RESUMEN
Methods to repeatedly, non-invasively, and quantitatively image gene expression in living animals are rapidly emerging and should fundamentally change studies of gene expression in vivo. We previously developed assays utilizing positron emission tomography (PET) to image reporter gene expression. In this paper we: (1) describe a new bi-directional, tetracycline-inducible system that can be used to pharmacologically induce target gene expression and to quantitatively image induced expression by using a PET reporter gene; (2) demonstrate the potential of this system in transient and stable cell transfection assays; and (3) demonstrate the ability to repetitively and quantitatively image tetracycline and tetracycline analog induction of gene expression in living animals. We utilize the dopamine type-2 receptor (D(2)R) and the mutant herpes-simplex virus type 1 thymidine kinase (HSV1-sr39tk) reporter genes to validate this system. We utilize microPET technology to show that quantitative tomographic imaging of gene induction is possible. We find a high correlation (r(2) = 0.98) between 'target' and reporter gene expression. This work establishes a new technique for imaging time-dependent variation of gene expression both from vectors with inducible promoters and in transgenic animals in which pharmacologic induction of gene expression must be monitored. These techniques may be applied both in gene therapy and for the study of gene expression in transgenic animals.
Asunto(s)
Genes Reporteros , Terapia Genética , Herpesvirus Humano 1/enzimología , Receptores de Dopamina D2/genética , Timidina Quinasa/genética , Tomografía Computarizada de Emisión/métodos , Transfección/métodos , Animales , Expresión Génica/efectos de los fármacos , Ingeniería Genética , Células HeLa , Humanos , Luciferasas/genética , Ratones , Ratones Desnudos , Ratones Transgénicos , Trasplante de Neoplasias , Regiones Promotoras Genéticas , Tetraciclina/farmacología , Factores de Tiempo , Células Tumorales Cultivadas , beta-Galactosidasa/genéticaRESUMEN
The dopamine D2 receptor (D2R) has been used in adenoviral delivery systems and in tumor cell xenografts as an in vivo reporter gene. D2R reporter gene expression has been non-invasively, repetitively and quantitatively imaged by positron emission tomography (PET), following systemic injection of a positron-labeled ligand (3-(2'-[18F]-fluoroethyl)-spiperone; FESP) and subsequent D2R-dependent sequestration. However, dopamine binding to the D2R can modulate cyclic AMP levels. For optimal utilization of D2R as a reporter gene, it is important to uncouple ligand-binding from Gi-protein-mediated inhibition of cAMP production. Mutation of Asp80 or Ser194 produces D2Rs that still bind [3H]spiperone in transfected cells. The D2R80A mutation completely eliminates the ability of the D2R to suppress forskolin-stimulated cAMP accumulation in response to dopamine, in cells transfected with a D2R80A expression plasmid and in cells infected with replication-defective adenovirus expressing D2R80A. The D2R194A mutation substantially reduces, but does not completely eliminate, dopamine modulation of cAMP levels. Cultured cells infected with adenoviruses expressing D2R and D2R80A demonstrated equivalent [3H]spiperone binding activity. Moreover, hepatic FESP sequestration is equivalent, following intravenous injection of adenoviruses expressing D2R and D2R80A. The D2R80A mutant, which can no longer modulate cAMP levels following ligand binding, has full capability as a PET reporter gene.
Asunto(s)
Genes Reporteros , Mutación Puntual , Receptores de Dopamina D2/genética , Adenoviridae/genética , Animales , Línea Celular , Colforsina/farmacología , AMP Cíclico/metabolismo , Dopamina/farmacología , Vectores Genéticos/administración & dosificación , Inyecciones Intravenosas , Hígado/metabolismo , Ratones , Unión Proteica , Ratas , Transducción de Señal , Espiperona/metabolismo , Tomografía Computarizada de Emisión , Células Tumorales CultivadasRESUMEN
Biodistribution, magnitude and duration of a therapeutic transgene's expression may be assessed by linking it to the expression of a positron emission tomography (PET) reporter gene (PRG) and then imaging the PRG's expression by a PET reporter probe (PRP) in living animals. We validate the simple approach of co-administering two distinct but otherwise identical adenoviruses, one expressing a therapeutic transgene and the other expressing the PRG, to track the therapeutic gene's expression. Two PET reporter genes, a mutant herpes simplex virus type 1 thymidine kinase (HSV1-sr39tk) and dopamine-2 receptor (D(2)R), each regulated by the same cytomegalovirus (CMV) promoter, have been inserted into separate adenoviral vectors (Ad). We demonstrate that cells co-infected with equivalent titers of Ad-CMV-HSV1-sr39tk and Ad-CMV-D(2)R express both reporter genes with good correlation (r(2) = 0.93). Similarly, a high correlation (r(2) = 0.97) was observed between the expression of both PRGs in the livers of mice co-infected via tail-vein injection with equivalent titers of these two adenoviruses. Finally, microPET imaging of HSV1-sr39tk and D(2)R expression with 9-(4-[(18)F]fluoro-3-hydroxymethylbutyl) guanine ([(18)F]FHBG) and 3-(2-[(18)F]fluoroethyl)spiperone ([(18)F]FESP), utilizing several adenovirus-mediated delivery routes, illustrates the feasibility of evaluating relative levels of transgene expression in living animals, using this approach.
Asunto(s)
Citomegalovirus/genética , Herpesvirus Humano 1/enzimología , Regiones Promotoras Genéticas , Receptores de Dopamina D2/genética , Timidina Quinasa/genética , Tomografía Computarizada de Emisión , Adenoviridae/genética , Animales , Células COS , Células Cultivadas , Estudios de Factibilidad , Expresión Génica , Vectores Genéticos/administración & dosificación , Guanina/análogos & derivados , Hígado/diagnóstico por imagen , Ratones , Ratones Endogámicos , Ratones Desnudos , Ratas , Espiperona/análogos & derivados , Factores de TiempoRESUMEN
An accurate, rapid, and versatile method for the analysis of enzyme kinetics using electrospray ionization mass spectrometry (ESI-MS) has been developed and demonstrated using fucosyltransferase V. Reactions performed in primary or secondary amine-containing buffers were diluted in an ESI solvent and directly analyzed without purification of the reaction products. Decreased mass resolution was used to maximize instrument sensitivity, and multiple reaction monitoring (MRM), in the tandem mass spectrometric mode, was used to enhance selectivity of detection. The approach allowed simultaneous monitoring of multiple processes, including substrate consumption, product formation, and the intensity of an internal standard. MRM gave an apparent K(m) for GDP-L-fucose (GDP-Fuc) of 50.4 +/- 5.5 microM and a k(cat) of 1.46 +/- 0.044 s(-1). Under the same conditions, the conventional radioactivity-based assay using GDP-[U-(14)C]Fuc as substrate gave virtually identical results: K(m) = 54.3 +/- 4.6 microM and k(cat) = 1.49 +/- 0.039 s(-1). The close correlation of the data showed that ESI-MS coupled to MRM is a valid approach for the analysis of enzyme kinetics. Consequently, this method represents a valuable alternative to existing analytic methods because of the option of simultaneously monitoring multiple species, the high degree of specificity, and rapid analysis times and because it does not rely on the availability of radioactive or chromogenic substrates.
Asunto(s)
Fucosiltransferasas/química , Amino Azúcares/química , Amino Azúcares/metabolismo , Sitios de Unión , Tampones (Química) , Secuencia de Carbohidratos , Transporte de Electrón , Fucosa/análogos & derivados , Fucosa/química , Fucosa/metabolismo , Fucosiltransferasas/metabolismo , Guanosina Difosfato/análogos & derivados , Guanosina Difosfato/química , Guanosina Difosfato/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Iones/química , Cinética , Antígeno Lewis X/análogos & derivados , Datos de Secuencia Molecular , Nucleótidos/química , Nucleótidos/metabolismo , Espectrometría de Masa por Ionización de Electrospray/métodos , Trisacáridos/química , Trisacáridos/metabolismoRESUMEN
UNLABELLED: We have synthesized and evaluated 8-[18F]fluoropenciclovir (FPCV) and compared it with 8-[18F]fluoroganciclovir (FGCV) for monitoring the expression of herpes simplex virus type 1 thymidine kinase (HSV1 -tk) reporter gene in cell culture and in vivo. METHODS: C6 rat glioma cells stably transfected with HSV1-tk (C6-stb-tk+) and control C6 cells were evaluated for their ability to accumulate FGCV versus FPCV. For in vivo studies, 15 mice were injected by tail vein with increasing levels of an adenoviral vector carrying HSV1-tk. Forty-eight hours later the mice were injected with FPCV and killed 3 h later. The percentage injected dose per gram (%ID/g) liver was then determined. Two additional mice were studied by microPET and autoradiography using FPCV to image adenoviral-mediated hepatic HSV1-tk reporter gene expression. A tumor-bearing mouse (C6 control and C6-stb-tk+) was imaged with FDG, FGCV, and FPCV. Two mice carrying tumors expressing two different reporter genes, HSV1-tk and dopamine type 2 receptor (D2R), were also imaged by microPET using FPCV (day 1) and 3-(2'-[18F]fluoroethyl)spiperone (FESP) (day 2). RESULTS: FPCV shows a significantly greater accumulation in C6-stb-tk+ cells than does FGCV (P < 0.05). Over identical ranges of adenoviral administration, mouse liver shows a higher %ID/g liver for FPCV (0%-9%) compared with our previously reported results with FGCV (0%-3%). In C6 control and C6-stb-tk+ tumor-bearing mice, FPCV has a greater accumulation than does FGCV for equal levels of HSV1-tk gene expression. In mice carrying tumors expressing either HSV1-tk or D2R reporter genes, there is a corresponding retention of FPCV and FESP, respectively. CONCLUSION: These results indicate that FPCV is a better reporter probe than is FGCV for imaging lower levels of HSV1 -tk gene expression in vivo. The results also reveal the ability to monitor the expression of two distinct reporter genes in the same animal using reporter probes specific for each gene.
Asunto(s)
Aciclovir/análogos & derivados , Antivirales , Radioisótopos de Flúor , Genes Reporteros , Herpesvirus Humano 1/genética , Timidina Quinasa/genética , Tomografía Computarizada de Emisión , Adenoviridae , Animales , Células Cultivadas , Expresión Génica , Vectores Genéticos , Guanina , Herpesvirus Humano 1/enzimología , Ratones , Ratas , Receptores de Dopamina D2/genéticaRESUMEN
Electrospray ionization mass spectrometry coupled to multiple reaction monitoring (ESI-MS/MRM) has been applied for the first time to analyze enzyme inhibitor kinetics. Specifically, a known competitive inhibitor, guanosine 5'-monophosphate (GMP), and a synthetic, transition-state analogue inhibitor, guanosine 5'-[1D-(1,3,4/2)-5-methyl-5-cyclohexene-1,2,3,4-tetrol 1-diphosphate] (1) have been characterized against recombinant fucosyltransferase (Fuc-T) V using ESI-MS/MRM. Dixon analysis with GMP yielded a signature plot for competitive inhibition. Nonlinear regression analysis gave a Ki of 211.8+/-24.7 microM. The conventional analysis using GDP-[U-14C]-Fuc yielded a similar Ki value of 235.6+/-59.4 microM, confirming the validity of the MS-based method. The synthetic inhibitor 1 showed potent competitive inhibition with a Ki of 25.6+/-2.8 microM. Although 1 possesses a chemically reactive allyl phosphate group, ESI-MS/MRM showed that there was no reduction in the concentration of 1 and no production of a predicted metabolite GDP during the assay. MS/MS also confirmed the absence of a possible pseudo-trisaccharide product. The results clearly show that 1 is neither a slow-reacting donor nor does it act as a suicide-type inhibitor toward Fuc-T V. ESI-MS/MRM is therefore a powerful tool for the kinetic characterization of enzyme inhibitors, providing complete disclosure of the mechanism of action of 1 as an inhibitor.
Asunto(s)
Inhibidores Enzimáticos/química , Guanosina/química , Fucosiltransferasas/antagonistas & inhibidores , Fucosiltransferasas/química , Guanosina/análogos & derivados , Guanosina Monofosfato/química , Indicadores y Reactivos , Cinética , Análisis de Regresión , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
The importance of analogues of lactosyl ceramides as basic structures of many natural glycosphingolipids provided a rationale for developing an efficient synthetic route to these compounds. We report herein a novel approach to synthesize several members of this family. Glycosylation of N-diphenylmethylene-spingosine, which exists in an imine-oxazolidine tautomeric mixture, with acetobromolactose under a modified Koenigs-Knorr condition yielded lactosyl beta-(1 --> 1) sphingosine, lactosyl beta-(1 --> 3) sphingosine and dilactosyl sphingosine in good yields. A similar glycosylation could be applicable to the synthesis of other glycosphingolipids.
Asunto(s)
Psicosina/análogos & derivados , Psicosina/síntesis química , Conformación de Carbohidratos , Secuencia de Carbohidratos , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Psicosina/química , Espectrometría de Masa Bombardeada por Átomos VelocesRESUMEN
Functional imaging by repeated noninvasive scans of specific (18)F tracer distribution using a high-resolution small-animal PET scanner, the microPET, assessed the time course of alterations in energy utilization and dopamine receptors in rats with unilateral striatal quinolinic acid lesions. Energy utilization ipsilateral to the lesion, determined using scans of 2-deoxy-2-[(18)F]fluoro-d-glucose uptake, was compromised severely 1 week after intrastriatal excitotoxin injections. When the same rats were imaged 5 and 7 weeks postlesion, decrements in energy metabolism were even more prominent. In contrast, lesion-induced effects on dopamine D(2) receptor binding were more progressive, with an initial upregulation of [3-(2'-(18)F]fluoroethyl)spiperone binding apparent 1 week postlesion followed by a decline 5 and 7 weeks thereafter. Additional experiments revealed that marked upregulation of dopamine D(2) receptors consequent to quinolinic acid injections could be detected as early as 3 days after the initial insult. Postmortem markers of striatal GABAergic neurons were assessed in the same rats 7 weeks after the lesion: expression of glutamic acid decarboxylase and dopamine D(1) receptor mRNA, as well as [(3)H]SCH-23,390 and [(3)H]spiperone binding to dopamine D(1) and D(2) receptors, respectively, detected prominent decrements consequent to the lesion. In contrast, by 7 weeks postlesion [(3)H]WIN-35,428 binding to dopamine transport sites within the striatum appeared to be enhanced proximal to the quinolinic acid injection sites. The results demonstrate that functional imaging using the microPET is a useful technique to explore not only the progressive neurodegeneration that occurs in response to excitotoxic insults, but also to examine more closely the intricacies of neurotransmitter activity in a small animal model of HD.
Asunto(s)
Cocaína/análogos & derivados , Cuerpo Estriado/metabolismo , Metabolismo Energético/fisiología , Enfermedad de Huntington/diagnóstico por imagen , Enfermedad de Huntington/metabolismo , Receptores de Dopamina D2/metabolismo , Tomografía Computarizada de Emisión/métodos , Animales , Autorradiografía , Benzazepinas/metabolismo , Benzazepinas/farmacología , Cocaína/metabolismo , Cocaína/farmacología , Cuerpo Estriado/patología , Modelos Animales de Enfermedad , Antagonistas de Dopamina/metabolismo , Antagonistas de Dopamina/farmacología , Inhibidores de Captación de Dopamina/metabolismo , Inhibidores de Captación de Dopamina/farmacología , Femenino , Fluorodesoxiglucosa F18 , Glutamato Descarboxilasa/genética , Enfermedad de Huntington/inducido químicamente , Hibridación in Situ , Degeneración Nerviosa/inducido químicamente , Degeneración Nerviosa/diagnóstico por imagen , Degeneración Nerviosa/metabolismo , Neurotoxinas/metabolismo , Ácido Quinolínico/farmacología , ARN Mensajero/análisis , Ensayo de Unión Radioligante , Ratas , Ratas Wistar , Receptores de Dopamina D1/análisis , Receptores de Dopamina D1/genética , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D2/análisis , Espiperona/metabolismo , Espiperona/farmacología , Sustancia Negra/metabolismo , Tritio , Ácido gamma-Aminobutírico/metabolismoRESUMEN
A vital step in transgenic animal study and gene therapy is the ability to assay the extent of transgene expression. Unfortunately, classic methods of assaying transgene expression require biopsies or death of the subject. We are developing techniques to noninvasively and repetitively determine the location, duration, and magnitude of transgene expression in living animals. This will allow investigators and clinicians to assay the effectiveness of their particular experimental and therapeutic paradigms. Of radionuclide (single photon emission computed tomography, positron emission tomography [PET]), optical (green fluorescent protein, luciferase), and magnetic (magnetic resonance imaging) approaches, only the radionuclide approach has sufficient sensitivity and quantitation to measure the expression of genes in vivo. We describe the instrumentation involved in high resolution PET scanning. We also describe the principles of PET reporter gene/reporter probe in vivo imaging, the development of two in vivo reporter gene imaging systems, and the validation of our ability to noninvasively, quantitatively, and repetitively image gene expression in murine viral gene transfer and transgenic models. We compare the two reporter gene systems and discuss their utility for the study of transgenic animals and gene therapies. Finally, we mention alternative approaches to image gene expression by using radiolabeled antibody fragments to image specific proteins and radiolabeled oligonucleotides to image RNA messages directly.
Asunto(s)
Expresión Génica/fisiología , Tomografía Computarizada de Emisión/métodos , Transgenes/genética , Animales , Animales Modificados Genéticamente , Genes Reporteros/genética , Humanos , RatonesRESUMEN
A variety of imaging technologies are being investigated as tools for studying gene expression in living subjects. Noninvasive, repetitive and quantitative imaging of gene expression will help both to facilitate human gene therapy trials and to allow for the study of animal models of molecular and cellular therapy. Radionuclide approaches using single photon emission computed tomography (SPECT) and positron emission tomography (PET) are the most mature of the current imaging technologies and offer many advantages for imaging gene expression compared to optical and magnetic resonance imaging (MRI)-based approaches. These advantages include relatively high sensitivity, full quantitative capability (for PET), and the ability to extend small animal assays directly into clinical human applications. We describe a PET scanner (microPET) designed specifically for studies of small animals. We review "marker/reporter gene" imaging approaches using the herpes simplex type 1 virus thymidine kinase (HSV1-tk) and the dopamine type 2 receptor (D2R) genes. We describe and contrast several radiolabeled probes that can be used with the HSV1-tk reporter gene both for SPECT and for PET imaging. We also describe the advantages/disadvantages of each of the assays developed and discuss future animal and human applications.
Asunto(s)
Cintigrafía/instrumentación , Cintigrafía/métodos , Transgenes/genética , Animales , Expresión Génica , Herpesvirus Humano 1/enzimología , Humanos , Ratones , Modelos Biológicos , Receptores de Dopamina D2/química , Receptores de Dopamina D2/genética , Timidina Quinasa/química , Timidina Quinasa/genética , Tomografía Computarizada de Emisión/métodosRESUMEN
Rapid imaging by antitumor antibodies has been limited by the prolonged targeting kinetics and clearance of labeled whole antibodies. Genetically engineered fragments with rapid access and high retention in tumor tissue combined with rapid blood clearance are suitable for labeling with short-lived radionuclides, including positron-emitting isotopes for positron-emission tomography (PET). An engineered fragment was developed from the high-affinity anticarcinoembryonic antigen (CEA) monoclonal antibody T84.66. This single-chain variable fragment (Fv)-C(H)3, or minibody, was produced as a bivalent 80 kDa dimer. The macrocyclic chelating agent 1,4,7, 10-tetraazacyclododecane-N,N',N", N"'-tetraacetic acid (DOTA) was conjugated to the anti-CEA minibody for labeling with copper-64, a positron-emitting radionuclide (t(1/2) = 12.7 h). In vivo distribution was evaluated in athymic mice bearing paired LS174T human colon carcinoma (CEA positive) and C6 rat glioma (CEA negative) xenografts. Five hours after injection with (64)Cu-DOTA-minibody, microPET imaging showed high uptake in CEA-positive tumor (17.9% injected dose per gram +/- 3.79) compared with control tumor (6.0% injected dose per gram +/- 1.0). In addition, significant uptake was seen in liver, with low uptake in other tissues. Average target/background ratios relative to neighboring tissue were 3-4:1. Engineered antibody fragments labeled with positron-emitting isotopes such as copper-64 provide a new class of agents for PET imaging of tumors.
Asunto(s)
Antígeno Carcinoembrionario/inmunología , Fragmentos de Inmunoglobulinas/inmunología , Animales , Especificidad de Anticuerpos , Radioisótopos de Cobre , Femenino , Humanos , Fragmentos de Inmunoglobulinas/genética , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/inmunología , Marcaje Isotópico , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Ratas , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Factores de Tiempo , Tomografía Computarizada de Emisión/métodos , Trasplante Heterólogo , Células Tumorales CultivadasRESUMEN
A new method for the preparation of 8-[(18)F]fluoroguanine derivatives based on a direct radiofluorination reaction has been developed. The radiofluorination of ganciclovir (1a) with [(18)F]F(2) was carried out in absolute ethanol in the presence of tetraethylammonium hydroxide at room temperature to give 8-[(18)F]fluoroganciclovir (3a) in an approximately 1% radiochemical yield. Similarly, 8-[(18)F]fluoropenciclovir (3b), 8-[(18)F]fluoroacyclovir (3c), and 8-[(18)F]fluoroguanosine (3d) were synthesized from penciclovir (1b), acyclovir (1c), and guanosine (1d), respectively, using [(18)F]F(2). The structural analyses of the final products (3a, 3b, 3c, and 3d) were carried out after (18)F decay by (1)H, (13)C, and (19)F nuclear magnetic resonance and high resolution mass spectroscopy.
Asunto(s)
Expresión Génica , Guanina/análogos & derivados , Guanina/síntesis química , Radiofármacos/síntesis química , Tomografía Computarizada de Emisión/métodos , Aciclovir/análogos & derivados , Aciclovir/química , Ganciclovir/química , Guanosina/química , Marcaje Isotópico , Espectroscopía de Resonancia MagnéticaRESUMEN
The ability to monitor reporter gene expression in living animals and in patients will permit longitudinal examinations both of somatically transferred DNA in experimental animals and patients and of transgenic constructs expressed in experimental animals. If investigators can non-invasively monitor the organ and tissue specificity, the magnitude and the duration of gene expression from somatically transferred DNA and from transgenes, conceptually new experimental paradigms will be possible. If clinicians can non-invasively monitor the location, extent and duration of somatically transferred genes, they will be better able to determine the correlations between expression of therapeutic genes and clinical outcomes. We have developed two reporter gene systems for in vivo reporter gene imaging in which the protein products of the reporter genes sequester positron-emitting reporter probes. The "PET reporter gene" dependent sequestration of the "PET reporter probes" is subsequently measured in living animals by Positron Emission Tomography (PET). We describe here the principles of PET reporter gene/PET reporter probe in vivo imaging, the development of two imaging systems, and the validation of their ability to non-invasively, quantitatively and repetitively image reporter gene expression in murine viral gene transfer and transgenic models.
Asunto(s)
Diagnóstico por Imagen/métodos , Regulación de la Expresión Génica/fisiología , Genes Reporteros/fisiología , Tomografía Computarizada de Emisión/métodos , AnimalesRESUMEN
Reporter genes (e.g. beta-galactosidase, chloramphenicol-acetyltransferase, green fluorescent protein, luciferase) play critical roles in investigating mechanisms of gene expression in transgenic animals and in developing gene delivery systems for gene therapy. However, measuring expression of these reporter genes requires biopsy or death. We now report a procedure to image reporter gene expression repetitively and non-invasively in living animals with positron emission tomography (PET), using the dopamine type 2 receptor (D2R) as a reporter gene and 3-(2'-[18F]fluoroethyl)spiperone (FESP) as a reporter probe. We use a viral delivery system to demonstrate the ability of this PET reporter gene/PET reporter probe system to image reporter gene expression following somatic gene transfer. In mice injected intravenously with replication-deficient adenovirus carrying a D2R reporter gene, PET in vivo measures of hepatic [18F] retention are proportional to in vitro measures of hepatic FESP retention, D2R ligand binding and D2R mRNA. We use tumor-forming cells carrying a stably transfected D2R gene to demonstrate imaging of this PET reporter gene/PET reporter probe system in 'tissues'. Tumors expressing the transfected D2R reporter gene retain substantially more FESP than control tumors. The D2R/FESP reporter gene/reporter probe system should be a valuable technique to monitor, in vivo, expression from both gene therapy vectors and transgenes.
Asunto(s)
Genes Reporteros , Hígado/diagnóstico por imagen , Receptores de Dopamina D2/genética , Tomografía Computarizada de Emisión , Adenoviridae/genética , Animales , Antagonistas de Dopamina , Colorantes Fluorescentes , Expresión Génica , Vectores Genéticos/administración & dosificación , Hígado/metabolismo , Masculino , Ratones , Ratones Desnudos , Ratones Transgénicos , Neoplasias Experimentales/diagnóstico por imagen , Neoplasias Experimentales/metabolismo , ARN Mensajero/análisis , Ensayo de Unión Radioligante , Espiperona/análogos & derivadosRESUMEN
5'-Deoxy-5'-fluoro-O4-methylthymidine was synthesized by the reaction of the corresponding 5'-O-tosylate with KF in the presence of Kryptofix [222] and coupled to a 5'-phosphoramidite-activated CPG-bound oligodeoxynucleotide. The sequence of reactions and purifications were accomplished within 4 h, a necessary condition of the development of radiofluorinated antisense oligodeoxynucleotide probe for use with PET.
Asunto(s)
Radioisótopos de Flúor , Oligonucleótidos Antisentido/síntesis química , Timidina/análogos & derivados , Compuestos Bicíclicos Heterocíclicos con Puentes , Quelantes , Oligonucleótidos Antisentido/farmacología , Timidina/síntesis química , Timidina/farmacología , Tomografía Computarizada de EmisiónRESUMEN
Sphingosine, a core unit of sphingolipids, has been shown to mediate intracellular signaling events. It is conceivable that sphingosine could act as an intracellular messenger as well as an intracellular modulator, if sphingosine were to be transferred intercellularly. Murine interleukin-2 (IL-2) dependent T-lymphocyte CTLL cells, murine fibroblasts Swiss 3R3 cells, and murine fibroblast BALB/C A31 cells metabolize exogenously added sphingosine to ceramide. Fumonisin B2, a mycotoxin produced by Fusarium moniliforme, blocks the conversion of sphingosine to ceramide. In the study described here, CTLL cells exhibiting the conversion of sphingosine to ceramide were used as recipient cells, whereas fumonisin B1-treated cells (A31 cells, Swiss 3T3 cells and CTLL cells), in which the conversion was blocked were used as donor cells. To demonstrate intercellular transfer of sphingosine through the conversion pathway of sphingosine to ceramide, fumonisin B1-treated donor cells incorporating radioactive sphingosine were co-incubated with CTLL cells and the ceramide response was examined. These experiments demonstrated that it is possible to prove the intercellular transfer of sphingosine by using different activities in the cellular conversion pathway of sphingosine to ceramide.
Asunto(s)
Ácidos Carboxílicos , Fumonisinas , Micotoxinas , Esfingosina/metabolismo , Células 3T3 , Aciltransferasas/antagonistas & inhibidores , Animales , Transporte Biológico , Ácidos Carboxílicos/farmacología , Línea Celular , Ceramidas/biosíntesis , Técnicas de Cocultivo , Inhibidores Enzimáticos/farmacología , Fibroblastos/metabolismo , Ratones , Ratones Endogámicos BALB C , Micotoxinas/farmacología , Esfingosina N-Aciltransferasa , Linfocitos T/metabolismoRESUMEN
We have investigated possible intercellular transfer of Sph between heterogeneous and homogeneous cells, utilizing different abilities on conversion of Sph into GlcCer. Murine IL-2-dependent T lymphocyte CTLL cells metabolize exogenously added Sph to GlcCer through Cer within 30 min, whereas fibroblast BALB/C A31 cells required more than 2 h. Thus, CTLL cells exhibiting the active conversion were used as recipient cells, whereas A31 cells where the conversion was slow and fumonisin B1 (inhibition of Cer biosynthesis)-treated CTLL cells being donor cells. To test intercellular transfer of Sph, donor cells incorporated radioactive Sph were coincubated with CTLL cells and GlcCer was examined. The present experiments described the possible transfer of Sph between heterogeneous (from A31 cells to CTLL cells) and homogeneous cells (from fumonisin B1-treated CTLL cells to CTLL cells).
Asunto(s)
Fumonisinas , Esfingosina/metabolismo , Animales , Comunicación Celular , Células Cultivadas , Inhibidores Enzimáticos/farmacología , Glucosilceramidas/metabolismo , Ratones , Micotoxinas/farmacologíaRESUMEN
Potential sphingosine (Sph) metabolites include phosphorylated, N-acylated, and N-methylated derivatives. Phosphorylated Sph, i.e., sphingosine 1-phosphate (Sph-1-P), may act as an autocrine stimulator of blood platelets, as it is abundantly stored in platelets and released extracellularly and its exogenous addition induces platelet activation. In this study, we evaluated Sph-1-P formation and its effects in human platelets in the presence of other Sph metabolites. On addition of [3H]Sph to intact platelets, the label was rapidly converted to Sph-1-P. This conversion into [3H]Sph-1-P was inhibited by N,N-dimethylsphingosine (DMS) in a dose-dependent manner, but not by other structurally related Sph derivatives, including ceramide. The inhibition of Sph-1-P formation by DMS was reproduced using a cell-free system (Sph kinase obtained from platelet cytosolic fractions) and much stronger than that by DL-threo-dihydrosphingosine, which had been considered to be the strongest inhibitor of Sph kinase. Administration of DMS to intact platelets resulted in a decrease in Sph-1-P mass and an increase in Sph mass. Furthermore, DMS inhibited the release of Sph-1-P from platelets stimulated with 12-O-tetradecanoylphorbol 13-acetate and inhibited platelet aggregation induced by exogenous addition of Sph-1-P. Collectively, our results indicate that DMS is useful as a Sph kinase inhibitor and that Sph-1-P actions as an autocrine stimulator of platelets are inhibited by DMS.