expresión Génica/métodos , Biblioteca de Genes , ARN Catalítico/genética , ARN Catalítico/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Apoptosis/efectos de los fármacos , Secuencia de Bases , Femenino , Humanos , Datos de Secuencia Molecular , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Recently, we developed a gene discovery system that can identify functional genes using a randomized hybrid ribozyme library. In this system, inhibition of the expression of a particular gene by active ribozymes was reflected by a change in a particular phenotype, the method allowed the identification of functional genes. In the case of identification of functional genes for apoptosis pathways, we identified many pro-apoptotic genes in TNF-alpha and Fas-mediated apoptosis pathways. In this study, we tried to identify the functional genes that are necessary for the retinoic acid (RA)-induced cell differentiation using randomized ribozyme and siRNA libraries. We succeeded to identify the several differentiation factors. Therefore, our gene discovery system based on randomized ribozyme and siRNA libraries are high potential to identify the differentiation and undifferentiation factors in the post genome era.