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1.
Mar Environ Res ; 76: 114-21, 2012 May.
Artículo en Inglés | MEDLINE | ID: mdl-22104963

RESUMEN

The potential toxicity of stannum dioxide (SnO2), cerium dioxide (CeO2) and iron oxide (Fe3O4) nanoparticles (NPs) in the marine environment was investigated using the sea urchin, Paracentrotus lividus, as an in vivo model. We found that 5 days after force-feeding of NPs in aqueous solutions, the three NPs presented different toxicity degrees, depending on the considered biomarkers. We examined: 1) the presence of the NPs in the coelomic fluid and the uptake into the immune cells (coelomocytes); 2) the cholinesterase activity and the expression of the stress-related proteins HSC70 and GRP78; 3) the morphological changes affecting cellular compartments, such as the endoplasmic reticulum (ER) and lysosomes. By Environmental Scanning Electron Microscope (ESEM) analysis, coupled with Energy Dispersive X-ray Spectroscopy (EDS) we found that NPs were uptaken inside coelomocytes. The cholinesterases activity, a well known marker of blood intoxication in vertebrates, was greatly reduced in specimens exposed to NPs. We found that levels of stress proteins were down-regulated, matching the observed ER and lysosomes morphological alterations. In conclusion, this is the first study which utilizes the sea urchin as a model organism for biomonitoring the biological impact of NPs and supports the efficacy of the selected biomarkers.


Asunto(s)
Nanopartículas del Metal/toxicidad , Óxidos/toxicidad , Paracentrotus/efectos de los fármacos , Contaminantes Químicos del Agua/toxicidad , Animales , Bioensayo , Biomarcadores/metabolismo , Colinesterasas/metabolismo , Activación Enzimática/efectos de los fármacos , Paracentrotus/citología , Paracentrotus/enzimología , Paracentrotus/metabolismo , Contaminantes Químicos del Agua/metabolismo
2.
Chem Biol Interact ; 187(1-3): 299-303, 2010 Sep 06.
Artículo en Inglés | MEDLINE | ID: mdl-20338157

RESUMEN

The NTera2/D1 (NT2) cell line, which was derived from a human teratocarcinoma, exhibits properties that are characteristics of a committed neuronal precursor at an early stage of differentiation. Its property to express a whole set of molecules related to the cholinergic neurotransmission system, including active acetylcholinesterase (AChE, EC 3.1.1.7) makes it a good alternative model for testing the effects of neurotoxic compounds, such as organophosphorus (OP) insecticides, whose primary target is the inhibition of AChE activity. Recent findings have elucidated the role of AChE in the modulation of apoptosis, but the mechanisms are still rather obscure. NT2 cells exposed to the OP insecticide diazinon at concentrations ranging between 10(-4) and 10(-5)M showed a time-dependent enhancement of cell death. When exposed at 10(-6)M diazinon showed higher cell viability than control samples up to 72 h, followed by a decreasing phase. The cell death caused by the exposures showed a number of features characteristic of apoptosis, including membrane and mitochondrial potential changes. We suggest the hypothesis that such behaviour is due to a dynamic balance between activated and blocked acetylcholine receptors that in turn trigger electrical events and caspase cascade.


Asunto(s)
Apoptosis/efectos de los fármacos , Diazinón/toxicidad , Insecticidas/toxicidad , Acetilcolina/metabolismo , Acetilcolinesterasa/metabolismo , Biomarcadores , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Citometría de Flujo , Humanos , Factores de Tiempo
3.
Cell Biol Toxicol ; 24(6): 587-601, 2008 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-18224450

RESUMEN

Presently, a large effort is being made worldwide to increase the sustainability of industrial development, while preserving not only the quality of the environment but also that of animal and human life. In this work, sea urchin early developmental stages were used as a model to test the effects of the organophosphate pesticide (diazinon) on the regulation of gene expression by immunohistochemical localization of the human regulatory protein against the human OTX2. Egg exposure to diazinon did not affect fertilization; however, at concentrations 10(-5)-10(-6) M, it did cause developmental anomalies, among which was the dose-dependent alteration of the intracellular distribution of a regulatory protein that is immunologically related to the human OTX2. The severe anomalies and developmental delay observed after treatment at 10(-5) M concentration are indicators of systemic toxicity, while the results after treatment at 10(-6) M suggest a specific action of the neurotoxic compound. In this second case, exposure to diazinon caused partial delivery of the protein into the nuclei, a defective translocation that particularly affected the blastula and gastrula stages. Therefore, the possibility that neurotoxic agents such as organophosphates may damage embryonic development is taken into account. Specifically, the compounds are known to alter cytoplasmic dynamics, which play a crucial role in regulating the distribution of intracellular structures and molecules, as well as transcription factors. Speculatively, basing our assumptions on Fura2 experiments, we submit the hypothesis that this effect may be due to altered calcium dynamics, which in turn alter cytoskeleton dynamics: the asters, in fact, appear strongly positive to the OTX2 immunoreaction, in both control and exposed samples. Coimmunoprecipitation experiments seem to supply evidence to the hypothesis.


Asunto(s)
Diazinón/toxicidad , Embrión no Mamífero/efectos de los fármacos , Modelos Biológicos , Sistema Nervioso/embriología , Factores de Transcripción Otx/metabolismo , Paracentrotus/efectos de los fármacos , Pruebas de Toxicidad , Animales , Discusiones Bioéticas , Calcio/metabolismo , Fenómenos Electrofisiológicos/efectos de los fármacos , Embrión no Mamífero/citología , Exposición a Riesgos Ambientales , Immunoblotting , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/metabolismo , Sistema Nervioso/efectos de los fármacos , Paracentrotus/citología , Paracentrotus/embriología , Transporte de Proteínas/efectos de los fármacos , Factores de Tiempo , Tubulina (Proteína)/metabolismo
4.
Eur J Histochem ; 51(4): 301-4, 2007.
Artículo en Inglés | MEDLINE | ID: mdl-18162460

RESUMEN

The teratocarcinoma cell line NTERA2 is recently used in a wide range of researches (from developmental biology to toxicology, for their ability to be induced to neural differentiation. In order to study the genetic potential of these cells, it is needed to use methods for gene silencing and/or mRNA interference, allowing cell viability and further differentiation. To check these features, we simultaneously tested the transfection efficiency of NTERA2, A549 and HeLa cells with Metafectene PRO (Biontex, Germany) and another optimal transfection reagent currently used in our Laboratory, using as a reporter gene the DsRed2 vector (Clontech, Mountain View, CA). Under our culture conditions for NTERA2 and HeLa cells, Metafectene PRO transfection method was found to possess high throughput performance, that allows low concentration rate and low exposure time to excitation light source, thus reducing both toxicity and phototoxicity.


Asunto(s)
Portadores de Fármacos/química , Lípidos/química , Liposomas/química , Teratocarcinoma/patología , Transfección/métodos , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Portadores de Fármacos/farmacología , Femenino , Formazáns/metabolismo , Células HeLa , Humanos , Lípidos/farmacología , Liposomas/farmacología , Proteínas Luminiscentes/química , Proteínas Luminiscentes/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Teratocarcinoma/metabolismo , Sales de Tetrazolio/metabolismo , Proteína Fluorescente Roja
6.
Chem Biol Interact ; 157-158: 305-16, 2005 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-16257396

RESUMEN

Organophosphate (OP) compounds exert inhibition on cholinesterase (ChE) activity by irreversibly binding to the catalytic site of the enzymes. For this reason, they are employed as insecticides for agricultural, gardening and indoor pest control. The biological function of the ChE enzymes is well known and has been studied since the beginning of the XXth century; in particular, acetylcholinesterase (AChE, E.C. 3.1.1.7) is an enzyme playing a key role in the modulation of neuromuscular impulse transmission. However, in the past decades, there has been increasing interest concerning its role in regulating non-neuromuscular cell-to-cell interactions mediated by electrical events, such as intracellular ion concentration changes, as the ones occurring during gamete interaction and embryonic development. An understanding of the mechanisms of the cholinergic regulation of these events can help us foresee the possible impact on environmental and human health, including gamete efficiency and possible teratogenic effects on different models, and help elucidate the extent to which OP exposure may affect human health. The chosen organophosphates were the ones mainly used in Europe: diazinon, chlorpyriphos, malathion, and phentoate, all of them belonging to the thionophosphate chemical class. This research has focused on the comparison between the effects of exposure on the developing embryos at different stages, identifying biomarkers and determining potential risk factors for sensitive subpopulations. The effects of OP oxonisation were not taken into account at this level, because embryonic responses were directly correlated to the changes of AChE activity, as determined by histochemical localisation and biochemical measurements. The identified biomarkers of effect for in vitro experiments were: cell proliferation/apoptosis as well as cell differentiation. For in vivo experiments, the endpoints were: developmental speed, size and shape of pre-gastrula embryos; developmental anomalies on neural tube, head, eye, heart. In all these events, we had evidence that the effects are mediated by ion channel activation, through the activation/inactivation of acetylcholine receptors (AChRs).


Asunto(s)
Colinesterasas/metabolismo , Desarrollo Embrionario/efectos de los fármacos , Compuestos Organofosforados/farmacología , Animales , Apoptosis/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Embrión de Pollo , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Factores de Tiempo
7.
Prog Mol Subcell Biol ; 39: 45-70, 2005.
Artículo en Inglés | MEDLINE | ID: mdl-17152693

RESUMEN

The early development of sea urchins has been thoroughly studied since the beginning of the 20th century thanks to the particular features of the model involving cell signalling, making it easy to follow the complex cell-to-cell interactions that lead to development. In this chapter, the prominent role of cell-to-cell communication in developmental events is discussed, as well as the role of intracellular ion changes that are in turn regulated by signal molecules belonging to the cholinergic system. The results seem to indicate that the zygote stage is the most suitable to study the role of the cholinergic system, as at this stage, a calcium spike can be evoked by exposure to acetylcholine (ACh) or to muscarinic drugs, at any time before the nuclear breakdown. The described outcomes also open a path to a new way of considering biomarkers. In fact, most environmental factors have the capacity to interfere with the cholinergic system: stress, wounds, inflammation and pollution in general. In particular, this offers a way to investigate the presence in the environment and the degree of aggressiveness of neurotoxic contaminants, such as organophosphate and carbamate pesticides, largely used in European countries for many purposes, including agricultural pest control and medical treatment. These drugs exert their function by interfering with the regulation of the cholinergic system and the consequent electrical events. Thus, the sea urchin zygote could represent a reliable model to be used in biosensors with the capacity to translate the effect of neurotoxic pesticides, and generally of stress-inducing contaminants, in living cell responses, such as electrical responses.


Asunto(s)
Desarrollo Embrionario , Monitoreo del Ambiente/métodos , Contaminantes Ambientales/análisis , Erizos de Mar/fisiología , Transducción de Señal , Acetilcolina/metabolismo , Animales , Técnicas Biosensibles , Comunicación Celular , Contaminantes Ambientales/toxicidad , Fertilización , Modelos Biológicos , Neurotoxinas/metabolismo
8.
Eur J Histochem ; 48(3): 235-43, 2004.
Artículo en Inglés | MEDLINE | ID: mdl-15590413

RESUMEN

Cholinergic neurotransmitter system molecules were found to play a role during fertilisation and early cell cycles of a large number of invertebrate and vertebrate organisms. In this study, we investigated the presence and possible function of choline acetyltransferase (ChAT, the biosynthetic enzyme of acetylcholine) in gametes of the sea urchin, Paracentrotus lividus, through localisation and functional studies. ChAT-like molecules were detected in oocytes, mature eggs and zygotes with indirect immunofluorescence methods. Positive immunoreactivity was found in the ovarian egg cytoplasm and surface as well as at the zygote surface. This suggests the eggs' capacity to autonomously synthesise acetylcholine (ACh), the signal molecule of the cholinergic system. Acetylcholinesterase (AChE, the lytic enzyme of acetylcholine) was also found in ovarian eggs, with a similar distribution; however, it disappeared after fertilisation. Ultrastructural ChAT localisation in sperms, which was carried out with the immuno-gold method, showed immunoreactivity in the acrosome of unreacted sperms and at the head surface of reacted sperms. In order to verify a functional role of ACh during fertilization and sea urchin development, in vivo experiments were performed. Exposure of the eggs before fertilisation to 1 mM ACh + 1 microM eserine caused an incomplete membrane depolarisation and consequently enhanced polyspermy, while lower concentrations of ACh caused developmental anomalies. The exposure of zygotes to 0,045 AChE Units/mL of sea water caused developmental anomalies as well, in 50% of the embryos. Altogether, these findings and other previously obtained results, suggest that the cholinergic system may subserve two different tasks during development, according to which particular type of ACh receptor is active during each temporal window. The first function, taking place in the course of fertilisation is a result of autonomously synthesised ACh in sperms, while the second function, taking place after fertilisation, is due to maternal ChAT molecules, assembled on the oolemma along with egg maturation and fertilisation processes.


Asunto(s)
Acetilcolina/biosíntesis , Acetilcolina/fisiología , Colina O-Acetiltransferasa/fisiología , Erizos de Mar/embriología , Erizos de Mar/crecimiento & desarrollo , Acetilcolinesterasa/farmacología , Animales , Colina O-Acetiltransferasa/ultraestructura , Femenino , Fertilización/efectos de los fármacos , Fertilización/fisiología , Inmunohistoquímica , Masculino , Modelos Biológicos , Oocitos/enzimología , Interacciones Espermatozoide-Óvulo/efectos de los fármacos , Interacciones Espermatozoide-Óvulo/fisiología , Espermatozoides/efectos de los fármacos , Espermatozoides/enzimología , Espermatozoides/ultraestructura , Cigoto/efectos de los fármacos , Cigoto/enzimología
9.
J Cell Biochem ; 84(1): 56-67, 2001.
Artículo en Inglés | MEDLINE | ID: mdl-11746516

RESUMEN

Recent reports have highlighted that phosphoinositide-specific phospholipase Cbeta1 expression is linked to neuronal differentiation in different experimental models. We sought to determine whether or not this is also true for nerve growth factor (NGF)-induced neuronal differentiation of rat PC12 cells. However, we did not find differences in the expression of both the forms of phosphoinositide-specific phospholipase Cbeta1 (a and b) during sympathetic differentiation of these cells. Also, PC12 cell clones stably overexpressing phosphoinositide-specific phospholipase Cbeta1 were not more susceptible to the differentiating effect of NGF. Furthermore, since it is well established that phosphoinositide-specific phospholipase Cbeta1 affects cell proliferation, we investigated whether or not PC12 cell clones stably overexpressing phosphoinositide-specific phospholipase Cbeta1 showed differences in survival to serum deprivation and cell cycle, when compared to wild type cells. Nevertheless, we did not find any differences in these parameters between wild type cells and the overexpressing clones. Interestingly, in PC12 cells the overexpressed phosphoinositide-specific phospholipase Cbeta1 did not localize to the nucleus, but by immunofluorescence analysis, was detected in the cytoplasm. Therefore, our findings may represent another important clue to the fact that only when it is located within the nucleus phosphoinositide-specific phospholipase Cbeta1 is able to influence cell proliferation.


Asunto(s)
Diferenciación Celular/efectos de los fármacos , Isoenzimas/metabolismo , Factor de Crecimiento Nervioso/farmacología , Células PC12/efectos de los fármacos , Fosfatidilinositoles/metabolismo , Fosfolipasas de Tipo C/metabolismo , Animales , Apoptosis/fisiología , Ciclo Celular/efectos de los fármacos , Ciclo Celular/fisiología , Diferenciación Celular/fisiología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Medio de Cultivo Libre de Suero/metabolismo , Citoplasma/metabolismo , Hidrólisis , Células PC12/enzimología , Células PC12/patología , Fosfolipasa C beta , Ratas
10.
FEBS Lett ; 505(1): 1-6, 2001 Sep 07.
Artículo en Inglés | MEDLINE | ID: mdl-11557031

RESUMEN

Although inositol lipids constitute only a very minor proportion of total cellular lipids, they have received immense attention by scientists since it was discovered that they play key roles in a wide range of important cellular processes. In the late 1980s, it was suggested that these lipids are also present within the cell nucleus. Albeit the early reports about the intranuclear localization of phosphoinositides were met by skepticism and disbelief, compelling evidence has subsequently been accumulated convincingly showing that a phosphoinositide cycle is present at the nuclear level and may be activated in response to stimuli that do not activate the inositol lipid metabolism localized at the plasma membrane. Very recently, intriguing new data have highlighted that some of the mechanisms regulating nuclear inositol lipid metabolism differ in a substantial way from those operating at the cell periphery. Here, we provide an overview of recent findings regarding the regulation of both nuclear phosphatidylinositol 3-kinase and phosphoinositide-specific phospholipase C-beta1.


Asunto(s)
Núcleo Celular/metabolismo , Inositol/metabolismo , Isoenzimas/metabolismo , Metabolismo de los Lípidos , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfolipasas de Tipo C/metabolismo , Animales , Humanos , Fosfatidilinositoles/metabolismo , Fosfolipasa C beta , Fosforilación
11.
FEBS Lett ; 486(3): 230-6, 2000 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-11119709

RESUMEN

Using NIH 3T3 cells, we have investigated nuclear phosphoinositide metabolism in response to insulin, a molecule which acts as a proliferating factor for this cell line and which is known as a powerful activator of the mitogen-activated protein (MAP) kinase pathway. Insulin stimulated inositol lipid metabolism in the nucleus, as demonstrated by measurement of the diacylglycerol mass produced in vivo and by in vitro nuclear phosphoinositide-specific phospholipase C (PI-PLC) activity assay. Despite the fact that nuclei of NIH 3T3 cells contained all of the four isozymes of the beta family of PI-PLC (i.e. beta1, beta2, beta3, and beta4), insulin only activated the beta1 isoform. Insulin also induced nuclear translocation of MAP kinase, as demonstrated by Western blotting analysis, enzyme activity assays, and immunofluorescence staining, and this translocation was blocked by the specific MAP kinase kinase inhibitor PD98059. By means of both a monoclonal antibody recognizing phosphoserine and in vivo labeling with [(32)P]orthophosphate, we ascertained that nuclear PI-PLC-beta1 (and in particular the b subtype) was phosphorylated on serine residues in response to insulin. Both phosphorylation and activation of nuclear PI-PLC-beta1 were substantially reduced by PD98059. Our results conclusively demonstrate that activation of nuclear PI-PLC-beta1 strictly depends on its phosphorylation which is mediated through the MAP kinase pathway.


Asunto(s)
Núcleo Celular/metabolismo , Insulina/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosfolipasas de Tipo C/metabolismo , Células 3T3 , Animales , Anticuerpos Monoclonales/metabolismo , Western Blotting , División Celular/efectos de los fármacos , Núcleo Celular/enzimología , Diglicéridos/metabolismo , Inhibidores Enzimáticos/farmacología , Flavonoides/farmacología , Técnica del Anticuerpo Fluorescente , Sustancias de Crecimiento/farmacología , Insulina/farmacología , Isoenzimas/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/fisiología , Ratones , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Fosfatidilinositol Diacilglicerol-Liasa , Fosfoinositido Fosfolipasa C , Fosforilación/efectos de los fármacos , Fosfoserina/inmunología , Fosfoserina/metabolismo , Transporte de Proteínas , Fracciones Subcelulares/metabolismo , Especificidad por Sustrato/fisiología
12.
Anticancer Res ; 20(5A): 3183-7, 2000.
Artículo en Inglés | MEDLINE | ID: mdl-11062741

RESUMEN

N-Acetylcysteine (NAC) is a drug bearing multiple preventive properties that can inhibit genotoxicity and carcinogenicity. NAC also inhibits invasion and metastasis of malignant cells, as well as tumor take. We recently demonstrated the effects of NAC on Kaposi's sarcoma cells supernatant-induced invasion in vitro and angiogenesis in vivo. Many anticancer agents act through cytotoxicity of rapidly proliferating cells and several antineoplastic drugs induce apoptosis of cancer cells. Since endothelial cells are the target for the inhibition of angiogenesis, we wanted to verify that NAC, while inhibiting tumor vascularization and endothelial cell invasion would not induce endothelial cell apoptosis. We tested the ability of NAC to modulate apoptosis and cytogenetic damage in vitro and to promote differentiation on a reconstituted basement membrane (matrigel) in two endothelial cell lines (EAhy926 and HUVE). Treatment with NAC protected endothelial cells from TGF-beta-induced apoptosis and paraquat-induced cytogenetic damage. Therefore, NAC acts as an antiangiogenic agent and, at the same time, appears to prevent apoptosis and oxygen-related genotoxicity in endothelial cells.


Asunto(s)
Acetilcisteína/farmacología , Antimutagênicos/farmacología , Apoptosis/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Inhibidores de la Angiogénesis/farmacología , Butionina Sulfoximina/farmacología , Línea Celular , Humanos , Paraquat/antagonistas & inhibidores , Paraquat/farmacología , Factor de Crecimiento Transformador beta/farmacología
13.
J Biol Chem ; 275(39): 30520-4, 2000 Sep 29.
Artículo en Inglés | MEDLINE | ID: mdl-10913438

RESUMEN

Phosphoinositide signaling resides in the nucleus, and among the enzymes of the cycle, phospholipase C (PLC) appears as the key element both in Saccharomyces cerevisiae and in mammalian cells. The yeast PLC pathway produces multiple inositol polyphosphates that modulate distinct nuclear processes. The mammalian PLCbeta(1), which localizes in the nucleus, is activated in insulin-like growth factor 1-mediated mitogenesis and undergoes down-regulation during murine erythroleukemia differentiation. PLCbeta(1) exists as two polypeptides of 150 and 140 kDa generated from a single gene by alternative RNA splicing, both of them containing in the COOH-terminal tail a cluster of lysine residues responsible for nuclear localization. These clues prompted us to try to establish the critical nuclear target(s) of PLCbeta(1) subtypes in the control of cell cycle progression. The results reveal that the two subtypes of PLCbeta(1) that localize in the nucleus induce cell cycle progression in Friend erythroleukemia cells. In fact when they are overexpressed in the nucleus, cyclin D3, along with its kinase (cdk4) but not cyclin E is overexpressed even though cells are serum-starved. As a consequence of this enforced expression, retinoblastoma protein is phosphorylated and E2F-1 transcription factor is activated as well. On the whole the results reveal a direct effect of nuclear PLCbeta(1) signaling in G(1) progression by means of a specific target, i.e. cyclin D3/cdk4.


Asunto(s)
Proteínas Portadoras , Proteínas de Ciclo Celular , Núcleo Celular/metabolismo , Proteínas de Unión al ADN , Fase G1/fisiología , Isoenzimas/metabolismo , Proteínas Proto-Oncogénicas , Fosfolipasas de Tipo C/metabolismo , Animales , Medio de Cultivo Libre de Suero , Ciclina D3 , Ciclina E/genética , Ciclina E/metabolismo , Quinasa 4 Dependiente de la Ciclina , Quinasas Ciclina-Dependientes/genética , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/genética , Ciclinas/metabolismo , Factores de Transcripción E2F , Factor de Transcripción E2F1 , Isoenzimas/genética , Ratones , Mutación , Señales de Localización Nuclear/genética , Fosfolipasa C beta , Fosforilación , Unión Proteica , Proteínas Recombinantes/metabolismo , Proteína de Retinoblastoma/metabolismo , Proteína 1 de Unión a Retinoblastoma , Transducción de Señal , Factor de Transcripción DP1 , Factores de Transcripción/metabolismo , Células Tumorales Cultivadas , Fosfolipasas de Tipo C/genética
14.
Int J Oncol ; 17(1): 75-81, 2000 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-10853021

RESUMEN

The vMIPs are chemokine-like proteins expressed by the Kaposi's sarcoma-associated herpesvirus (KSHV/HHV8) during the lytic phase of viral infection. vMIP-I activates CCR8, a chemokine receptor expressed by Th2 lymphocytes and cultured monocytes. vMIP-II is an agonist for CCR3, a receptor expressed by eosinophils, and an antagonist for several other chemokine receptors. Both are highly angiogenic in the chick chorio-allantoic membrane. We designed and tested three 26-mer peptides, derived from vMIP-I (pK-I), from vMIP-II (pK-II) and from the control MIP-1alpha (pM), spanning key residues of chemokines. pK-I, pK-II and pM all were able to activate a strong chemotactic response in monocytes, higher than parental vMIP-I and II. This corresponded to induction of calcium fluxes in these cells, typical of chemokines. Interestingly, pK-II and pM were also active on PMN neutrophils. In vivo studies (matrigel sponge and rabbit cornea models) showed that pK-I retains the strong angiogenic potential exerted by vMIP-I, while pK-II and pM induced an inflammatory response, probably mediated by PMN recruitment. Our observations indicate that chemokine-derived peptides can show biological activity at pharmacological concentrations. pK-I, in particular, displays the angiogenic activity of full-length vMIP-I, while all peptides appear to have acquired additional properties, stimulating new cellular targets.


Asunto(s)
Canales de Calcio/fisiología , Quimiotaxis de Leucocito/efectos de los fármacos , Herpesvirus Humano 8/fisiología , Proteínas Inflamatorias de Macrófagos/farmacología , Neovascularización Fisiológica/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Receptores de Quimiocina/fisiología , Proteínas Virales , Alantoides/irrigación sanguínea , Secuencia de Aminoácidos , Animales , Calcio/sangre , Canales de Calcio/efectos de los fármacos , Quimiocinas CC/fisiología , Embrión de Pollo , Corion/irrigación sanguínea , Córnea/irrigación sanguínea , Granulocitos/efectos de los fármacos , Granulocitos/fisiología , Herpesvirus Humano 8/genética , Técnicas In Vitro , Proteínas Inflamatorias de Macrófagos/química , Proteínas Inflamatorias de Macrófagos/genética , Datos de Secuencia Molecular , Neovascularización Patológica , Neutrófilos/efectos de los fármacos , Neutrófilos/fisiología , Conejos , Receptores CCR8
15.
Biochem Biophys Res Commun ; 270(3): 992-6, 2000 Apr 21.
Artículo en Inglés | MEDLINE | ID: mdl-10772939

RESUMEN

Certain chemokines inhibit HIV replication through binding to cell surface receptors which also act as viral coreceptors. Based on our previous observations that HIV-1 Tat can interact with alpha- and beta-chemokine receptors, we investigated the potential effect of extracellular Tat (ecTat) on infection and replication of CCR5-dependent (R5) and CXCR4-using (X4) HIV-1 strains in primary activated peripheral blood mononuclear cells (PBMC) of uninfected donors. Receptor desensitization and binding competition studies were used to determine chemokine receptor binding by ecTat. Standard HIV replication assays based on reverse transcriptase (RT) activity determination in culture supernatants of PBMC and real time PCR for HIV-1 gag DNA were used to determine potential effects on early (entry or RT) steps of infection. ecTat bound to CXCR4 expressing monocytes and mitogen-activated PBMC, and competed with the natural ligand of CXCR4, SDF-1alpha (stromal cell-derived factor-1alpha) in calcium mobilization assays. EcTat inhibited replication of the X4 HIV-1 (LAI/IIIB strain) in activated PBMC at concentrations close to those of SDF-1alpha, whereas it only modestly interfered with R5 HIV-1 (BaL) replication in PBMC. Both SDF-1alpha and ecTat inhibited accumulation of X4 HIV-1 gag DNA, indicating interference with viral entry and/or RT. Our data show the surprising and counter-intuitive observation that ecTat selectively represses X4 HIV replication. This could favour spreading of R5 viruses, a condition observed in vivo immediately after transmission and in the early asymptomatic phase of infection.


Asunto(s)
Productos del Gen tat/farmacología , VIH-1/fisiología , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/virología , Monocitos/inmunología , Monocitos/virología , Receptores CXCR4/fisiología , Calcio/metabolismo , Células Cultivadas , ADN Viral/análisis , Productos del Gen tat/metabolismo , Seronegatividad para VIH , VIH-1/efectos de los fármacos , VIH-1/genética , Humanos , Técnicas In Vitro , Cinética , Fragmentos de Péptidos/farmacología , Receptores CXCR4/efectos de los fármacos , Receptores CXCR4/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Productos del Gen tat del Virus de la Inmunodeficiencia Humana
16.
Cell Death Differ ; 7(1): 112-7, 2000 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-10713726

RESUMEN

During the early hours after exposure to differentiation inducing agents, Friend erythroleukaemia cells undergo alterations which commit them to cessation of growth and development of the characteristics of differentiation. Our current experiments have compared the expression and activity of phosphoinositide 3-kinase (PI 3-kinase) in control cells with cells undergoing differentiation which has been induced by dimethyl sulfoxide (DMSO). When the cultures were initiated with stationary phase cells and DMSO was added at the time of seeding, PI 3-kinase activity was stimulated in both treated and control cells during the first 3 h from seeding. This event appears to be a rate limiting step in commitment since pretreatment of cells with 10 microM LY294002 or down-regulation of p85 expression prior to adding DMSO completely prevents commitment to erythropoiesis. Accordingly, PI 3-kinase inhibition during the commitment period prevents DNA-binding of the transcription factor GATA-1, essential for erythroid differentiation. However, once cells are committed to differentiate, PI 3-kinase activity and expression dramatically decreases along with the differentiation programme, to become barely detectable after 96 h. Remarkably, LY294002 treatment leads to accumulation of cell in G1 phase and prevents DMSO-dependent cyclin D3 induction. Based on these data, we suggest that PI 3-kinase is rate limiting for the completion of the first round cycle of cell division required for initiation of erythrocytic differentiation. On the other hand, the late decrease of PI 3-kinase associated with the differentiation process seems to be part of the programmed shut off of genes not needed in mature erythrocytes.


Asunto(s)
Diferenciación Celular , Inhibidores de las Quinasa Fosfoinosítidos-3 , Androstadienos/farmacología , Ciclo Celular , Cromonas/farmacología , Ciclina D3 , Ciclinas/metabolismo , Inhibidores Enzimáticos/farmacología , Humanos , Leucemia Eritroblástica Aguda , Morfolinas/farmacología , Proteínas Quinasas S6 Ribosómicas/metabolismo , Células Tumorales Cultivadas , Wortmanina
17.
Lab Invest ; 79(9): 1151-9, 1999 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-10496534

RESUMEN

The thiol N-acetylcysteine (NAC) is a chemopreventive agent that acts through a variety of mechanisms and can prevent in vivo carcinogenesis. We have previously shown that NAC inhibits invasion and metastasis of malignant cells as well as tumor take. Neovascularization is critical for tumor mass expansion and metastasis formation. We investigated whether a target of the anti-cancer activity of NAC could be the inhibition of the tumor angiogenesis-associated phenotype in vitro and in vivo using the potent angiogenic mixture of Kaposi's sarcoma cell products as a stimulus. Two endothelial (EAhy926 and human umbilical vein endothelial [HUVE]) cell lines were utilized in a panel of assays to test NAC ability in inhibiting chemotaxis, invasion, and gelatinolytic activity in vitro. NAC treatment of EAhy926 and HUVE cells in vitro dose-dependently reduced their ability to invade a reconstituted basement membrane, an indicator of endothelial cell activation. Invasion of HUVE cells was inhibited with an ID50 of 0.24 mM NAC, whereas inhibition of chemotaxis required a 10 fold higher doses, indicating that invasion is a preferential target. NAC inhibited the enzymatic activity and conversion to active forms of the gelatinase produced by endothelial cells. The matrigel in vivo assay was used for the evaluation of angiogenesis; NAC strongly inhibited neovascularization of the matrigel sponges in response to Kaposi's sarcoma cell products. NAC prevented angiogenesis while preserving endothelial cells, implying that it could be safely used as an anti-angiogenic treatment.


Asunto(s)
Acetilcisteína/farmacología , Endotelio Vascular/efectos de los fármacos , Neovascularización Patológica , Animales , Línea Celular , Quimioprevención , Endotelio Vascular/patología , Gelatinasas/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Invasividad Neoplásica
18.
Int J Biol Markers ; 14(4): 268-71, 1999.
Artículo en Inglés | MEDLINE | ID: mdl-10669958

RESUMEN

We have extensively studied the effects of N-acetylcysteine (NAC), a cytoprotective drug that can prevent in vivo carcinogenesis. Here we review our findings NAC completely inhibits gelatinolytic activity of metalloproteases and chemotactic and invasive activities of tumor cells. In addition, NAC reduces the number of lung metastases when malignant murine melanoma cells are injected into nude mice. NAC treatment decreases the weight of primary tumors and produces a dose-related increase in tumor latency. Moreover, oral administration of NAC reduces the formation of spontaneous metastases. In experimental metastasis assays, we have found a synergistic reduction in the number of lung metastases after treatment with doxorubicin (DOX) and NAC in nude mice. In tumorigenicity and spontaneous metastasis assays, the combined administration of DOX and oral NAC again has shown synergistic effects on the frequency and weight of primary tumors and local recurrences and completely prevented the formation of lung metastases. The addition of NAC to endothelial cells strongly reduces their invasive activity in response to angiogenic stimuli. NAC inhibited the degradation and release of radiolabeled type IV collagen by activated endothelial cells, indicating that NAC blocks gelatinase activity. Oral administration of NAC reduces the angiogenic response induced by KS tumor cell products, confirming the ability of NAC to inhibit the invasive activity of endothelial cells in vivo and thereby blocking angiogenesis.


Asunto(s)
Acetilcisteína/farmacología , Inhibidores de la Angiogénesis/farmacología , Invasividad Neoplásica/prevención & control , Animales , Doxorrubicina/farmacología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/patología , Humanos , Ratones , Metástasis de la Neoplasia
19.
Proc Natl Acad Sci U S A ; 95(22): 13153-8, 1998 Oct 27.
Artículo en Inglés | MEDLINE | ID: mdl-9789057

RESUMEN

The HIV-1 Tat protein is a potent chemoattractant for monocytes. We observed that Tat shows conserved amino acids corresponding to critical sequences of the chemokines, a family of molecules known for their potent ability to attract monocytes. Synthetic Tat and a peptide (CysL24-51) encompassing the "chemokine-like" region of Tat induced a rapid and transient Ca2+ influx in monocytes and macrophages, analogous to beta-chemokines. Both monocyte migration and Ca2+ mobilization were pertussis toxin sensitive and cholera toxin insensitive. Cross-desensitization studies indicated that Tat shares receptors with MCP-1, MCP-3, and eotaxin. Tat was able to displace binding of beta-chemokines from the beta-chemokine receptors CCR2 and CCR3, but not CCR1, CCR4, and CCR5. Direct receptor binding experiments with the CysL24-51 peptide confirmed binding to cells transfected with CCR2 and CCR3. HIV-1 Tat appears to mimic beta-chemokine features, which may serve to locally recruit chemokine receptor-expressing monocytes/macrophages toward HIV producing cells and facilitate activation and infection.


Asunto(s)
Calcio/metabolismo , Quimiocinas/farmacología , Quimiotaxis de Leucocito/fisiología , Productos del Gen tat/inmunología , Productos del Gen tat/farmacología , Monocitos/fisiología , Fragmentos de Péptidos/farmacología , Linfocitos T/fisiología , Secuencia de Aminoácidos , Células Cultivadas , Quimiocinas/química , Quimiotaxis de Leucocito/efectos de los fármacos , Citometría de Flujo , Productos del Gen tat/química , VIH-1/inmunología , VIH-1/fisiología , Humanos , Macrófagos/efectos de los fármacos , Macrófagos/fisiología , Datos de Secuencia Molecular , Monocitos/efectos de los fármacos , Fragmentos de Péptidos/inmunología , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Productos del Gen tat del Virus de la Inmunodeficiencia Humana
20.
Int J Cancer ; 72(6): 1088-94, 1997 Sep 17.
Artículo en Inglés | MEDLINE | ID: mdl-9378544

RESUMEN

Retinoblastomas appear to be derived from a multipotential stem cell of the retina, due to alterations of the Rb1 gene. These tumors arise only within a discrete time frame during childhood, prior to terminal differentiation of the retinal precursor cells. Treatment of retinoblastoma cells with certain agents can induce a partial differentiation of cell types resembling those of the mature retina, such as rod and cone photoreceptors, glia, conventional neurons and pigment epithelia. We have tested the effects of 8-Cl-cAMP, a synthetic analog of cAMP which preferentially binds to and activates the RII subunit of protein kinase A on the Y-79 retinoblastoma cell line in vitro. Y-79 cells treated with 8-Cl-cAMP produced short, branching processes and showed a substantial increase in staining for neuron-specific enolase, a marker for conventional neuronal differentiation. In contrast, dibutyryl-cAMP gives a strong increase in the glial marker glial acidic fibrillary protein. Y-79 cell proliferation was strongly inhibited by 8-Cl-cAMP at concentrations as low as 5-25 microM. 8-Cl-cAMP significantly increased the rate of apoptosis of Y-79 cells in a dose-dependent manner. It also modulated expression of the RI regulatory subunit of intracellular cAMP-dependent protein kinase A, which is produced in abnormal quantities by Y-79 cells. A decrease in protein production was observed, with no clear effect on the RI subunit mRNA expression, suggesting that RI regulation occurs post-transcriptionally.


Asunto(s)
8-Bromo Monofosfato de Adenosina Cíclica/análogos & derivados , Apoptosis/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Bucladesina/farmacología , Niño , Proteínas Quinasas Dependientes de AMP Cíclico/biosíntesis , Proteínas Quinasas Dependientes de AMP Cíclico/química , Fragmentación del ADN , Neoplasias del Ojo , Humanos , Cinética , Sustancias Macromoleculares , Fosfopiruvato Hidratasa/biosíntesis , ARN Mensajero/biosíntesis , Retinoblastoma , Transcripción Genética/efectos de los fármacos , Células Tumorales Cultivadas
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