RESUMEN
Effective use of microarray technology in clinical and regulatory settings is contingent on the adoption of standard methods for assessing performance. The MicroArray Quality Control project evaluated the repeatability and comparability of microarray data on the major commercial platforms and laid the groundwork for the application of microarray technology to regulatory assessments. However, methods for assessing performance that are commonly applied to diagnostic assays used in laboratory medicine remain to be developed for microarray assays. A reference system for microarray performance evaluation and process improvement was developed that includes reference samples, metrics and reference datasets. The reference material is composed of two mixes of four different rat tissue RNAs that allow defined target ratios to be assayed using a set of tissue-selective analytes that are distributed along the dynamic range of measurement. The diagnostic accuracy of detected changes in expression ratios, measured as the area under the curve from receiver operating characteristic plots, provides a single commutable value for comparing assay specificity and sensitivity. The utility of this system for assessing overall performance was evaluated for relevant applications like multi-laboratory proficiency testing programs and single-laboratory process drift monitoring. The diagnostic accuracy of detection of a 1.5-fold change in signal level was found to be a sensitive metric for comparing overall performance. This test approaches the technical limit for reliable discrimination of differences between two samples using this technology. We describe a reference system that provides a mechanism for internal and external assessment of laboratory proficiency with microarray technology and is translatable to performance assessments on other whole-genome expression arrays used for basic and clinical research.
Asunto(s)
Técnicas de Laboratorio Clínico/normas , Perfilación de la Expresión Génica/normas , Análisis de Secuencia por Matrices de Oligonucleótidos/normas , ARN/genética , Animales , Perfilación de la Expresión Génica/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Especificidad de Órganos , Control de Calidad , ARN/análisis , ARN/normas , Ratas , Estándares de Referencia , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Ketamine is a widely used pediatric anesthetic recently reported (C. Ikonomidou et al., 1999, Science 283, 70-74) to enhance neuronal death in neonatal rats. To confirm and extend these results, we treated four groups of PND 7 rats with seven sc doses, one every 90 min, of either saline, 10 mg/kg ketamine, 20 mg/kg ketamine, or a single dose of 20 mg/kg ketamine. The repeated doses of 20 mg/kg ketamine increased the number of silver-positive (degenerating) neurons in the dorsolateral thalamus to a degree comparable to previous results (Ikonomidou et al., 1999, Science 283, 70-74), i.e., 28-fold vs. 31-fold respectively. However, blood levels of ketamine immediately after the repeated 20 mg/kg doses were about 14 micrograms/ml, about seven-fold greater than anesthetic blood levels in humans (J. M. Malinovsky et al., 1996, Br. J. Anaesth. 77, 203-207; R. A. Mueller and R. Hunt, 1998, Pharmacol. Biochem. Behav. 60, 15-22). Levels of ketamine in blood following exposure to the multiple 10 mg/kg doses of ketamine or to a single 20 mg/kg dose ranged around 2-5 micrograms/ml; although these blood levels are close to an anesthetic level in humans, they failed to produce neurodegeneration. To investigate the mode of ketamine-induced neuronal death, coronal sections were stained with both Fluoro-Jade B (a green fluorescent stain selective for neurodegeneration) and DAPI (a blue DNA stain), as well as for caspase-3 (using an antisera labeled red with rhodamine). These histochemical results confirmed the developmental neurotoxicity of ketamine, demonstrated that Fluoro-Jade B (FJ-B), like silver methods, successfully stained degenerating neurons in neonatal rats, and indicated that ketamine acts by increasing the rate of neuronal apoptosis.
Asunto(s)
Apoptosis/efectos de los fármacos , Antagonistas de Aminoácidos Excitadores/toxicidad , Ketamina/toxicidad , Sistema Nervioso/crecimiento & desarrollo , Sistema Nervioso/patología , Neuronas/patología , Síndromes de Neurotoxicidad/patología , Animales , Animales Recién Nacidos , Encéfalo/patología , Química Encefálica/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Antagonistas de Aminoácidos Excitadores/sangre , Femenino , Fluoresceínas , Colorantes Fluorescentes , Ketamina/sangre , Masculino , Sistema Nervioso/efectos de los fármacos , Neuronas/efectos de los fármacos , Compuestos Orgánicos , Ratas , Ratas Sprague-Dawley , Tinción con Nitrato de Plata , Tálamo/patologíaRESUMEN
A first-pass approach incorporating high-field magnetic resonance imaging (MRI) was used for rapid detection of neuropathologic lesions in fixed rat brains. This inherently 3-dimensional and nondestructive technique provides high-resolution, high-contrast images of fixed neuronal tissue in the absence of sectioning or staining. This technique, magnetic resonance microscopy (MRM), was used to identify diverse lesions in 2 well-established rat neurotoxicity models. The intrinsic contrast in the images delineated lesions that were identified using a battery of histologic stains, some of which would not be used in routine screening. Furthermore, the MRM images provided the locations of lesions, which were verified upon subsequent sectioning and staining of the same samples. The inherent contrast generated by water properties is exploited in MRM by choosing suitable pulse sequences, or proton stains. This approach provides the potential for a comprehensive initial MRM screen for neurotoxicity in preclinical models with the capability for extrapolation to clinical analyses using classical MRI.
Asunto(s)
Procesamiento de Imagen Asistido por Computador/métodos , Enfermedades del Sistema Nervioso/patología , Síndromes de Neurotoxicidad/patología , Animales , Encéfalo/patología , Ácido Kaínico/análogos & derivados , Ácido Kaínico/toxicidad , Imagen por Resonancia Magnética , Microscopía , Neurotoxinas/toxicidad , Oxidopamina/toxicidad , Enfermedad de Parkinson Secundaria/inducido químicamente , Enfermedad de Parkinson Secundaria/patología , Ratas , Simpaticolíticos/toxicidadRESUMEN
A novel fluorescence-based method was developed to measure HIV envelope glycoprotein (env)-CD4-mediated cell fusion. This method measures the spread of a fluorescent dye as the cytosolic compartments of adjacent cells become contiguous upon cell-to-cell fusion. Calcein-labeled CD4+ Sup-T1 cells were seeded onto a monolayer of unlabeled TF228.1.16 cells, which stably express env, the gp120-gp41 complex. Changes in the following parameters were measured using a stage-scanning laser microscope: total fluorescent area, average fluorescent area, and average shape factor. Anti-CD4 monoclonal antibodies, anti-Leu3a, and OKT4E were shown to block fusion in a dose-dependent manner, while OKT4 had no effect. Aurin tricarboxylic acid, a compound that interferes with the binding of anti-Leu3a mAb and gp120 to CD4+ human peripheral blood lymphocytes, T20, a peptide that interferes with gp41, and cytochalasin D, a microfilament disrupter, all blocked fusion in a dose-dependent manner. This semiautomated assay can be used to quickly assess the effectiveness of compounds acting at different sites to block CD4 and env initiated cell-to-cell fusion.
Asunto(s)
Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/virología , Proteína gp120 de Envoltorio del VIH/análisis , Proteína gp41 de Envoltorio del VIH/análisis , VIH-1/aislamiento & purificación , Anticuerpos Monoclonales , Linfocitos T CD4-Positivos/inmunología , Fusión Celular/fisiología , Línea Celular , Citocalasina D/farmacología , Fluoresceínas/farmacocinética , Colorantes Fluorescentes/farmacocinética , Células Gigantes/fisiología , VIH-1/crecimiento & desarrollo , Humanos , Procesamiento de Imagen Asistido por Computador , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Factores de Tiempo , Replicación Viral/efectos de los fármacosRESUMEN
The binding between the HIV surface protein, gp120, and the CD4 coreceptor is known to be initiated by electrostatic interactions. Because of the ability of chlorpromazine to interact with proteins by charge transfer, we tested several derivatives for their ability to block binding of HIV to CD4+ cells. We have shown that 7,8-dioxo-chlorpromazine blocks binding of fluorescein isothiocyanate-labeled anti-Leu3a and rgp120 to peripheral human blood T4 cells and blocks syncytia formation between gp120- and CD4-expressing cells. We also found that 7,8-dioxo-chlorpromazine blocks HIV infectivity of H9 cells and acts synergistically with zidovudine.
Asunto(s)
Fármacos Anti-VIH/farmacología , Clorpromazina/farmacología , VIH/efectos de los fármacos , Linfocitos T CD4-Positivos/metabolismo , Línea Celular , Clorpromazina/análogos & derivados , Proteína gp120 de Envoltorio del VIH/metabolismo , Humanos , Reacción en Cadena de la Polimerasa , Linfocitos T/virologíaRESUMEN
Pharmacologically active in vivo doses of P-glycoprotein (Pgp) blockers, specifically verapamil, Cremophor EL and PSC833 cause toxicity in addition to that from the concomitantly used cancer chemotherapeutic drugs. It was shown before that these blockers cause different types of toxicities in vivo. We found that these 3 chemically distinct Pgp blockers exert different biophysical effects on the membranes of L1210 MDR cells. They also affect the general metabolism of these cells differently, but all block affinity labeling of Pgp. We could also show that the combination of suboptimal doses of these blockers can restore the uptake of the Pgp substrate rhodamine 123 into L1210MDR, 3T3MDR and KB-VI cells and can reduce the survival rate of these cells when treated in combination with daunorubicin. Our results suggest that the combination of suboptimal doses of these Pgp blockers may be advantageous in clinical practice.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/antagonistas & inhibidores , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Bloqueadores de los Canales de Calcio/farmacología , Membrana Celular/metabolismo , Ciclosporinas/farmacología , Glicerol/análogos & derivados , Verapamilo/farmacología , División Celular/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Glicerol/farmacología , Humanos , Leucemia , Transfección , Células Tumorales CultivadasRESUMEN
The substrate specificity of the P-glycoprotein (P-170), a multidrug transporter, was studied using N-acylated daunorubicin derivatives and four MDR1 cDNA transfected cell lines. Results showed that N-acetyl-daunorubicin is a substrate, but the longer fatty acid derivatives, N-octanoyl and N-dodecanoyl daunorubicins, are not. This conclusion was reached by flow cytometric drug uptake assay, cell proliferation assays, and confocal microscopy. It was concluded that the longer fatty acid derivatives interact with plasma membranes in a way that affected P-glycoprotein function.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Daunorrubicina/análogos & derivados , Resistencia a Múltiples Medicamentos/genética , Células 3T3 , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/antagonistas & inhibidores , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Animales , División Celular/efectos de los fármacos , Ciclosporina/farmacología , Daunorrubicina/metabolismo , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Humanos , Ratones , Transfección , Células Tumorales CultivadasRESUMEN
Instances when T cell activation via the T cell receptor/CD3 complex is suppressed by anti-CD4 Abs are generally attributed either to the topological separation of CD4-p56lck from CD3, or their improper apposition. Photobleaching fluorescence resonance energy transfer measurements permitted direct analysis of these alternatives on human peripheral blood lymphocytes. Distinction between changes of relative antigen densities or positioning was made possible by simultaneously recording donor and acceptor fluorescence in the energy transfer experiment performed on homogeneous populations of flow-sorted cells. We show here that CD4 stays in the molecular vicinity of CD3, while anti-CD3 stimulation is suppressed by anti-CD4 or cross-linked HIV gp120. Our data suggest that cross-linking of CD4 through particular epitopes is capable of inhibiting activation driven by Abs binding to specific sites on CD3 without major topological sequestration of the Ags, in such a way that additional positive signals will also be affected. Thus, these and other related cases of negative signaling via CD4 may be interpreted in terms of functional uncoupling rather than a wide physical separation of CD4 from the T cell receptor-CD3 complex.
Asunto(s)
Complejo CD3/química , Antígenos CD4/química , Fenómenos Biofísicos , Biofisica , Linfocitos T CD4-Positivos/química , Linfocitos T CD4-Positivos/inmunología , Reactivos de Enlaces Cruzados , Transferencia de Energía , Epítopos , Humanos , Técnicas In Vitro , Fotoquímica , Espectrometría de FluorescenciaRESUMEN
Several accessory molecules were shown to play important roles in T cell functions and be in close proximity to the T cell receptor (TcR/CD3). The L-selectin molecule (Leu8, LAM1-1, LECAM1) also plays an important role in lymphocyte homing and proliferation. We were interested in determining the proximity of this molecule to the TcR/CD3 complex on live peripheral human T cells. Using a fluorescence energy transfer method, designed to study individual cells, we could show that L-selectin is within 170 A of the TcR/CD3 complex. Monoclonal antibody directed against the LAM1-1 (Leu8) epitope of the L-selectin molecule suppressed the mitogenic activity of antibodies specific for various CD3 epitopes in vitro. Intracellular Ca2+ mobilization obtained with wt31 followed by cross-linking antibody or with anti-CD3 was not influenced by anti-Leu8 antibody. Also antibody directed against the LAM1-1 epitope did not influence the binding of the mitogenic antibodies, as shown by fluorescence-based flow cytometry. Therefore, we suggest that binding of TcR/CD3 bound mitogenic antibodies to accessory cell Fc receptors may be hindered by antibodies bound to the close proximity L-selectin molecules.
Asunto(s)
Moléculas de Adhesión Celular/análisis , Complejo Receptor-CD3 del Antígeno de Linfocito T/análisis , Linfocitos T/ultraestructura , Complejo CD3/inmunología , Calcio/metabolismo , Moléculas de Adhesión Celular/inmunología , División Celular , Replicación del ADN , Transferencia de Energía , Epítopos/inmunología , Humanos , Selectina L , Rayos Láser , Activación de Linfocitos , Muromonab-CD3/inmunología , Fotoquímica , Unión Proteica , Receptores Fc/metabolismo , Transducción de Señal , Espectrometría de Fluorescencia , Linfocitos T/inmunologíaRESUMEN
It has previously been shown that crosslinking of the CD4 molecule, either with anti-Leu3a mAb or with gp120 (the HIV coat protein) plus anti-gp120 mAb, suppresses activation induced by wt31, a TcR/CD3-specific mAb. This suppression was associated with hindrance of the necessary association of the p56lck kinase bearing CD4 molecule with the TcR/CD3 complex. In this paper we demonstrate that this crosslinking-induced suppression can be bypassed by perturbing the microfilament system of CD4+ cells by pretreatment with 1 microM cytochalasin D. Using the fluorescence resonance energy transfer method, we have shown that the cytochalasin D-affected increase of mitogenesis is not due to changes in the TcR/CD3 to CD4 distance. Likewise, other membrane biophysical parameters, membrane potential and lateral diffusion of surface receptors, cannot be associated with these cytochalasin D-affected mitogenic changes. Cytochalasin D treatment elevates intracellular Ca2+ levels induced by wt31 mAb plus crosslinking and generates a TcR/CD3-dependent signal which is cyclosporin sensitive.
Asunto(s)
Antígenos CD4/fisiología , Linfocitos T CD4-Positivos/fisiología , Calcio/fisiología , Citocalasina D/farmacología , Transducción de Señal/inmunología , Anticuerpos Monoclonales , Linfocitos T CD4-Positivos/efectos de los fármacos , Ciclosporina/farmacología , Citometría de Flujo , Fluorescencia , Humanos , Activación de Linfocitos/efectos de los fármacos , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Fotoquímica , Complejo Receptor-CD3 del Antígeno de Linfocito T/efectos de los fármacos , Complejo Receptor-CD3 del Antígeno de Linfocito T/fisiología , Receptores de Interleucina-2/inmunología , Transducción de Señal/efectos de los fármacosRESUMEN
We have developed an in vitro system to model the interactions of drugs used to treat transplant rejection. This system consists of stimulation of human lymphocytes with a primary mitogen (anti-T-cell receptor complex antibodies (OKT3 or wt31)) and treatment with a primary immunosuppressive drug (ISD) (Cyclosporine A (CsA) or FK-506)). This is later followed by stimulation with a secondary mitogen (Interleukin-2 or anti-CD28), and treatment with a second ISD. This system allows a variety of concentrations and compounds to be rapidly tested. We have used this system to study the effect of various compounds when used as either primary or secondary ISDs. Our results show that when CsA is used as the primary ISD, further proliferation can be inhibited by rapamycin, mycophenolic acid, or suramin. When FK-506 is the primary ISD, inhibition of proliferation by rapamycin is variable depending on the primary and secondary mitogens. If rapamycin is the primary ISD, both CsA and FK-506 show antagonistic interactions. These results suggest that the order in which combinations of ISDs are administered in transplantation may have significant effects on the clinical outcome.
Asunto(s)
Inmunosupresores/farmacología , Linfocitos/efectos de los fármacos , Anticuerpos Monoclonales/inmunología , Benzoquinonas , Antígenos CD28/inmunología , Células Cultivadas , Ciclosporina/farmacología , Interacciones Farmacológicas , Humanos , Inmunosupresores/administración & dosificación , Interleucina-2/inmunología , Lactamas Macrocíclicas , Ácido Micofenólico/farmacología , Polienos/farmacología , Quinonas/farmacología , Receptores de Antígenos de Linfocitos T/inmunología , Rifabutina/análogos & derivados , Sirolimus , Suramina/farmacología , Tacrolimus/farmacologíaAsunto(s)
Anticuerpos , Inmunohistoquímica/métodos , Rayos Láser , Microscopía Fluorescente/métodos , Animales , Fenómenos Biofísicos , Biofisica , Muerte Celular , Membrana Celular/metabolismo , Separación Celular/métodos , Transferencia de Energía , Citometría de Flujo/métodos , Colorantes Fluorescentes , Humanos , Inmunohistoquímica/instrumentación , Proteínas de la Membrana/metabolismo , Microscopía Fluorescente/instrumentación , Óptica y Fotónica/instrumentaciónRESUMEN
Brief treatment of human peripheral blood lymphocytes with the potential anti-HIV compound aurintricarboxylic acid (ATA) prompts the selective release of already bound L-selectin-specific anti-Leu8 and anti-LAM1-1 antibodies from the cells. Two other anti-LAM1 antibodies, anti-LAM1-3 and anti-LAM1-5 stay antigen-bound at the same time. Interestingly, the ATA-sensitive anti-Leu8 strongly competes with the ATA-resistant anti-LAM1-3 for binding. Photobleaching fluorescence resonance energy transfer (pFRET) measurements on flow-sorted cells suggests that these two antibodies compete for the same epitope, while anti-LAM1-5-FITC and anti-Leu8-PE bind to distinct sites, although they also compete for binding. Combining the data on competition, pFRET and ATA effect, we suggest that the ATA sensitive anti-Leu8 and resistant anti-LAM1-3 bind to overlapping but non-identical epitopes. This remarkably specific effect may be exploited for designing anti-inflammatory drugs that modulate leukocyte adhesion.
Asunto(s)
Reacciones Antígeno-Anticuerpo/efectos de los fármacos , Ácido Aurintricarboxílico/farmacología , Moléculas de Adhesión Celular/inmunología , Linfocitos/efectos de los fármacos , Sitios de Unión de Anticuerpos , Unión Competitiva , Citometría de Flujo , Humanos , Selectina LRESUMEN
The MDRI protein is an energy-dependent transport protein responsible for the multi-drug resistance seen in many tumors. A variety of drugs have been shown to inhibit the function of this pump, including compounds known to block various ion channels. The mouse lymphoma cell line L5178Y has been transduced with the human mdrI gene. Using this cell line, we have tested a number of compounds to determine whether there is a correlation between the ability to block a specific type of ion channel, or shift membrane potential, and the ability to act as an MDR-reversing agent using the fluorescent substrates Rhodamine 123 and daunorubicin as test compounds. Our results show no apparent correlation between the ability to block a specific ion channel and reversal of MDR transport ability. We have found active MDR inhibitors in compounds that affect K+, Na+, Ca++, H+, but not Cl- channels. Our data suggest that Cl- channel activity may be distinct from MDR activity. Several immunosuppressive compounds and analogs were also tested and found to be active reversing agents. Measurements suggest a significant difference in resting membrane potential between the L5178YvMDR line and the L5178Y parental cell line used in these experiments. No correlation was found between the ability of drugs to alter membrane potential and to inhibit MDR transport activity. Our results suggest that MDR transport function may be independent of the physiological movement of ions and show that a wide variety of compounds can inhibit MDR transport.
Asunto(s)
Proteínas Portadoras/efectos de los fármacos , Proteínas Portadoras/fisiología , Inmunosupresores/farmacología , Canales Iónicos/efectos de los fármacos , Canales Iónicos/metabolismo , Glicoproteínas de Membrana/efectos de los fármacos , Glicoproteínas de Membrana/fisiología , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP , Secuencia de Aminoácidos , Animales , Benzofuranos/farmacología , Transporte Biológico Activo/efectos de los fármacos , Proteínas Portadoras/metabolismo , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Membrana Celular/fisiología , Daunorrubicina/metabolismo , Daunorrubicina/farmacocinética , Resistencia a Medicamentos , Éteres Cíclicos/farmacología , Citometría de Flujo , Colorantes Fluorescentes , Concentración de Iones de Hidrógeno , Líquido Intracelular/química , Leucemia L5178/tratamiento farmacológico , Leucemia L5178/metabolismo , Linfoma de Células T/tratamiento farmacológico , Linfoma de Células T/metabolismo , Glicoproteínas de Membrana/metabolismo , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Ratones , Datos de Secuencia Molecular , Potasio , Canales de Potasio/efectos de los fármacos , Canales de Potasio/metabolismo , Rodamina 123 , Rodaminas/metabolismo , Rodaminas/farmacocinética , Células Tumorales CultivadasRESUMEN
Aurintricarboxylic acid (ATA) has been shown to block the binding site for both HIV gp120 and mAb anti-Leu 3a on CD4. We have unexpectedly found that brief treatment with > or = 1 micrograms/ml ATA rapidly disengages another mAb, OKT4E, after it has been bound to CD4 on human PBL. OKT4E is specific for a discontinuous epitope overlapping the MHC class II-binding region in the N-terminal CD4 domain. Interestingly, among 10 other mAb tested, only anti-Leu 8, specific for a leukocyte homing receptor is also quickly released from the cells by ATA treatment. Disengagement of the OKT4E mAb is also seen on a CD4-positive cell line (HPB-ALL) and with recombinant soluble CD4 (sCD4) bound to immobilized OKT4E. In all of these cases, disengagement is prevented if OKT4E is cross-linked, or the Leu 3a site is blocked by the mAb, but not by gp120. Photobleaching fluorescence resonance energy transfer (pFRET) measurements suggest that OKT4E is released as an indirect consequence of ATA-evoked conformational changes of CD4. Similar changes were detected as a result of gp120 binding to PBL. These data raise the possibility of a novel type of immunomodulation: induced disengagement of a bound ligand from its Ag.
Asunto(s)
Reacciones Antígeno-Anticuerpo/efectos de los fármacos , Ácido Aurintricarboxílico/farmacología , Antígenos CD4/química , Anticuerpos Monoclonales/inmunología , Antígenos CD4/metabolismo , Humanos , Técnicas In Vitro , Ligandos , Fluidez de la Membrana , Microscopía Fluorescente , Conformación ProteicaRESUMEN
Several tannins with anti-HIV activity have been described previously (Nonaka et al., J Nat Prod 53: 587-595, 1990). We have shown that the tannins chebulinic acid and punicalin were able to block the binding of HIV rgp120 to CD4. These compounds were not toxic to stimulated human peripheral blood lymphocytes at concentrations ten times above their maximal effective concentration.
Asunto(s)
Antivirales/farmacología , Antígenos CD4/metabolismo , Proteína gp120 de Envoltorio del VIH/metabolismo , Taninos/farmacología , Diseño de Fármacos , Humanos , Linfocitos/efectos de los fármacos , Linfocitos/inmunología , Unión Proteica/efectos de los fármacos , Proteínas Recombinantes/metabolismoRESUMEN
The donor photobleaching method (T. M. Jovin and D. J. Arndt-Jovin. 1989. Annu. Rev. Biophys. Biophys. Chem. 18:271-308.) has been adapted to an ACAS 570 (laser scanning microscope) system to measure fluorescence resonance energy transfer (FRET) on individual human peripheral blood T cells. Photobleaching was completed in approximately 100 ms in our case and it followed double-exponential kinetics. The energy transfer efficiency (E) was approximately 20% between the CD4 epitopes OKT4-FITC and Leu-3a-PE as well as between OKT4E-FITC and OKT4-PE. E was approximately 8% between OKT4-FITC and Leu-4-PE (alpha CD3) and barely detectable (approximately 4%) from OKT4-FITC to Leu-5b-PE (alpha CD2). The E values obtained by the photobleaching method were highly reproducible both in repeated measurement of identical samples and in experiments with different batches of cells and were in agreement with the flow cytometric donor quenching measurements. As expected, E measured between primary and secondary layers of antibodies increased (from approximately 14% to approximately 28%) when F(ab')2 fragments were substituted for whole antibody molecules as the donor. On a T cell line we mapped the distance between the idiotypic determinant of the T cell receptor (TcR) and the Leu-4 epitope of CD3 as proximal as E = 28%, as compared to E = 4% between a framework TcR epitope and Leu-4. In the latter case, however, approximately 40% less Leu-4 was bound suggesting that the antigen binding site of TcR is in close proximity with one of the two CD3 epsilon chains, which hence are not equivalent.
Asunto(s)
Epítopos/análisis , Linfocitos/inmunología , Receptores de Antígenos de Linfocitos T/análisis , Anticuerpos Monoclonales , Línea Celular , Fluoresceína-5-Isotiocianato , Humanos , Fragmentos Fab de Inmunoglobulinas , Rayos Láser , Leucemia de Células T , Microscopía Electrónica de Rastreo/métodos , Microscopía Fluorescente/métodos , Receptores de Antígenos de Linfocitos T/inmunología , Espectrometría de FluorescenciaRESUMEN
The multidrug-resistant gene (MDR1) encodes an energy-dependent drug efflux pump (P-glycoprotein) for many anti-cancer drugs. We have studied the intracellular distribution of rhodamine 123 (R123), daunorubicin (DN), and doxorubicin (DOX) in cells expressing a human MDR1 gene. The distribution of these fluorescent drugs was measured by laser scanning microscopy and confocal microscopy. We devised a new method for analysis of fluorescence line scan data to determine the intracellular distribution of fluorescent probes. This method and confocal microscopy showed that R123, DN, and DOX are localized to both plasma membrane and intracellular compartments in multidrug-resistant cells. When the cells are treated with verapamil, an inhibitor of the multidrug transporter, the amount of DOX, DN, and R123 associated with the cell rises. After inhibition, the relative distribution of DOX and DN between the cell surface and intracellular structures does not change dramatically. However, R123 tends to relocalize to intracellular sites from predominantly plasma membrane sites, indicating that this dye behaves differently than the anti-cancer drugs. These results show the subcellular distributions of R123, DN, and DOX in plasma membrane, cytoplasm, and intracellular membrane systems, but do not allow definitive distinctions among existing models of how P-glycoprotein affects the distribution of drugs.
Asunto(s)
Daunorrubicina/metabolismo , Doxorrubicina/metabolismo , Resistencia a Medicamentos/fisiología , Rodaminas/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP , Animales , Línea Celular , Daunorrubicina/análisis , Doxorrubicina/análisis , Resistencia a Medicamentos/genética , Humanos , Células KB , Rayos Láser , Glicoproteínas de Membrana/genética , Ratones , Microscopía Fluorescente/métodos , Rodamina 123 , Rodaminas/análisis , Transfección , Verapamilo/farmacologíaRESUMEN
The immunosuppressive drugs FK-506 and mycophenolic acid (MPA) have recently been described, but their mode(s) of action are not well understood. We have compared them to cyclosporine A (CsA) in several assays. We have shown that CsA (1 microgram/ml), MPA (0.1 microgram/ml), and FK-506 (0.5 microgram/ml) all induce a state of unresponsiveness to anti-CD3 stimulation as measured by [3H]-thymidine uptake. This suggests that the target of these drugs may be present only after mitogenic stimulation. These drugs also cause a hyperpolarization of the plasma membrane of lymphocytes. This effect is blocked by quinine or verapamil. All three immunosuppressors only slightly modulate the increase in intracellular Ca++ caused by Con-A or by anti-CD3 stimulation but do not affect Ca++ levels alone. They also decrease expression of IL-2 receptors on alpha CD3-stimulated lymphocytes. Similarities in their modes of action, as measured by these biophysical and cell biological tests, indicate the possibility that these three drugs will show similarities in their clinical performance.