RESUMEN
Chromium (Cr) compounds are widely used in alloys manufacturing and forming processes. One of the main concerns in the use of cobalt-chromium (Co-Cr) alloy-based implants is the long-term fate of Co and Cr ions in the blood, organs, and urine of patients. Our previous studies have shown that Cr(III) forms complexes in different cell culture media, whereas Cr(VI) does not form any detectable structure under the same conditions. Because Cr(VI) is known to be more toxic than Cr(III), we hypothesized that the presence of serum proteins in the molecular structure of Cr(III) may be responsible for the difference in toxicity. We investigated the interaction of the Cr(III) complexes with serum proteins and their internalization by U937 macrophage-like cells. By using a proteomic approach, we showed that in the presence of fetal bovine serum, Cr(III) complexes interacted only with albumin, whereas they interacted mainly with albumin, transferrin, and immunoglobulins (Ig) in the presence of human serum (HS). Cr(III) complexes were more easily engulfed by U937 macrophage-like cells when they were formed with HS. To the best of our knowledge, this is the first report on the formation of Cr(III) complexes in the presence of serum proteins and the interaction of these complexes with U937 macrophage-like cells. (c) 2010 Wiley Periodicals, Inc. J Biomed Mater Res, 2010.
Asunto(s)
Proteínas Sanguíneas/metabolismo , Cromo/metabolismo , Proteoma , Secuencia de Aminoácidos , Animales , Proteínas Sanguíneas/química , Proteínas Sanguíneas/genética , Bovinos , Cromo/química , Humanos , Espectrometría de Masas , Unión Proteica , Estrés Mecánico , Resistencia a la Tracción , Células U937/metabolismo , Células U937/ultraestructuraRESUMEN
We demonstrate the use of electron microscopy as a powerful characterization tool to identify and locate antibody-conjugated composite organic-inorganic nanoparticle (COINs) surface enhanced Raman scattering (SERS) nanoparticles on cells. U937 leukemia cells labeled with antibody CD54-conjugated COINs were characterized in their native, hydrated state using wet scanning electron microscopy (SEM) and in their dehydrated state using high-resolution SEM. In both cases, the backscattered electron (BSE) detector was used to detect and identify the silver constituents in COINs due to its high sensitivity to atomic number variations within a specimen. The imaging and analytical capabilities in the SEM were further complemented by higher resolution transmission electron microscopy (TEM) images and scanning Auger electron spectroscopy (AES) data to give reliable and high-resolution information about nanoparticles and their binding to cell surface antigens.
Asunto(s)
Nanopartículas del Metal/ultraestructura , Microscopía Electrónica de Rastreo/métodos , Microscopía Electrónica de Transmisión/métodos , Células U937/ultraestructura , Humanos , Inmunoconjugados/química , Inmunoconjugados/ultraestructura , Molécula 1 de Adhesión Intercelular/química , Nanopartículas del Metal/química , Plata/química , Espectrometría Raman/métodosRESUMEN
Excessive accumulation of phospholipids leads to phospholipidosis (PL), which disrupts cellular functions, in extreme cases leading to acute or chronic disease. Citalopram and many other cationic amphiphilic drugs (CADs) have been shown to cause PL both in vitro and in vivo. Recent toxicogenomic studies suggest four hypothetical mechanisms for PL (lysosomal enzyme transport decrease, lysosomal phospholipase activity decrease, phospholipids biosynthesis increase or cholesterol biosynthesis increase). However, the post-genomic steps remain largely unknown and proteomic analyses hold significant promise for defining mechanisms of PL induction. In this study U937 monoblastoid cells were exposed to citalopram hydrobromide for 24 h (0, 20, 100 or 200 microM as citalopram free base) and then harvested for whole cell proteomic analysis using 2-D gel electrophoresis, or transmission electron microscopy (TEM). Protein spots that were significantly altered versus controls were analysed by MALDI-TOF mass spectrometry. Up-regulated proteins were Glyoxalase-I (Glo 1) and 3-Hydroxy-3-methylglutaryl-Coenzyme A synthase 1 (HMGCS1) in cells with PL shown by TEM (favouring the cholesterol biosynthesis increase hypothesis for citalopram induced PL). Other altered proteins were catalase (up-regulated), beta-actin (up-regulated) and 14-3-3 protein (down-regulated). The function of several of the successfully identified proteins indicates a potential perturbed lipid metabolism.
Asunto(s)
Colesterol/biosíntesis , Citalopram/farmacología , Fosfolípidos/metabolismo , Proteómica/métodos , Inhibidores Selectivos de la Recaptación de Serotonina/farmacología , Células U937/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Electroforesis en Gel Bidimensional , Humanos , Hidroximetilglutaril-CoA Sintasa/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Células U937/metabolismo , Células U937/ultraestructura , Regulación hacia ArribaRESUMEN
Oxysterols, mainly those oxidized at the C7 position, induce a complex mode of cell death exhibiting some characteristics of apoptosis associated with a rapid induction of lipid rich multilamellar cytoplasmic structures (myelin figures) observed in various pathologies including atherosclerosis. The aim of this study was to determine the relationships between myelin figure formation, cell death, and lipid accumulation in various cell lines (U937, THP-1, MCF-7 [caspase-3 deficient], A7R5) treated either with oxysterols (7-ketocholesterol [7KC], 7beta-hydroxycholesterol, cholesterol-5alpha,6alpha-epoxide, cholesterol-5beta,6beta-epoxide, 25-hydroxycholesterol) or cytotoxic drugs (etoposide, daunorubicin, tunicamycin, rapamycin). Cell death was assessed by the measurement of cellular permeability with propidium iodide, characterization of the morphological aspect of the nuclei with Hoechst 33342, and identification of myelin figures by transmission electron microscopy. Nile Red staining (distinguishing neutral and polar lipids) was used to identify lipid content by flow cytometry and spectral imaging microscopy. Whatever the cells considered, myelin figures were only observed with cytotoxic oxysterols (7KC, 7beta-hydroxycholesterol, cholesterol-5beta, 6beta-epoxide), and their formation was not inhibited by the broad spectrum caspase inhibitor z-VAD-fmk. When U937 cells were treated with oxysterols or cytotoxic drugs, polar lipid accumulation was mainly observed with 7KC and 7beta-hydroxycholesterol. The highest polar lipid accumulation, which was triggered by 7KC, was counteracted by z-VAD-fmk. These findings demonstrate that myelin figure formation is a caspase-independent event closely linked with the cytotoxicity of oxysterols, and they highlight a relationship between caspase activity and polar lipid accumulation.
Asunto(s)
Apoptosis/efectos de los fármacos , Caspasas/metabolismo , Inhibidores Enzimáticos/farmacología , Cetocolesteroles/farmacología , Metabolismo de los Lípidos , Vaina de Mielina/efectos de los fármacos , Inhibidores de Caspasas , Permeabilidad de la Membrana Celular/efectos de los fármacos , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Humanos , Vaina de Mielina/metabolismo , Vaina de Mielina/ultraestructura , Propidio , Células U937/efectos de los fármacos , Células U937/metabolismo , Células U937/ultraestructuraRESUMEN
Recently we discovered a bacterial strain (MS-02-063) that produces large amounts of red pigment (PG-L-1). Among the cell lines tested, U937 cells showed the highest susceptibility to PG-L-1 toxicity. PG-L-1 induced typical apoptotic nuclear morphological changes, and single cell gel electrophoresis revealed that PG-L-1 caused DNA fragmentation in U937 cells. In PG-L-1 treated U937 cells, the acidic compartment such as lysosomes disappeared, suggesting that PG-L-1-induced disorder of intracellular pH compartmentalization might trigger apoptotic signal. Since p38 MAP kinase inhibitor specifically prevented the PG-L-1 mediated cell death, p38 MAP kinase may be involved in the cytotoxic mechanism. In fact, immunoblot analysis of p38 MAP kinase revealed that phosphorylation of p38 MAP kinase occurred in PG-L-1-treated U937 cells. In addition to the activity to induce apoptotic cell death as reported in several PG family members, our chemiluminescence analysis suggested that PG-L-1 inhibited superoxide generation by 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated U937 cells in a dose-dependent manner. Since PG-L-1 had no effect on the chemiluminescence response caused by xanthine oxidase/hypoxanthine system, PG-L-1 acts on the enzyme system responsible for O(2)(-) generation rather than direct scavenging toward O(2)(-). Our results suggest that PG-L-1 causes multiple biochemical effects on the target cells such as increase in pH in acidic intracellular compartment, activation of p38 MAP kinase, inhibition of O(2)(-) generation, and eventually induces apoptotic cell death.
Asunto(s)
Antibacterianos/farmacología , Apoptosis , Gammaproteobacteria/metabolismo , Prodigiosina/análogos & derivados , Prodigiosina/toxicidad , Naranja de Acridina , Antibacterianos/toxicidad , Compartimento Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Fragmentación del ADN/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Humanos , Concentración de Iones de Hidrógeno , Immunoblotting , Mediciones Luminiscentes , Prodigiosina/metabolismo , Serratia marcescens/metabolismo , Superóxidos/antagonistas & inhibidores , Superóxidos/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Células U937/efectos de los fármacos , Células U937/metabolismo , Células U937/ultraestructura , Proteínas Quinasas p38 Activadas por Mitógenos/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Human monocytic leukemia U937 cells readily undergo apoptosis when exposed to various stimuli, including inhibition of protein synthesis, oxidative stress, antitumoral agents, etc. The sequential, step-by-step morphological changes in U937 cells that occur during the apoptotic program are largely determined by the activation of a specific class of proteases, the caspases. The action of these proteases were followed at the ultrastructural level. From our observations 1) no unique morphological feature exists during apoptosis, even in the same cell type; 2) the extent of the morphological modifications are inducer- and dose-dependent; 3) double or triple treatments amplify the morphological modifications with a single inducer, but not the rate of apoptosis; and 4) in the case of double treatment the second inducer has to have a cytoplasmatic target because damage to the cytoplasm occurs before nuclear modifications become visible. These data should facilitate a more objective evaluation of apoptosis in conditions where antiproliferative drugs, like antiblastic or immunosuppressive molecules, are used to monitor the efficiency of treatment.
Asunto(s)
Apoptosis/efectos de los fármacos , 3-Yodobencilguanidina/farmacología , Antineoplásicos/farmacología , Apoptosis/fisiología , Cicloheximida/farmacología , Humanos , Peróxido de Hidrógeno/farmacología , Oxidantes/farmacología , Paclitaxel/farmacología , Inhibidores de la Síntesis de la Proteína/farmacología , Puromicina/farmacología , Células U937/ultraestructuraRESUMEN
Several cationic amphiphilic compounds are known to induce phospholipidosis, a condition primarily characterized by excessive accumulation of phospholipids in different cell types, giving the affected cells a finely foamy appearance. Excessive storage of lamellar membranous intralysosomal inclusion bodies is the hallmark for phospholipidosis on the electron microscopic level. In case of alveolar phospholipidosis, foamy macrophages accumulate within the alveolar spaces of the lung. Based on such findings in a one-year toxicity study with gepirone in rats, we studied the molecular properties of this compound and compounds suspected of being phospholipidosis inducers by means of physicochemical calculations. Physicochemical molecular calculations of molecular weight, ClogP (partition coefficient octanol/water), logD at pH 7.4, and pKa were performed, for the cationic amphiphilic compounds chlorpromazine, amiodarone, imipramine, propranolol and fluoxetine, and for the structurally related compounds 1-phenylpiperazine (1-PHP), gepirone (and its major metabolites, 3-OH-gepirone and 1-pyrimidinylpiperazine [1-PP]), and buspirone. ClogP and calculated pKa cluster differently for the amphiphilic drugs compared to the chemical series of piperazines. In line with this analysis, lamellar inclusion bodies were found in an in vitro validation experiment in the human monoblastoid cell line U-937, incubated for 96 h at 10 microg/mL with cationic amphiphilic drugs (amiodarone, imipramine, or propranolol). No such lamellar inclusion bodies were seen for any of the compounds from the chemical series of piperazines including gepirone and its metabolites. The data presented support the use of simple physicochemical calculations of ClogP and pKa to discriminate rapidly between compounds suspected of being phospholipidosis inducers. Finally, the discriminative power of these physicochemical ClogP and pKa calculations to predict phospholipidosis-inducing potential was further validated by extension of the set of compounds.
Asunto(s)
Antidepresivos/química , Antidepresivos/toxicidad , Química Física , Lipidosis/inducido químicamente , Pirimidinas/química , Pirimidinas/toxicidad , Administración Oral , Animales , Antidepresivos/administración & dosificación , Fenómenos Químicos , Dieta , Relación Dosis-Respuesta a Droga , Femenino , Células Espumosas/efectos de los fármacos , Células Espumosas/metabolismo , Células Espumosas/patología , Humanos , Lipidosis/metabolismo , Lipidosis/patología , Masculino , Fosfolípidos/metabolismo , Alveolos Pulmonares/efectos de los fármacos , Alveolos Pulmonares/metabolismo , Alveolos Pulmonares/patología , Pirimidinas/administración & dosificación , Ratas , Ratas Sprague-Dawley , Pruebas de Toxicidad , Células U937/efectos de los fármacos , Células U937/metabolismo , Células U937/ultraestructuraRESUMEN
We carried out a screening for drugs that can induce apoptosis in human monocytic leukemia U937 cells. In the screening, we found that 8-nitrocaffeine induces cell death distinct from typical apoptosis. Morphological and biochemical analysis revealed that reactive oxygen species mediates the 8-nitrocaffeine-induced necrotic cell death.
Asunto(s)
Apoptosis/efectos de los fármacos , Cafeína/farmacología , Especies Reactivas de Oxígeno/metabolismo , Cafeína/análogos & derivados , Muerte Celular/efectos de los fármacos , Etopósido/farmacología , Humanos , Células U937/efectos de los fármacos , Células U937/patología , Células U937/ultraestructuraRESUMEN
When studied in vitro, mitochondrial permeability transition (MPT) is associated with an increase in mitochondrial permeability to solutes up to 1500 Da in mass and a loss of electrical potential difference across the inner mitochondrial membrane (Deltapsimit). The MPT has been implicated as being important in cellular calcium homeostasis, autophagy and cell death via necrosis and apoptosis. Thus, it is important to develop a valid technique for accurate measurement of this phenomenon in intact cells. We developed a procedure for the detection of MPT in intact cells that avoids the disadvantages associated with earlier approaches. In this new technique, unmodified (green-fluorescent) calcein is simultaneously introduced into the cytosol of millions of cells by the process of pinocytic loading and, to identify the position of individual mitochondria and to measure Deltapsimit, the cells are counter-stained with a red-fluorescing potentiometric dye. Using this approach with a variety of cell types, we demonstrate that cytosolic calcein is excluded from normal polarized mitochondria but enters them during MPT. This technique may be valuable in studies investigating the cellular functions of MPT.
Asunto(s)
Fluoresceínas/análisis , Colorantes Fluorescentes/análisis , Microscopía Confocal , Mitocondrias/metabolismo , Pinocitosis , Células HeLa/metabolismo , Células HeLa/ultraestructura , Humanos , Membranas Intracelulares/metabolismo , Células Jurkat/metabolismo , Células Jurkat/ultraestructura , Mitocondrias/ultraestructura , Permeabilidad , Rodaminas/análisis , Células U937/metabolismo , Células U937/ultraestructuraRESUMEN
Molecular cytogenetics is helpful to identify complex and cryptic genomic changes in malignancy. Human leukemic cell lines are an important tool for advancements of biological research on malignant cells, one critical step being characterization of genomic changes. We used fluorescence in situ hybridization and comparative genomic hybridization to refine karyotypic interpretation of the diffuse histiocytic lymphoma derived U937-1 cell line. From this integrated approach, chromosome material involved in nine karyotypic markers and in unbalanced translocations could be identified. Moreover, a previously undetected amplicon emerged within band 6p21. The U937-I is a new in vitro model to study genome amplification and unknown recombinations in leukemic cells, such as those involving the centromeric region of chromosome 1.
Asunto(s)
Cromosomas Humanos Par 6/genética , Amplificación de Genes , Hibridación Fluorescente in Situ , Hibridación de Ácido Nucleico , Células U937/ultraestructura , Aneuploidia , Cromosomas Humanos/genética , Cromosomas Humanos Par 1/genética , Cromosomas Humanos Par 1/ultraestructura , Cromosomas Humanos Par 6/ultraestructura , Humanos , CariotipificaciónRESUMEN
Exposure of U937 human leukemic cells to the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) induces their differentiation into monocyte/macrophage-like cells. This terminal differentiation is associated with a resistant phenotype to apoptosis induced by the topoisomerase II inhibitor etoposide. The inhibition occurs upstream of the mitochondrial release of cytochrome c and the activation of procaspase-2, -3, -6, -7, -8, and -9. By using cell-free systems, it was demonstrated that the mitochondrial pathway to cell death that involves mitochondrial membrane depolarization, cytochrome c release and cytosolic activation of procaspases by cytochrome c/dATP remains functional in TPA-differentiated U937 cells. Accordingly, 2 drugs recently shown to target the mitochondria, namely lonidamine and arsenic trioxide, bypass the resistance of TPA-differentiated U937 cells to classical anticancer drugs. Cell death induced by the 2 compounds is associated with mitochondrial membrane depolarization, release of cytochrome c and Smac/Diablo from the mitochondria, activation of caspases, poly(ADP-ribose) polymerase cleavage and internucleosomal DNA fragmentation. Moreover, the decreased glutathione content associated with the differentiation process amplifies the ability of arsenic trioxide to activate the mitochondrial pathway to cell death. Similar results were obtained by comparing undifferentiated and TPA-differentiated human HL60 leukemic cells. These data demonstrate that mitochondria-targeting agents bypass the resistance to classical anticancer drugs induced by TPA-mediated leukemic cell differentiation. (Blood. 2001;97:3931-3940)
Asunto(s)
Apoptosis/efectos de los fármacos , Arsenicales/farmacología , Indazoles/farmacología , Leucemia/patología , Mitocondrias/efectos de los fármacos , Óxidos/farmacología , Acetato de Tetradecanoilforbol/farmacología , Antineoplásicos/farmacología , Trióxido de Arsénico , Caspasas/efectos de los fármacos , Caspasas/fisiología , Diferenciación Celular/efectos de los fármacos , Sistema Libre de Células/efectos de los fármacos , Sistema Libre de Células/enzimología , Resistencia a Medicamentos , Etopósido/farmacología , Humanos , Mitocondrias/enzimología , Proteínas Proto-Oncogénicas c-bcl-2/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Células U937/efectos de los fármacos , Células U937/enzimología , Células U937/ultraestructuraRESUMEN
Fluorescence in situ hybridization (FISH) combined with high-resolution cytometry was used to determine the topographic characteristics of the centromeric heterochromatin (of the chromosomes 6, 8, 9, 17) and the tumor suppressor gene TP53 (which is located on chromosome 17) in cells of the human leukemia cell lines ML-1 and U937. Analysis was performed on cells that were either untreated or irradiated with gamma rays and incubated for different intervals after exposure. Compared to untreated cells, homologous centromeres and the TP53 genes were found closer to each other and also closer to the nuclear center 2 h after irradiation. The spatial relationship between genetic elements returned to that of the unirradiated controls during the next 2-3 h. Statistical evaluation of our experimental results shows that homologous centromeres and the homologous genes are positioned closer to each other 2 h after irradiation because they are localized closer to the center of the nucleus (probably due to more pronounced decondensation of the chromatin related to repair). This radial movement of genetic loci, however, is not connected with repair of DSBs by processes involving homologous recombination, because the angular distribution of homologous sequences remains random after irradiation.
Asunto(s)
Núcleo Celular/ultraestructura , Cromosomas Humanos/ultraestructura , Genes/efectos de la radiación , Leucemia/patología , Células Madre Neoplásicas/ultraestructura , Células U937/ultraestructura , Núcleo Celular/química , Núcleo Celular/efectos de la radiación , Centrómero/química , Centrómero/efectos de la radiación , Centrómero/ultraestructura , Cromosomas Humanos/química , Cromosomas Humanos/efectos de la radiación , Daño del ADN , Reparación del ADN , Rayos gamma , Heterocromatina/química , Heterocromatina/efectos de la radiación , Heterocromatina/ultraestructura , Humanos , Procesamiento de Imagen Asistido por Computador , Hibridación Fluorescente in Situ , Leucemia/genética , Células Madre Neoplásicas/química , Células Madre Neoplásicas/efectos de la radiación , Recombinación Genética , Células U937/química , Células U937/efectos de la radiaciónRESUMEN
Within five minutes of macrophage infection by Legionella pneumophila, the bacterium responsible for Legionnaires' disease, elements of the rough endoplasmic reticulum (RER) and mitochondria attach to the surface of the bacteria-enclosed phagosome. Connecting these abutting membranes are tiny hairs, which are frequently periodic like the rungs of a ladder. These connections are stable and of high affinity - phagosomes from infected macrophages remain connected to the ER and mitochondria (as they were in situ) even after infected macrophages are homogenized. Thin sections through the plasma and phagosomal membranes show that the phagosomal membrane is thicker (72+/-2 A) than the ER and mitochondrial membranes (60+/-2 A), presumably owing to the lack of cholesterol, sphingolipids and glycolipids in the ER. Interestingly, within 15 minutes of infection, the phagosomal membrane changes thickness to resemble that of the attached ER vesicles. Only later (e.g. after six hours) does the ER-phagosome association become less frequent. Instead ribosomes stud the former phagosomal membrane and L. pneumophila reside directly in the rough ER. Examination of phagosomes of various L. pneumophila mutants suggests that this membrane conversion is a four-stage process used by L. pneumophila to establish itself in the RER and to survive intracellularly. But what is particularly interesting is that L. pneumophila is exploiting a poorly characterized naturally occurring cellular process.
Asunto(s)
Membranas Intracelulares/microbiología , Membranas Intracelulares/fisiología , Legionella pneumophila/fisiología , Legionella pneumophila/ultraestructura , Fagosomas/microbiología , Fagosomas/fisiología , Proteínas Bacterianas/fisiología , Proteínas Portadoras/genética , Proteínas Portadoras/fisiología , Fraccionamiento Celular , Membrana Celular/genética , Membrana Celular/microbiología , Membrana Celular/fisiología , Membrana Celular/ultraestructura , Bacterias Grampositivas/patogenicidad , Bacterias Grampositivas/fisiología , Bacterias Grampositivas/ultraestructura , Humanos , Membranas Intracelulares/ultraestructura , Legionella pneumophila/genética , Metabolismo de los Lípidos , Lisosomas/fisiología , Proteínas de la Membrana/genética , Proteínas de la Membrana/fisiología , Microscopía Electrónica , Mitocondrias/genética , Mitocondrias/microbiología , Mitocondrias/fisiología , Mitocondrias/ultraestructura , Chaperonas Moleculares/genética , Chaperonas Moleculares/fisiología , Mutación , Orgánulos , Fagosomas/genética , Fagosomas/ultraestructura , Ribosomas/fisiología , Factores de Tiempo , Células U937/microbiología , Células U937/fisiología , Células U937/ultraestructuraRESUMEN
The mixed-lineage leukemia gene (MLL) is associated with more than 25 chromosomal translocations involving band 11q23 in diverse subtypes of human acute leukemia. Conditional expression of a 50 kDa amino terminal fragment spanning the AT hook motifs of MLL (MLL3AT) causes cell cycle arrest, upregulation of p21Cip1 and p27KiP1 and partial monocytic differentiation of the monoblastic U937 cell line, suggesting a major role for MLL3AT in MLL-AF9-induced myelomonocytic differentiation. In this study, we analyzed the subcellular localization of conditionally expressed MLL3AT in both U937 and HeLa cell lines. Immunofluorescence staining, confocal laser scanning microscopy and immunoelectron microscopy indicated that MLL3AT, like endogenous MLL, localized in the nucleoplasm in a punctate pattern of distribution, including regions attached to the nuclear envelope and the periphery of the nucleolus. We found that MLL3AT and endogenous MLL were present in interphase nuclear matrices and colocalized with topoisomerase II to mitotic chromosomal scaffolds. Nucleoplasm and nucleolar localization was observed even for MLL-AF9 and MLL-AF4 conditionally expressed chimeric proteins, suggesting a common target conferred by the amino terminus of MLL to many if not all the chimeric MLL proteins. The nuclear matrix/scaffold association suggests a role for the amino terminus of MLL in the modulation of chromatin structure, leading to epigenetic effects on the maintenance of gene expression.
Asunto(s)
Nucléolo Celular/metabolismo , Estructuras Cromosómicas/metabolismo , Proteínas de Unión al ADN/metabolismo , Matriz Nuclear/metabolismo , Señales de Clasificación de Proteína , Proto-Oncogenes , Factores de Transcripción , Transporte Activo de Núcleo Celular , Secuencias de Aminoácidos , Ciclo Celular , Proteínas Cromosómicas no Histona/metabolismo , ADN-Topoisomerasas de Tipo II/análisis , Proteínas de Unión al ADN/fisiología , Técnica del Anticuerpo Fluorescente Indirecta , Células HeLa/metabolismo , Células HeLa/ultraestructura , N-Metiltransferasa de Histona-Lisina , Humanos , Microscopía Confocal , Microscopía Inmunoelectrónica , Mitosis , Proteína de la Leucemia Mieloide-Linfoide , Proteínas de Neoplasias/metabolismo , Señales de Localización Nuclear , Estructura Terciaria de Proteína , Transporte de Proteínas , Proteínas Recombinantes de Fusión/metabolismo , Transfección , Células U937/metabolismo , Células U937/ultraestructuraRESUMEN
We previously reported a novel monoclonal antibody (MAb), designated mNI-11, recognizing an adhesion-associated antigen distinct from any previously reported ones. In this article, this adhesion-associated antigen with a molecular weight of about 97 kDa was found to be strongly expressed on human umbilical vein endothelial cells (HUVECs) by fluorescence-activated cell sorter (FACS) analysis. Expression of this antigen on HUVECs was slightly increased in response to the exposure to tumor necrosis factor-alpha (TNF-alpha) or phorbol myristate acetate (PMA). As a biological function exerted by this antigen, it was of great interest that immobilized mNI-11 directly and rapidly enhanced the spread formation of HUVECs, whereas MAbs binding other adhesion-associated antigens such as mNI-58A (anti-CD11a), L130 (anti-CD18), L133.1 (anti-CD31), L178 (anti-CD44), L25.3 (anti-CD49d), or LB-2 (anti-CD54) did not carry such activity under the same conditions. The HUVECs spread formation enhanced by mNI-11 was completely blocked in the presence of a microfilament formation inhibitor, cytochalasin D (CyD), a Ca2+ calmodulin inhibitor, W-7, EDTA, and was partially blocked by a microtubule formation inhibitor, nocodazole, a protein kinase C (PKC) inhibitor, H-7, and a protein synthesis inhibitor, cycloheximide (CHX). However, a protein tyrosine kinase (PTK) inhibitor, genistein, did not affect the spread formation under the same conditions. Taken together, it was suggested that the spread formation of HUVECs enhanced by mNI-11 was mainly associated with the influx of Ca2+ and microfilament reorganization. In addition, the novel property associated with mNI-11 to enhance the spread formation of HUVECs was possibly mediated through its reaction against a unique epitope on HUVECs.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Moléculas de Adhesión Celular/inmunología , Endotelio Vascular/citología , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacología , Citoesqueleto de Actina/efectos de los fármacos , Citoesqueleto de Actina/fisiología , Citoesqueleto de Actina/ultraestructura , Anticuerpos Monoclonales/aislamiento & purificación , Anticuerpos Monoclonales/farmacología , Especificidad de Anticuerpos , Calcio/fisiología , Calmodulina/antagonistas & inhibidores , Moléculas de Adhesión Celular/fisiología , Tamaño de la Célula , Células Cultivadas/efectos de los fármacos , Células Cultivadas/ultraestructura , Quelantes/farmacología , Cicloheximida/farmacología , Citocalasina D/farmacología , Ácido Edético/farmacología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/inmunología , Inhibidores Enzimáticos/farmacología , Epítopos/inmunología , Citometría de Flujo , Genisteína/farmacología , Células HL-60/efectos de los fármacos , Células HL-60/ultraestructura , Humanos , Células K562/efectos de los fármacos , Células K562/ultraestructura , Microtúbulos/efectos de los fármacos , Nocodazol/farmacología , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/fisiología , Inhibidores de la Síntesis de la Proteína/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/fisiología , Sulfonamidas/farmacología , Acetato de Tetradecanoilforbol/farmacología , Factor de Necrosis Tumoral alfa/farmacología , Células U937/efectos de los fármacos , Células U937/ultraestructura , Venas UmbilicalesRESUMEN
2',3'-Dideoxycytidine is a powerful in vitro inhibitor of human immunodeficiency virus and is currently used in the treatment of acquired immunodeficiency syndrome. A long-term exposure of U937 monoblastoid cells to dideoxycytidine induces the selection of drug-resistant cells (U937-R). In previous studies, we investigated some important biochemical properties and functional activities, such as basal respiration, protein kinase C activity, superoxide anion release, and the level of reduced glutathione, which were found to be higher in the drug-resistant cell line, compared to the parental one. In the present study, we evaluated the response of the two cell lines to the induction of apoptosis by treatment with staurosporine and okadaic acid, which interfere with the protein kinase and phosphatase pathways, respectively. Moreover, knowing that GSH plays a crucial role in the regulation of nitric oxide-dependent apoptosis, U937-R and parental lines have been treated with SIN-1, which is known to generate significant amounts of O2 and nitric oxide. Resistant and parental cells have been analysed by light and electron microscopy and agarose gel electrophoresis of isolated DNA has been performed. The obtained results demonstrate a different susceptibility of U937-R cell line to apoptosis induced with the three triggers. U937-R cells show more advanced apoptotic features if compared with parental cells, after staurosporine treatment. Differently, the okadaic acid does not induce a different behaviour in the two models. On the contrary, the agent SIN-1 determines an increased number of apoptotic cells in the U937 line. The results suggest that a higher level of protein kinase C and glutathione could prevent programmed cell death in U937-R.
Asunto(s)
Fármacos Anti-VIH/farmacología , Apoptosis/efectos de los fármacos , Zalcitabina/farmacología , ADN/análisis , Resistencia a Medicamentos , Electroforesis en Gel de Agar , Inhibidores Enzimáticos/farmacología , Glutatión/metabolismo , Humanos , Molsidomina/análogos & derivados , Molsidomina/farmacología , Donantes de Óxido Nítrico/farmacología , Ácido Ocadaico/farmacología , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas , Estaurosporina/farmacología , Células U937/efectos de los fármacos , Células U937/metabolismo , Células U937/ultraestructuraRESUMEN
Phosphatidylinositide 3-kinase (PI3K) is a key enzyme implicated in intracellular signaling of diverse cellular responses including receptor-mediated responses and neutrophil activation. Several PI3K subunits have been cloned and shown to be localized to plasma membrane receptors, the cytosol, or intracellular vesicles or caveolae. We report the localization of PI3K to a distinct intracellular site, cytoplasmic lipid bodies, in leukocytes. In U937 monocyte cells, PI3K p85 regulatory and p110beta catalytic subunits were localized to lipid bodies by immunocytochemistry and/or immunoblotting and enzyme assays of subcellular fractions. In RAW murine macrophages, p55, p85alpha, and p85beta PI3K subunits were present at isolated lipid bodies. PI3K p85 was also shown to colocalize and, by co-immunoprecipitation, to be physically associated with phosphorylated Lyn kinase in lipid bodies induced to form in human polymorphonuclear leukocytes. These findings, therefore, indicate a novel site for PI3K compartmentalization and suggest that PI3K-mediated signaling is active within cytoplasmic lipid bodies in leukocytes.
Asunto(s)
Ácidos Araquidónicos/metabolismo , Citoplasma/enzimología , Macrófagos/enzimología , Complejos Multienzimáticos/metabolismo , Proteínas de Neoplasias/metabolismo , Neutrófilos/enzimología , Fosfatidilinositol 3-Quinasas/metabolismo , Células U937/enzimología , Animales , Dimerización , Humanos , Sustancias Macromoleculares , Macrófagos/ultraestructura , Ratones , Neutrófilos/ultraestructura , Fosfatidilinositol 3-Quinasas/química , Multimerización de Proteína , Fracciones Subcelulares/enzimología , Células U937/ultraestructura , Familia-src Quinasas/metabolismoRESUMEN
We describe a simple procedure to prepare cultured cells in suspension to analyse elemental content at the cellular level by electron probe X-ray microanalysis. Cells cultured in suspension were deposited onto polycarbonate tissue, culture plate well inserts, centrifuged at low g, washed to remove the extracellular medium, cryofixed and freeze-dried, and analysed in the scanning mode of a scanning electron microscope. We tested the effect of different washing solutions (150 mM ammonium acetate, 300 mM sucrose, and distilled water) on the elemental content of cultured cells in suspension. The results demonstrated that distilled water was the best washing solution to prepare cultured cells. In addition, the low Na content, high K content and high K/Na ratio of the cells indicated that this procedure, based on the centrifugation at low g followed by cryopreparation, constitutes a satisfactory method to prepare cultured cells in suspension. We also investigated the effects of different accelerating voltages on X-ray signal collection. The results showed that moderate accelerating voltages, i.e. 10-11 kV, should be used to analyse whole cells in the scanning mode of the scanning electron microscope. We show that this method of preparation makes it possible to prepare cryosections of the cultured cells, thus permitting analysis of the elemental content at the subcellular level, i.e. nucleus, cytoplasm and mitochondria, using a scanning transmission electron microscope.
Asunto(s)
Criopreservación/métodos , Microanálisis por Sonda Electrónica/métodos , Microscopía Electrónica/métodos , Células U937/química , Compartimento Celular , Humanos , Microscopía Electrónica de Rastreo/métodos , Células U937/ultraestructuraRESUMEN
Apoptotic micronuclei have been studied, in different cell types, from a morphologic and functional point of view. Conventional electron microscopy, in various staining conditions, selective cytochemistry for DNA, and freeze fracture for the analysis of chromatin fiber organization and size were performed. In situ TdT and Pol I immunofluorescent techniques were carried out to detect double- and single-strand DNA breaking points by confocal laser scanning microscopy. Apoptotic cell ultrathin cryosections were also performed and were analysed by field emission in lens scanning electron microscopy. Double/single strand massively cleaved DNA was detected in micronuclei, with a highly supercoiled, uniformly packed, very dense arrangement.