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1.
Planta Med ; 84(17): 1255-1264, 2018 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-29906814

RESUMEN

Magnoflorine, a major bioactive metabolite isolated from Tinospora crispa, has been reported for its diverse biochemical and pharmacological properties. However, there is little report on its underlying mechanisms of action on immune responses, particularly on macrophage activation. In this study, we aimed to investigate the effects of magnoflorine, isolated from T. crispa on the pro-inflammatory mediators generation induced by LPS and the concomitant NF-κB, MAPKs, and PI3K-Akt signaling pathways in U937 macrophages. Differentiated U937 macrophages were treated with magnoflorine and the release of pro-inflammatory mediators was evaluated through ELISA, while the relative mRNA expression of the respective mediators was quantified through qRT-PCR. Correspondingly, western blotting was executed to observe the modulatory effects of magnoflorine on the expression of various markers related to NF-κB, MAPK and PI3K-Akt signaling activation in LPS-primed U937 macrophages. Magnoflorine significantly enhanced the upregulation of TNF-α, IL-1ß, and PGE2 production as well as COX-2 protein expression. Successively, magnoflorine prompted the mRNA transcription level of these pro-inflammatory mediators. Magnoflorine enhanced the NF-κB activation by prompting p65, IκBα, and IKKα/ß phosphorylation as well as IκBα degradation. Besides, magnoflorine treatments concentration-dependently augmented the phosphorylation of JNK, ERK, and p38 MAPKs as well as Akt. The immunoaugmenting effects were further confirmed by investigating the effects of magnoflorine on specific inhibitors, where the treatment with specific inhibitors of NF-κB, MAPKs, and PI3K-Akt proficiently blocked the magnoflorine-triggered TNF-α release and COX-2 expression. Magnoflorine furthermore enhanced the MyD88 and TLR4 upregulation. The results suggest that magnoflorine has high potential on augmenting immune responses.


Asunto(s)
Aporfinas/farmacología , Factores Inmunológicos/farmacología , Inflamación/inducido químicamente , Lipopolisacáridos/farmacología , Factor 88 de Diferenciación Mieloide/metabolismo , Transducción de Señal/efectos de los fármacos , Células U937/efectos de los fármacos , Citocinas/metabolismo , Dinoprostona/metabolismo , Humanos , Immunoblotting , Reacción en Cadena en Tiempo Real de la Polimerasa , Células U937/fisiología
2.
PLoS One ; 11(8): e0161494, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-27536992

RESUMEN

SIRT6 is a protein deacetylase, involved in various intracellular processes including suppression of glycolysis and DNA repair. Aldose Reductase (AR), first enzyme of polyol pathway, was proposed to be indirectly associated to these SIRT6 linked processes. Despite these associations, presence of SIRT6 based regulation of AR still remains ambiguous. Thus, regulation of AR expression by SIRT6 was investigated under hyperosmotic stress. A unique model of osmotic stress in U937 cells was used to demonstrate the presence of a potential link between SIRT6 and AR expression. By overexpressing SIRT6 in HeLa cells under hyperosmotic stress, its role on upregulation of AR was revealed. In parallel, increased SIRT6 activity was shown to upregulate AR in U937 cells under hyperosmotic milieu by using pharmacological modulators. Since these modulators also target SIRT1, binding of the inhibitor, Ex-527, specifically to SIRT6 was analyzed in silico. Computational observations indicated that Ex-527 may also target SIRT6 active site residues under high salt concentration, thus, validating in vitro findings. Based on these evidences, a novel regulatory step by SIRT6, modifying AR expression under hyperosmotic stress was presented and its possible interactions with intracellular machinery was discussed.


Asunto(s)
Aldehído Reductasa/metabolismo , Células HeLa/fisiología , Presión Osmótica/fisiología , Sirtuinas/fisiología , Células U937/fisiología , Simulación por Computador , Regulación Enzimológica de la Expresión Génica/fisiología , Células HeLa/enzimología , Células HeLa/metabolismo , Humanos , Immunoblotting , Técnicas In Vitro , Simulación del Acoplamiento Molecular , Células U937/enzimología , Células U937/metabolismo , Regulación hacia Arriba
3.
Environ Toxicol Pharmacol ; 32(2): 285-95, 2011 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-21843810

RESUMEN

Benzene is a well-known environmental pollutant that can induce hematotoxicity, aplastic anemia, acute myelogenous leukemia, and lymphoma. Benzene toxicity is likely mediated through metabolites induced by means of multiple pathways. Although benzene metabolites are known to induce oxidative stress and disrupt the cell cycle, the mechanism underlying leukemogenesis is not fully understood. The aim of this study was to analyze the genome-wide expression profiles of human promyelocytic leukemia HL-60 cells that had been exposed to benzene and its metabolites. This was carried out using whole human genome oligonucleotide microarrays to ascertain potential biomarkers. Genes that were differentially expressed (>1.5-fold and p-values <0.05) after exposure to benzene (BZ), hydroquinone (HQ), and 1,4-benzoquinone (BQ) were then classified with GO, KEGG and GSEA pathway annotation. All genes that were identified were then functionally categorized as being involved in the cell cycle, the p53 signaling pathway, apoptosis, the MAPK signaling pathway, or the T cell receptor signaling pathway. Functionally important genes were further validated by means of real-time RT-PCR. The results showed that EGR1, PMAIP1, AR, CCL2, CD69, HSPA8, SLC7A11, HERPUD1, ELK1, and MKI57 genes altered their expression profiles. Similar expression profiles were also found in human erythromyeloblastoid leukemia K562 cells and in human leukemic monocyte lymphoma U937 cells. In conclusion, gene expression profiles along with GO, KEGG and GSEA pathway annotation analysis have provided an insight into the leukemogenesis as well as highlighted potential gene-based biomarkers of human leukemia cell lines when they are exposed to benzene and its metabolites.


Asunto(s)
Benceno/toxicidad , Benzoquinonas/toxicidad , Expresión Génica/efectos de los fármacos , Células HL-60/efectos de los fármacos , Hidroquinonas/toxicidad , Células K562/efectos de los fármacos , Células U937/efectos de los fármacos , Benceno/metabolismo , Benzoquinonas/metabolismo , Biomarcadores/metabolismo , Análisis por Conglomerados , Contaminantes Ambientales/metabolismo , Contaminantes Ambientales/toxicidad , Perfilación de la Expresión Génica , Células HL-60/fisiología , Humanos , Hidroquinonas/metabolismo , Células K562/fisiología , Leucemia/genética , Análisis por Micromatrices , Transducción de Señal/genética , Células U937/fisiología
4.
J Cell Physiol ; 224(3): 775-85, 2010 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-20432471

RESUMEN

Caffeine attenuated invasion of human leukemia U937 cells with characteristic of decreased protein expression and mRNA levels of matrix metalloproteinase-2 (MMP-2) and MMP-9. Down-regulation of MMP-2 and MMP-9 in U937 cells was abrogated by abolishment of caffeine-elicited increase in intracellular Ca(2+) concentration and ROS generation. Pretreatment with BAPTA-AM (Ca(2+) chelator) and N-acetylcysteine (ROS scavenger) abolished caffeine-induced ERK inactivation and p38 MPAK activation. Moreover, caffeine treatment led to MAPK phosphatase-1 (MKP-1) down-regulation and protein phosphatase 2A catalytic subunit (PP2Ac) up-regulation, which were involved in cross-talk between p38 MAPK and ERK. Transfection of constitutively active MEK1 or pretreatment with SB202190 (p38 MAPK inhibitor) restored MMP-2 and MMP-9 protein expression in caffeine-treated cells. Caffeine treatment repressed ERK-mediated c-Fos phosphorylation but evoked p38 MAPK-mediated c-Jun phosphorylation. Knock-down of c-Fos and c-Jun by siRNA reflected that c-Fos counteracted the effect of c-Jun on MMP-2/MMP-9 down-regulation. Taken together, our data indicate that MMP-2/MMP-9 down-regulation in caffeine-treated U937 cells is elicited by Ca(2+)/ROS-mediated suppression of ERK/c-Fos pathway and activation of p38 MAPK/c-Jun pathway.


Asunto(s)
Cafeína/farmacología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Proteínas Proto-Oncogénicas c-fos/metabolismo , Células U937/efectos de los fármacos , Animales , Calcio/metabolismo , Regulación hacia Abajo , Activación Enzimática , Humanos , Inhibidores de Fosfodiesterasa/farmacología , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/fisiología , Células U937/fisiología
5.
BMB Rep ; 42(2): 96-100, 2009 Feb 28.
Artículo en Inglés | MEDLINE | ID: mdl-19250610

RESUMEN

Aromatic (ar)-turmerone from turmeric oil displays anti-tumorigenesis activity that includes inhibited cell proliferation. This study investigated ar-turmerone-mediated apoptotic protein activation in human lymphoma U937 cells. Ar-turmerone treatment inhibited U937 cell viability in a concentration-dependent fashion, with inhibition exceeding 84%. Moreover, the treatment produced nucleosomal DNA fragmentation and the percentage of sub-diploid cells increased in a concentration-dependent manner; both are hallmarks of apoptosis. The apoptotic effect of ar-turmerone was associated with the induction of Bax and p53 proteins, rather than Bcl-2 and p21. Activation of mitochondrial cytochrome c and caspase-3 demonstrated that the activation of caspases accompanied the apoptotic effect of ar-turmerone, which mediated cell death. These results suggest that the apoptotic effect of ar-turmerone on U937 cells may involve caspase-3 activation through the induction of Bax and p53, rather than Bcl-2 and p21.


Asunto(s)
Curcuma/química , Cetonas , Extractos Vegetales/química , Aceites de Plantas , Tolueno/análogos & derivados , Células U937 , Antioxidantes/química , Apoptosis/efectos de los fármacos , Caspasa 3/metabolismo , Supervivencia Celular/efectos de los fármacos , Citocromos c/metabolismo , Activación Enzimática , Humanos , Cetonas/química , Cetonas/farmacología , Aceites de Plantas/química , Aceites de Plantas/farmacología , Sesquiterpenos , Tolueno/química , Tolueno/farmacología , Células U937/efectos de los fármacos , Células U937/fisiología , Proteína X Asociada a bcl-2/genética , Proteína X Asociada a bcl-2/metabolismo
6.
Int J Hyperthermia ; 24(8): 613-22, 2008 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-18608577

RESUMEN

In this study, to better understand the molecular mechanism underlying cellular responses to mild hyperthermia, we investigated gene expression patterns and genetic networks in human myelomonocytic lymphoma U937 cells using high-density oligonucleotide microarrays and computational gene expression analysis tools. The cells were incubated at 41 degrees C for 30 min (mild hyperthermia treatment) and then at 37 degrees C for 0-6 h. Although the mild hyperthermia treatment of the cells did not induce apoptosis, significant increases in the protein expression levels of heat shock proteins (HSPs), namely, Hsp27, Hsp40 and Hsp70, were observed following the activation of heat shock factor-1. Of the 22,283 probe sets analyzed, 423 probe sets were up-regulated and 515 probe sets were down-regulated by >1.5-fold in the cells 3 h post-treatment. Computational gene network analysis demonstrated that the significant genetic network A that contained many HSPs such as DNAJB1, HSPA1A, and HSPA1B was associated with cellular function and maintenance, post-transcriptional modification, or protein folding. Moreover, the significant genetic network B whose core contained v-myc myelocytomatosis viral oncogene homolog (MYC) was associated with cell morphology, cell cycle, and cellular development. The expression levels of nine selected genes were comparable to those determined by microarray analysis with real-time quantitative PCR assay. The present results indicate that mild hyperthermia affects the expression of a large number of genes and provides additional novel insights into the molecular basis of mild hyperthermia in cells.


Asunto(s)
Fiebre/fisiopatología , Regulación Neoplásica de la Expresión Génica , Redes Reguladoras de Genes , Linfoma/genética , Células U937/fisiología , Perfilación de la Expresión Génica , Humanos , Datos de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos
7.
Toxicol Appl Pharmacol ; 220(2): 113-24, 2007 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-17306317

RESUMEN

The human myeloid cell line U937 was evaluated as an in vitro test system to identify contact sensitizers in order to develop alternatives to animal tests for the cosmetic industry. Specific culture conditions (i.e., presence of interleukin-4, IL-4) were applied to obtain a dendritic cell-like phenotype. In the described test protocol, these cells were exposed to test chemicals and then analyzed by flow cytometry for CD86 expression and by quantitative real-time reverse transcriptase-polymerase chain reaction for IL-1beta and IL-8 gene expressions. Eight sensitizers, three non-sensitizers and five oxidative hair dye precursors were examined after 24-, 48- and 72-h exposure times. Test item-specific modulations of the chosen activation markers (CD86, IL-1beta and IL-8) suggest that this U937 activation test could discriminate test items classified as contact sensitizers or non-sensitizers in the local lymph node assay in mice (LLNA). More specifically, a test item can be considered as a potential sensitizer when it significantly induced the upregulation of the expression of at least two markers. Using this approach, we could correctly evaluate the dendritic cell (DC) activation potential for 15 out of 16 tested chemicals. We conclude that the U937 activation test may represent an useful tool in a future in vitro test battery for predicting sensitizing properties of chemicals.


Asunto(s)
Alérgenos/toxicidad , Dermatitis por Contacto/patología , Células U937/fisiología , Animales , Antígeno B7-2/inmunología , Supervivencia Celular , Medios de Cultivo , Células Dendríticas/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Fluoresceína-5-Isotiocianato , Colorantes Fluorescentes , Tinturas para el Cabello/toxicidad , Humanos , Interleucina-1beta/biosíntesis , Interleucina-8/metabolismo , Ensayo del Nódulo Linfático Local , ARN/biosíntesis , ARN/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
8.
Methods Mol Med ; 122: 123-48, 2006.
Artículo en Inglés | MEDLINE | ID: mdl-16511979

RESUMEN

Human monocytes and macrophages, which are also called mononuclear phagocytes, represent a major arm of the innate immune system. These cells not only protect against infection but are also central to tissue remodeling and production of chemokines, cytokines, and growth factors. Tissue macrophages reside in the human placenta and uterine decidua throughout pregnancy, where they comprise part of the host defense network and facilitate placental and extraembryonic development. The purpose of this chapter is to describe methods for establishing useful models of human uteroplacental macrophages: (1) differentiated U937 myelomonocytic cells, (2) peripheral blood monocytes, (3) peripheral blood monocyte-derived macrophages, (4) decidual macrophages, and (5) placental macrophages.


Asunto(s)
Técnicas de Cultivo de Célula/métodos , Separación Celular/métodos , Macrófagos/fisiología , Placenta/inmunología , Útero/inmunología , Medios de Cultivo , Endometrio/inmunología , Femenino , Humanos , Leucocitos Mononucleares , Macrófagos/citología , Monocitos/citología , Embarazo , Células U937/fisiología
9.
Life Sci ; 75(5): 599-609, 2004 Jun 18.
Artículo en Inglés | MEDLINE | ID: mdl-15158369

RESUMEN

Several observational studies have shown that estrogen replacement therapy decreases cardiovascular mortality and morbidity in postmenopausal women. However, The Women's Health Initiative (WHI) study has found that women receiving estrogen plus progestin had a significantly higher risk of breast cancer, coronary heart disease, stroke, and pulmonary embolus. In the present study, we examined whether estrogen prevents mechanisms that relate to plaque formation by inhibiting monocyte adhesion to endothelial cells. ECV304 cells, an endothelial cell line that normally expresses minimal estrogen receptor (ER)alpha, were transfected with an ERalpha expression plasmid. Treatment with tumor necrosis factor (TNF)-alpha increased expression of vascular cell adhesion molecule (VCAM)-1 mRNA, activation of nuclear factor-kappaB (NF-kappaB), and U937 cell adhesion in ECV304 cells. These effects of TNF-alpha were not significantly inhibited by pretreatment of native ECV304 cells with 17beta-estradiol (E(2)). In ECV304 cells overexpressing ERalpha, E(2) significantly inhibited the effects of TNF-alpha on NF-kappaB activation, VCAM-1 expression, and U937 cell adhesion. These findings suggest E(2) suppresses inflammatory cell adhesion to vascular endothelial cells that possess functional estrogen receptors. The mechanism of suppression may involve inhibition of NF-kappaB-mediated up-regulation of VCAM-1 expression induced by atherogenic stimuli. E(2) may prevent plaque formation, as first stage of atheroscrelosis through inhibiting adhesion monocytes to endothelial cell. Actions of estrogen replacement therapy can be assessed in terms of densities of functional ERalpha.


Asunto(s)
Endotelio Vascular/efectos de los fármacos , Estradiol/farmacología , Monocitos/efectos de los fármacos , Receptores de Estradiol , Adhesión Celular/efectos de los fármacos , Adhesión Celular/fisiología , Línea Celular , Relación Dosis-Respuesta a Droga , Endotelio Vascular/metabolismo , Endotelio Vascular/patología , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Monocitos/metabolismo , Monocitos/patología , FN-kappa B/biosíntesis , FN-kappa B/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Estradiol/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transfección , Factor de Necrosis Tumoral alfa/farmacología , Células U937/efectos de los fármacos , Células U937/fisiología , Regulación hacia Arriba , Molécula 1 de Adhesión Celular Vascular/genética , Molécula 1 de Adhesión Celular Vascular/metabolismo
10.
Biosens Bioelectron ; 19(4): 331-6, 2003 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-14615091

RESUMEN

A single-cell detector is described that combines the natural signal amplification of whole-cell biosensors with the flexibility and specificity of immunological recognition. An immune cell that expresses receptors for the constant region of immunoglobulin G (IgG) is loaded with a Ca(2+)-indicating dye and with antibodies directed against the protein of interest. Introduction of a multivalent protein antigen causes cross-linking of the receptors, which results in a detectable increase in the concentration of cytosolic Ca(2+). Some immune cell lines respond to stimulation with oscillations in their cytosolic Ca(2+) levels that complicate their use as detectors. The human monocytic cell line U-937, when treated with the cytokine interferon-gamma, produces a large, short-lived Ca(2+) signal in response to cross-linking of its high-affinity IgG receptors. U-937 was therefore chosen for development as an immunity-based detector. Human and rabbit antibodies are found to effectively stimulate the cell, causing a prompt and transient response. The cell is able to respond to repeated stimulation, though the response diminishes during rapid stimulation. Ovalbumin can be detected in micromolar concentrations. Possible fundamental constraints on the size of a detectable analyte are discussed.


Asunto(s)
Técnicas Biosensibles/métodos , Calcio/análisis , Técnicas de Cultivo de Célula/métodos , Inmunoensayo/métodos , Ovalbúmina/análisis , Proteínas/análisis , Espectrometría de Fluorescencia/métodos , Células U937/fisiología , Humanos , Ovalbúmina/inmunología , Proteínas/inmunología , Sensibilidad y Especificidad
11.
J Leukoc Biol ; 74(5): 846-56, 2003 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-12960250

RESUMEN

Expression of phosphatidylserine (PS) on the surface of both macrophages and their apoptotic targets is required for efficient phagocytosis. Monocytes, the precursors of macrophages, do not express PS on their surface and do not efficiently phagocytose apoptotic cells. We report here that PS appears on the surface of both human monocytic U937 cells and primary human monocytes as they differentiate in culture and acquire the ability to phagocytose apoptotic thymocytes. Phagocytosis was blocked by pretreating either the apoptotic target or the phagocyte with annexin V to mask PS and was CD14-dependent. Expression of PS, like other events characteristic of differentiating monocytes such as Mac-1 expression, was independent of the agent used to induce differentiation and was insensitive to the addition of caspase inhibitors. These results demonstrate that PS is expressed on monocytes as part of their differentiation program and is independent of apoptosis.


Asunto(s)
Apoptosis/fisiología , Monocitos/citología , Fagocitosis/fisiología , Fosfatidilserinas/biosíntesis , Linfocitos T/citología , Linfocitos T/fisiología , Diferenciación Celular , Humanos , Macrófagos/citología , Macrófagos/fisiología , Monocitos/fisiología , Acetato de Tetradecanoilforbol/farmacología , Células U937/citología , Células U937/efectos de los fármacos , Células U937/fisiología
12.
Int J Radiat Oncol Biol Phys ; 49(5): 1369-79, 2001 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-11286845

RESUMEN

PURPOSE: The relationship between apoptosis induced by 42 degrees C and 44 degrees C hyperthermia alone or in combination with verapamil and changes in intracellular Ca(2+) concentration ([Ca(2+)]i) was investigated in U937 cells. METHODS: Apoptosis induced by hyperthermia was assessed according to DNA fragmentation, nuclear morphologic changes, and expression of phosphatidylserine on the outside plasma cell membrane. These changes were measured by flow cytometry. The [Ca(2+)]i of individual cells after hyperthermia was monitored by a digital image-analyzing technique using Fura-2. RESULTS: Hyperthermia-induced apoptosis reached a plateau after 6 h and was found to be both time and temperature-dependent. DNA fragmentation was maximum at 44 degrees C after 30 min. Verapamil enhanced the apoptosis induced by 42 degrees C and 44 degrees C hyperthermia in normal cells and by 44 degrees C hyperthermia in thermotolerant cells. The number of cells containing higher [Ca(2+)]i (more than 200 nM) was significantly increased by hyperthermia and further elevated by the addition of verapamil in both normal and thermotolerant cells. Apoptosis induced by hyperthermia was markedly decreased by an intracellular Ca(2+) chelator, BAPTA-AM, in a dose-dependent manner. CONCLUSION: These results indicate that [Ca(2+)]i increase plays a crucial role in apoptosis induced by hyperthermia and the combined treatment with verapamil in normal and thermotolerant U937 cells. Furthermore, hyperthermia-combined drug therapy has potential significance in cancer therapy.


Asunto(s)
Apoptosis/fisiología , Bloqueadores de los Canales de Calcio/farmacología , Calcio/metabolismo , Hipertermia Inducida , Verapamilo/farmacología , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Membrana Celular/efectos de la radiación , Quelantes/farmacología , Fragmentación del ADN , Relación Dosis-Respuesta a Droga , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Humanos , Fosfatidilserinas/metabolismo , Radiobiología , Temperatura , Factores de Tiempo , Células U937/efectos de los fármacos , Células U937/fisiología , Células U937/efectos de la radiación
13.
J Cell Sci ; 114(Pt 24): 4637-50, 2001 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-11792828

RESUMEN

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/ultraestructura
14.
Int J Radiat Oncol Biol Phys ; 47(3): 767-77, 2000 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-10837963

RESUMEN

PURPOSE: Apoptosis is currently being evaluated for its importance as a pathway of radiation-induced cell death. However, the difference in the mechanisms between premitotic and postmitotic apoptosis following X-irradiation remains not well understood. We show here that the human monoblastoid cell line U937 can be induced to undergo these two different types of apoptosis. METHODS AND MATERIALS: U937 cells were irradiated at a dose of 5 or 20 Gy, and the DNA fragmentation rate was measured by both flow cytometric analysis and gel electrophoresis. Activation of caspase-3 was detected by Western blot analysis and fluorogenic assay using acetyl-Asp-Glu-Val-Asp-7-amino-4-methyl-coumarin (Ac-DEVD-AMC). Detection of mitochondrial transmembrane potential (DeltaPsi) was performed by using Rho123. Chasing of S-phase fraction following X-irradiation was performed after labeling with 5-bromo-2'-deoxyuridine (BrdU). Thymidine was used for synchronization of the cells. Inhibition of caspase-3 activity was achieved by Acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO). RESULTS: Time courses of the apoptotic rates, caspase activation, and DeltaPsi indicated that two different types of cell death were induced by the different X-ray doses. High-dose X-ray (20 Gy) induced a rapid and strong apoptosis, whereas low-dose X-ray (5 Gy) induced a slow and mild apoptosis. Cell-cycle analyses revealed that there was cell death before cell division in the former apoptosis but the cells must be dying after cell division in the latter apoptosis. By means of cell-cycle synchronization, the S-phase cells proved to be the most sensitive fraction to premitotic apoptosis, but an obvious difference in the susceptibility to cell death among the cell-cycle phases was not observed in postmitotic apoptosis. Ac-DEVD-CHO treatment effectively blocked caspase activity and premitotic apoptosis, but it failed to block postmitotic apoptosis. CONCLUSIONS: Irradiation of U937 cells at different X-ray doses induced two different types of apoptotic cell death, premitotic apoptosis and postmitotic apoptosis, which are characterized by the time course and cell-cycle specificity. Decision concerning these two types of apoptotic cell death may be made by the difference in the magnitude of cell damage following X-irradiation.


Asunto(s)
Apoptosis/efectos de la radiación , Caspasas/metabolismo , Células U937/efectos de la radiación , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Caspasa 3 , Inhibidores de Caspasas , Ciclo Celular/fisiología , Ciclo Celular/efectos de la radiación , Inhibidores de Cisteína Proteinasa/farmacología , Fragmentación del ADN , ADN de Neoplasias/análisis , ADN de Neoplasias/efectos de la radiación , Activación Enzimática , Citometría de Flujo , Humanos , Potenciales de la Membrana/efectos de la radiación , Mitosis , Oligopéptidos/farmacología , Dosis de Radiación , Tolerancia a Radiación , Células U937/efectos de los fármacos , Células U937/fisiología
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