RESUMEN

Indirubin, an isomer of indigo, is a reported inhibitor of cyclin-dependent kinases (CDKs) and glycogen synthase kinase-3 (GSK-3) as well as an agonist of the aryl hydrocarbon receptor (AhR). Indirubin is the active ingredient of a traditional Chinese medicinal recipe used against chronic myelocytic leukemia. Numerous indirubin analogs have been synthesized to optimize this promising kinase inhibitor scaffold. We report here on the cellular effects of 7-bromoindirubin-3'-oxime (7BIO). In contrast to its 5-bromo- and 6-bromo- isomers, and to indirubin-3'-oxime, 7BIO has only a marginal inhibitory activity towards CDKs and GSK-3. Unexpectedly, 7BIO triggers a rapid cell death process distinct from apoptosis. 7-Bromoindirubin-3'-oxime induces the appearance of large pycnotic nuclei, without classical features of apoptosis such as chromatin condensation and nuclear fragmentation. 7-Bromoindirubin-3'-oxime-induced cell death is not accompanied by cytochrome c release neither by any measurable effector caspase activation. Furthermore, the death process is not altered either by the presence of Q-VD-OPh, a broad-spectrum caspase inhibitor, or the overexpression of Bcl-2 and Bcl-XL proteins. Neither AhR nor p53 is required during 7BIO-induced cell death. Thus, in contrast to previously described indirubins, 7BIO triggers the activation of non-apoptotic cell death, possibly through necroptosis or autophagy. Although their molecular targets remain to be identified, 7-substituted indirubins may constitute a new class of potential antitumor compounds that would retain their activity in cells refractory to apoptosis.

Asunto(s)

Muerte Celular/efectos de los fármacos , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Indoles/farmacología , Oximas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Clorometilcetonas de Aminoácidos/farmacología , Animales , Proteína Quinasa CDC2/antagonistas & inhibidores , Caspasas/fisiología , Ciclo Celular/efectos de los fármacos , Línea Celular , Línea Celular Tumoral/efectos de los fármacos , Línea Celular Tumoral/enzimología , Núcleo Celular/ultraestructura , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Inhibidores de Cisteína Proteinasa/farmacología , Femenino , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Humanos , Indoles/síntesis química , Indoles/química , Masculino , Ratones , Oximas/síntesis química , Oximas/química , Fosforilación , Procesamiento Proteico-Postraduccional , Proteínas Proto-Oncogénicas c-bcl-2/fisiología , Quinolinas/farmacología , Proteínas Recombinantes de Fusión/antagonistas & inhibidores , Factor de Transcripción STAT3/metabolismo , Spodoptera , Estrellas de Mar , Relación Estructura-Actividad , Porcinos , Proteína p53 Supresora de Tumor/fisiología , Proteína bcl-X/fisiología

RESUMEN

An undescribed fungus of the genus Microsphaeropsis, isolated from the Mediterranean sponge Aplysina aerophoba, produces two new betaenone derivatives (1, 2) and three new 1,3,6, 8-tetrahydroxyanthraquinone congeners (5-7). The structures of the compounds were established on the basis of NMR spectroscopic and mass spectrometric data and by CD spectroscopy. This is the first report wherein the (1)H and (13)C NMR data of the betaenone congeners are fully and unambiguously assigned on the basis of two-dimensional NMR spectroscopy. Furthermore, we describe the first elucidation of the absolute configuration of 1-(2'-anthraquinonyl)ethanols such as 5 and 6, by quantum chemical calculation of their circular dichroism (CD) and comparison with experimentally measured spectra. Moreover, it was shown that compounds 1, 5, 6, and 7 are inhibitors of PKC-epsilon, CDK4, and EGF receptor tyrosine kinases.

Asunto(s)

Antraquinonas/química , Inhibidores Enzimáticos/síntesis química , Cetonas/química , Hongos Mitospóricos/química , Naftoles/química , Poríferos/microbiología , Inhibidores de Proteínas Quinasas , Animales , Dicroismo Circular , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Conformación Proteica

RESUMEN

Neurothelin/HT7, a transmembrane glycoprotein of the immunoglobulin superfamily, is a marker of blood-brain barrier (BBB)-forming endothelial cells. We have studied the expression of neurothelin in tumors grown on the chorioallantoic membrane (CAM) of chick embryos. We inoculated each 3-5 x 10(6) rat C6 glioma, rat 10AS pancreatic carcinoma, human A375 melanoma, and human mammary duct adenoma cells on the CAM of 10-day-old chick embryos. The tumors were harvested on day 17. All four tumor cell lines formed solid tumors which were supplied by vessels of CAM origin. Foci of bleeding were regularly observed within the tumors. All four tumors induced the expression of neurothelin/HT7 (but not of glucose transporter-1) in tumor endothelial cells, whereas expression in adjacent endothelial cells of normal CAM did not occur. Confocal laser scanning microscopy revealed that the pattern of neurothelin expression in tumor endothelial cells was different from that in normal central nervous system (CNS) endothelium, but the relative molecular weight of neurothelin, studied by western blot analysis, was the same in brain and in tumors. It has been shown that, with increasing malignancy, vessels of CNS tumors lose their morphological characteristics, and BBB markers such as the glucose transporter-1 are downregulated. Our results show that, in contrast, the BBB marker, neurothelin, is expressed de novo in tumor endothelial cells. Potential common functions of neurothelin in endothelial cells of the CNS and tumors are discussed.

Asunto(s)

Antígenos CD , Antígenos de Neoplasias , Antígenos de Superficie , Proteínas Aviares , Proteínas Sanguíneas , Barrera Hematoencefálica , Alantoides , Animales , Basigina , Biomarcadores , Biomarcadores de Tumor/inmunología , Encéfalo/irrigación sanguínea , Embrión de Pollo , Corion , Inducción Embrionaria , Endotelio/química , Endotelio/citología , Endotelio/embriología , Endotelio Vascular/química , Regulación de la Expresión Génica , Transportador de Glucosa de Tipo 1 , Humanos , Immunoblotting , Glicoproteínas de Membrana/biosíntesis , Glicoproteínas de Membrana/genética , Microcirculación , Microscopía Confocal , Proteínas de Transporte de Monosacáridos/inmunología , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Trasplante de Neoplasias , Neoplasias Experimentales/irrigación sanguínea , Neoplasias Experimentales/química , Ratas , Factores de Tiempo , Células Tumorales Cultivadas/trasplante

RESUMEN

We have used the avian chorioallantoic membrane (CAM) to study the interaction of tumor cells with the lymphatics in vivo. The vascular endothelial growth factor-C (VEGF-C) has been shown to be lymphangiogenic. We have therefore grown VEGF-C-expressing human A375 melanoma cells on the CAM. These tumors induced numerous lymphatics at the invasive front, and compressed or destroyed VEGF receptor (R)-3-positive lymphatics were observed within the solid tumors. The lymphatics in the CAM and in the A375 melanomas could also be demonstrated with an antibody against Prox 1, a highly specific marker of lymphatic endothelial cells. Proliferation studies revealed a BrdU labeling index of 11.6% of the lymphatic endothelial cells in the tumors and at their margins. A great number of melanoma cells invaded the lymphatics. Such interactions were not observed with VEGF-C-negative Malme 3 M melanoma cells. Lymphangiogenesis was inhibited to some extent when A375 melanoma cells were transfected with cDNA encoding soluble VEGFR-3 (sflt4), and the BrdU labeling index of the lymphatics in these tumors was 3.9%. Invasion of lymphatics and growth of blood vascular capillaries were not inhibited by the transfection. Therefore, tumor-induced lymphangiogenesis seems to be dependent to some extent on VEGF-C/flt4 interactions, but invasion of lymphatics seems to be a distinct mechanism.

Asunto(s)

Sistema Linfático/patología , Melanoma Experimental/patología , Alantoína/metabolismo , Animales , Northern Blotting , División Celular , Núcleo Celular/ultraestructura , Embrión de Pollo , Coturnix , Técnica del Anticuerpo Fluorescente Indirecta , Regulación Neoplásica de la Expresión Génica/genética , Proteínas de Homeodominio/biosíntesis , Humanos , Inmunohistoquímica , Hibridación in Situ , Sistema Linfático/ultraestructura , Melanoma Experimental/genética , Melanoma Experimental/ultraestructura , Ratones , Ratones Desnudos , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores de Factores de Crecimiento/metabolismo , Transfección , Células Tumorales Cultivadas , Proteínas Supresoras de Tumor , Receptor 3 de Factores de Crecimiento Endotelial Vascular

RESUMEN

16S rDNA polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used to generate restriction profiles of the reference strains, including the American Type Culture Collection type strains, of oral Actinomyces spp., i.e., A. israelii, A. gerencseriae, A. naeslundii genospecies 1 and 2, A. odontolyticus, A. meyeri and A. georgiae, and 23 Actinomyces strains isolated from human dental plaque. The 16S rRNA gene sequences from isolated genomic DNA samples were amplified by PCR. The PCR products were purified and characterized by single digestion with four restriction endonucleases, i.e., MnlI, HaeIII, CfoI, or HpaII. Among them, MnlI was found to discriminate the respective reference strains. The clinical isolates were assigned to one of the species, i.e., A. gerencseriae, A. naeslundii genospecies 1 and 2 and A. odontolyticus, on the basis of their restriction profiles by single digestion with MnlI. Thus, 16S rDNA PCR-RFLP, using MnlI, is a rapid and reliable method for the differentiation of oral Actinomyces spp.

Asunto(s)

Actinomyces/clasificación , ADN Ribosómico/genética , Placa Dental/microbiología , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , ARN Ribosómico 16S/genética , Actinomyces/genética , Técnicas de Tipificación Bacteriana , Secuencia de Bases , Enzimas de Restricción del ADN/metabolismo , Genoma Bacteriano , Humanos , Reproducibilidad de los Resultados

RESUMEN

The tolerance to acid and the acid-neutralizing activity of three important periodontopathic bacteria, Porphyromonas gingivalis, Prevotella intermedia and Fusobacterium nucleatum were studied. P. gingivalis strains grew only at neutral pH and did not utilize glucose, whereas strains of P. intermedia and F. nucleatum could grow under acidic conditions and increased their growth by utilizing glucose. P. gingivalis tended to raise the culture pH during growth. P. intermedia and F. nucleatum raised the culture pH during growth in the absence of glucose, while in the presence of glucose they decreased the pH. Resting cell suspensions of all the bacteria raised the pH in the presence of tryptone and casamino acids. Acid-neutralizing activity was confirmed by measuring base production at a fixed pH with a pH-stat. During neutralization, the cells produced cytotoxic substrates, ammonia and organic acids (butyric, isobutyric and isovaleric acids by P. gingivalis; isovaleric and succinic acids by P. intermedia; propionic and butyric acids by F. nucleatum). These findings suggest that deamination of amino acids into ammonia and organic acids occurs simultaneously with base production, resulting in acid neutralization. These results could partially explain the survival of P. intermedia and F. nucleatum in both supragingival and subgingival plaque and the apparent restriction of P. gingivalis to subgingival plaque. The former bacteria may aid in creation of an environment fostering colonization of subgingival plaque by P. gingivalis.

Asunto(s)

Fusobacterium nucleatum/crecimiento & desarrollo , Porphyromonas gingivalis/crecimiento & desarrollo , Prevotella intermedia/crecimiento & desarrollo , Medios de Cultivo , Placa Dental/microbiología , Fusobacterium nucleatum/metabolismo , Glucosa/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Porphyromonas gingivalis/metabolismo , Prevotella intermedia/metabolismo

RESUMEN

Inhibition of protein kinase C (PKC) is discussed as a new approach for overcoming multidrug resistance (MDR) in cancer chemotherapy. For evaluation of this concept we applied the bisindolylmaleimide GF 109203X, which shows a highly selective inhibition of PKC isozymes alpha, beta 1, beta 2, gamma, delta and epsilon in vitro. The efficacy of this compound in modulation of MDR was examined using several P-glycoprotein (P-gp)-overexpressing cell lines including a MDR1-transfected HeLa clone, and was compared with the activities of dexniguldipine-HCI (DNIG) and dexverapamil-HC1 (DVER), both of which essentially act via binding to P-gp. As PKC alpha has been suggested to play a major role in P-gp-mediated MDR, cell lines exhibiting different expression levels of this PKC isozyme were chosen. On crude PKC preparations or in a cellular assay using a cfos(-711)CAT-transfected NIH 3T3 clone, the inhibitory qualities of the bisindolylmaleimide at submicromolar concentrations were demonstrated. At up 1 microM final concentrations of the PKC inhibitor GF 109203X, a concentration at which many PKC isozymes should be blocked substantially, no cytotoxic or MDR-reversing effects whatsoever were seen, as monitored by 72 h tetrazolium-based colorimetric MTT assays or a 90 min rhodamine 123 accumulation assay. Moreover, depletion of PKC alpha by phorbol ester in HeLa-MDR1 transfectants had no influence on rhodamine 123 accumulation after 24 or 48 h. MDR reversal activity of GF 109203X was seen at higher final drug concentrations, however. Remarkably, [3H]vinblastine-sulphate binding competition experiments using P-gp-containing crude membrane preparations demonstrated similar dose dependencies as found for MDR reversion by the three modulators, i.e. decreasing efficacy in the series dexniguldipine-HCl > dexverapamil-HCl > GF 109203X. Similar interaction with the P-gp in the micromolar concentration range was revealed by competition of GF 109203X with photoincorporation of [3H]azidopine into P-gp-containing crude membrane preparations. No significant effect of the PKC inhibitor on MDR1 expression was seen, which was examined by cDNA-PCR. Thus, the bisindolylmaleimide GF 109203X probably influences MDR mostly via direct binding to P-gp. Our work identifies the bisindolylmaleimide GF 109203X as a new type of drug interacting with P-gp directly, but does not support the concept of a major contribution of PKC to a P-gp-associated MDR, at least using the particular cellular model systems and the selective, albeit general, PKC inhibitor GF 109203X.

Asunto(s)

Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/fisiología , Resistencia a Múltiples Medicamentos , Inhibidores Enzimáticos/farmacología , Indoles/farmacología , Maleimidas/farmacología , Proteína Quinasa C/antagonistas & inhibidores , Células 3T3 , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Animales , Azidas/metabolismo , Dihidropiridinas/metabolismo , Dihidropiridinas/farmacología , Expresión Génica , Humanos , Ratones , Fosforilación , Verapamilo/farmacología

RESUMEN

Vascular endothelial growth factor (VEGF) was identified as an endothelial cell specific mitogen that induces angiogenesis and vascular permeability in vivo. VEGF is a homodimeric protein which contains three intramolecular and two intermolecular disulfide bridges. Here, we report on an efficient procedure for recombinant production of VEGF isoforms VEGF121 and VEGF165 in Escherichia coli. The proteins were solubilized from inclusion bodies, refolded, and purified by chromatographic methods. The final protein products were almost completely in the dimeric conformation, bound to VEGF receptor FLT1 with a Kd of 30 pM, stimulated proliferation of human umbilical vein endothelial cells half-maximally at a concentration of 30 pM, and induced in vivo neovascularization and vascular permeability on the chicken chorioallantoic membrane.

Asunto(s)

Factores de Crecimiento Endotelial/biosíntesis , Factores de Crecimiento Endotelial/farmacología , Endotelio Vascular/citología , Linfocinas/biosíntesis , Linfocinas/farmacología , Neovascularización Fisiológica/efectos de los fármacos , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores de Factores de Crecimiento/metabolismo , Proteínas Recombinantes/biosíntesis , Alantoides/irrigación sanguínea , Animales , Unión Competitiva , Permeabilidad Capilar/efectos de los fármacos , Células Cultivadas , Embrión de Pollo , Corion/irrigación sanguínea , Clonación Molecular , Factores de Crecimiento Endotelial/química , Endotelio Vascular/efectos de los fármacos , Escherichia coli , Humanos , Cinética , Linfocinas/química , Sustancias Macromoleculares , Conformación Proteica , Pliegue de Proteína , Receptores de Factores de Crecimiento Endotelial Vascular , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacología , Venas Umbilicales , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial Vascular

RESUMEN

A system was developed by which 2',7'-bis(carboxyethyl)-4 or 5-carboxyfluorescein could be used to monitor intracellular pH at the same time that proton excretion was being measured. Streptococcal cells were loaded with the dye, and after the addition of glucose protons were excreted and the intracellular pH increased quickly and remained higher than the extracellular pH of 7.0. The excretion of protons stopped and the intracellular pH returned to the original level when glucose was depleted. The intracellular level of ATP remained high during glucose metabolism and decreased with the depletion of glucose. At extracellular pH of 5.5, and 5.0, the intracellular pH of fasting cells was higher than the extracellular pH value. After addition of glucose there were initial lags of proton excretion and of increases in intracellular pH at the acidic extracellular pH values. In the presence of fluoride, a lag in proton excretion and a simultaneous decrease in intracellular pH were observed, indicating a partial and transient inhibition of proton-ATPase activity.

Asunto(s)

Fluoruros/farmacología , Glucólisis/fisiología , Streptococcus mutans/metabolismo , Streptococcus sanguis/metabolismo , Adenosina Trifosfato/análisis , Medios de Cultivo/química , Fluoresceínas , Colorantes Fluorescentes , Glucosa/metabolismo , Glucólisis/efectos de los fármacos , Concentración de Iones de Hidrógeno , Líquido Intracelular/química , Líquido Intracelular/metabolismo , Transporte Iónico/efectos de los fármacos , Transporte Iónico/fisiología , Bombas de Protones/metabolismo , ATPasas de Translocación de Protón/antagonistas & inhibidores , ATPasas de Translocación de Protón/metabolismo , Espectrometría de Fluorescencia , Streptococcus mutans/efectos de los fármacos , Streptococcus sanguis/efectos de los fármacos

RESUMEN

Inhibition of the rate of acid production from glucose by the cells of Streptococcus mutans NCIB 11723 was directly related to the concentrations of 0.075 to 0.20 mM chlorhexidine. Lactate production was inhibited to a greater extent than acetate and formate. Quantification of glycolytic intermediates revealed that the steps in glycolysis inhibited by chlorhexidine were the reactions catalyzed by phosphofructokinase and glyceraldehyde 3-phosphate dehydrogenase and/or phosphoglycerate kinase. However, the activities of these enzymes were not decreased in cells treated with the inhibitor. It was demonstrated that chlorhexidine caused leakage of metabolites from the cells. Our results indicate that the decreased rate of glycolysis caused by chlorhexidine is due to the leakage of metabolic intermediates and not to direct effects on enzymes involved in glycolysis by S. mutans NCIB 11723.

Asunto(s)

Antiinfecciosos Locales/farmacología , Permeabilidad de la Membrana Celular/efectos de los fármacos , Clorhexidina/farmacología , Glucólisis/efectos de los fármacos , Streptococcus mutans/efectos de los fármacos , Acetatos/metabolismo , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Formiatos/metabolismo , Fructosadifosfatos/metabolismo , Fructosafosfatos/metabolismo , Glucosa-6-Fosfato , Glucofosfatos/metabolismo , Gliceraldehído 3-Fosfato/metabolismo , Ácidos Glicéricos/metabolismo , Líquido Intracelular/efectos de los fármacos , Líquido Intracelular/metabolismo , Lactatos/metabolismo , NAD/metabolismo , Estadísticas no Paramétricas , Streptococcus mutans/metabolismo

RESUMEN

Protein kinase C (PKC) regulates a variety of signal transduction events implicated in the pathogenesis of inflammation, including the biosynthesis of inflammatory cytokines and superoxide and the activation of phospholipase A2. Because of the significant role of PKC in these inflammatory processes, we evaluated a specific and potent inhibitor of C kinase for efficacy in several in vitro and in vivo murine models of inflammation. Unlike the relatively nonspecific kinase inhibitor staurosporine, the bisindolylmaleimide 3-[1-[-3-(dimethylaminopropyl]-1H-indol-3-yl]- 4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione monohydrochloride (Gö 6850) demonstrated increased selectivity for C kinase in purified enzyme assays (respective IC50 values (microM) for Gö 6850 and staurosporine: protein kinase C (0.032, 0.009); myosin light-chain kinase (0.6, 0.01); protein kinase G (4.6, 0.018); protein kinase A (33, 0.04); tyrosine kinase1 (94, 0.4); tyrosine kinase2 (> 100, > 1)). Topically applied Gö 6850 inhibited phorbol myristate acetate-induced edema, neutrophil influx and vascular permeability in murine epidermis in a dose- and time-dependent manner at levels comparable to indomethacin. In a murine model of delayed type hypersensitivity, Gö 6850 inhibited dinitrofluorobenzene-induced contact dermatitis with and ID50 value of 150 micrograms/ear. Cellular studies in mouse peritoneal macrophages demonstrated that Gö 6850 was a potent inhibitor of phorbol myristate acetate-induced prostaglandin E2 production. Superoxide production in phorbol myristate acetate-stimulated murine neutrophils was also inhibited by Gö 6850 (IC50 = 88 nM).(ABSTRACT TRUNCATED AT 250 WORDS)

Asunto(s)

Antiinflamatorios no Esteroideos/farmacología , Antiinflamatorios/farmacología , Inhibidores Enzimáticos/farmacología , Indoles/farmacología , Maleimidas/farmacología , Proteína Quinasa C/antagonistas & inhibidores , Animales , Ácido Araquidónico/antagonistas & inhibidores , Dinoprostona/metabolismo , Femenino , Hipersensibilidad Tardía/tratamiento farmacológico , Macrófagos Peritoneales/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Neutrófilos/efectos de los fármacos , Superóxidos/metabolismo , Acetato de Tetradecanoilforbol/antagonistas & inhibidores

RESUMEN

We studied the mechanism by which the antimicrobial compound sucrose monolaurate inhibits Streptococcus mutans NCTC 10449 by determining its effect on the rate of acid production from glucose and sucrose and the intracellular and extracellular levels of glycolytic intermediates. Sucrose monolaurate was more effective than either sodium laurate or sodium fluoride in inhibiting acid production at pH 7.0 from glucose. Inhibition of acid production was the same when either glucose or sucrose was the carbon source and in the presence or absence of oxygen. Quantitative analysis of various glycolytic intermediates revealed that the steps inhibited by sucrose monolaurate were the reactions catalyzed by phosphofructokinase and glyceraldehyde 3-phosphate dehydrogenase and/or phosphoglycerate kinase. Since the activities of these enzymes in cell-free extracts were not decreased by the addition of sucrose monolaurate, the inhibition of acid production could not be ascribed to direct effects on the enzymes. A decrease in the rate of acid production with corresponding elevations in the extracellular levels of glycolytic intermediates indicates that sucrose monolaurate inhibits S. mutans by altering the permeability of the cell membrane, which causes a loss of important metabolites.

Asunto(s)

Antiinfecciosos Locales/farmacología , Glucólisis/efectos de los fármacos , Streptococcus mutans/efectos de los fármacos , Sacarosa/análogos & derivados , Ácidos Carboxílicos/metabolismo , Permeabilidad de la Membrana Celular/efectos de los fármacos , Sistema Libre de Células/enzimología , Concentración de Iones de Hidrógeno , Ácidos Láuricos/farmacología , Pruebas de Sensibilidad Microbiana , Fluoruro de Sodio/farmacología , Estadísticas no Paramétricas , Streptococcus mutans/metabolismo , Sacarosa/farmacología

RESUMEN

The newly identified drug transporter MRP is functionally linked to a multiple drug resistance independent from P-glycoprotein. Resistance modifiers for this type of MDR are rare at present. We analyzed the modulating effect of the highly selective bisindolylmaleimide PKC inhibitor GF 109203X on the MRP overexpressing human MDR sublines HL60/AR and GLC4/ADR. Applying a 72 hour MTT-assay we demonstrate a complete reversal of the vincristine resistance of HL60/AR cells. Adriamycin resistance of HL60/AR, or vincristine resistance of GLC4/ADR were partially reversed. Furthermore, rhodamine 123 efflux from HL60/AR was strongly modulated by GF 109203X. Since the PKC inhibitor did not significantly influence MRP gene expression at the mRNA level which was examined by cDNA-PCR, our results suggest either a direct interaction of the compound with MRP or/and an indirect influence on MRP activity via altering the phosphorylation status of the transporter.

Asunto(s)

Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/biosíntesis , Resistencia a Múltiples Medicamentos/fisiología , Expresión Génica/efectos de los fármacos , Indoles/farmacología , Maleimidas/farmacología , Proteína Quinasa C/antagonistas & inhibidores , Antimetabolitos Antineoplásicos/metabolismo , Secuencia de Bases , Línea Celular , Supervivencia Celular/efectos de los fármacos , Cartilla de ADN , Relación Dosis-Respuesta a Droga , Doxorrubicina/toxicidad , Resistencia a Múltiples Medicamentos/genética , Humanos , Cinética , Leucemia Promielocítica Aguda , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Rodamina 123 , Rodaminas/metabolismo , Células Tumorales Cultivadas , Vincristina/toxicidad

RESUMEN

The inhibitory effects of three novel staurosporine-derived compounds were tested with five different types of protein kinases, including protein kinase C (PKC). IC50 values of two of these compounds were found to be 300 to > 5000 times lower for PKC alpha beta gamma (a mixture of the PKC isoenzymes alpha, beta and gamma) than for any of the other protein kinases. The inhibitory action of the most selective inhibitor was tested also with the Ca(2+)-unresponsive PKC isoenzyme delta and was found to suppress PKC alpha beta gamma and PKC delta differentially. The highly specific PKC inhibitors are active both in cell culture and in vivo. They inhibit the PKC-catalyzed phosphorylation of the specific PKC substrate MARCKS in Swiss-3T3 fibroblasts and the okadaic acid-induced edema of the mouse ear. However, the more complex biological processes triggered by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate in mouse skin, such as inflammation, stimulation of cellular hyperproliferation and tumor promotion, remain largely unaffected upon topical application of these compounds.

Asunto(s)

Alcaloides/uso terapéutico , Anticarcinógenos/uso terapéutico , Isoenzimas/antagonistas & inhibidores , Proteína Quinasa C/antagonistas & inhibidores , Neoplasias Cutáneas/inducido químicamente , Neoplasias Cutáneas/prevención & control , Acetato de Tetradecanoilforbol/antagonistas & inhibidores , Células 3T3/efectos de los fármacos , Animales , Femenino , Ratones , Ratones Endogámicos , Piel/efectos de los fármacos , Estaurosporina , Especificidad por Sustrato , Acetato de Tetradecanoilforbol/toxicidad

RESUMEN

TGF-beta 1 purified from human platelets induces colony formation of rat fibroblasts reversibly when administered together with EGF. TGF-beta 1 has been reported by us to lead to stable transformation of rat fibroblasts, to activate latent Epstein-Barr virus, to exhibit tumor promoting activity for murine fibroblasts, and to cause the release of apoptosis-inducing signals from normal cells which are directed specifically against transformed cells. The use of recombinant TGF-beta 1 in this study showed that all the effects measured were indeed due to TGF-beta 1. In order to get information on intracellular signal pathways induced by TGF-beta 1, specific inhibitors of protein kinase C (PKC) were applied. Induction of EBV antigens by TGF-beta 1 was dependent on the function of PKC, whereas induction of apoptosis in transformed cells by TGF-beta 1-treated normal cells was independent of the action of PKC.

RESUMEN

Cyclosporin A (CsA) has been reported to inhibit phorbol myristate acetate (PMA)-induced superoxide anion (O2-) formation in human neutrophils and murine macrophages. We found that CsA inhibited O2- formation in HL-60 cells induced by PMA (30 nM) and phorbol dibutyrate (200 nM) with a half-maximal effect at 1 and 0.75 microM, respectively. One possible target of CsA action is protein kinase C (PKC) [EC 2.7.1.37] since phorbol esters activate this kinase. However, CsA did not inhibit PMA-mediated reduction of histamine-induced rises in cytosolic Ca2+ concentration in, and PMA-induced differentiation of, HL-60 cells and platelet aggregation. CsA did not reduce the activity of various recombinant c-PKC isoenzymes (alpha, beta 1 and gamma), n-PKC isoenzymes (delta and epsilon), an a-PKC isoenzyme (zeta) nor of PKC purified from rat brain in vitro. These data show that CsA inhibits phorbol ester-induced O2- formation in HL-60 cells but not other phorbol ester-mediated events and that inhibition by CsA of O2- formation cannot readily be attributed to direct PKC inhibition. We also show that CsA does not change the activity of nucleoside diphosphate kinase [EC 2.7.4.6] in HL-60 membranes nor the latter's physical properties.

Asunto(s)

Ciclosporina/farmacología , Fagocitos/efectos de los fármacos , Proteína Quinasa C/antagonistas & inhibidores , Superóxidos/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Animales , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Humanos , Ratones , Neutrófilos/metabolismo , Fagocitos/metabolismo , Agregación Plaquetaria/efectos de los fármacos , Ratas

RESUMEN

The role of protein kinase C (PKC) isoenzymes in the regulation of cell functions is largely unknown. We studied the effects of 2-(1H-indol-3-yl)-3- [1-(3-dimethylaminopropyl)-1H-indol-3-yl]-maleinimide (Gö 6850), a selective inhibitor of c- and n-PKC isoenzymes, and 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo[2 ,3- a]pyrrolo[3, 4-c]-carbazole (Gö 6976), an inhibitor of c-PKC isoenzymes, on various human neutrophil functions. Gö 6850 inhibited 4 beta-phorbol 12-myristate 13-acetate (PMA)-, 1,2-dicaprylyl- glycerol- and chemotactic peptide-induced superoxide anion formation with half-maximal effects at 100 nM, 240 nM and 850 nM, respectively. Gö 6850 reverted PMA-mediated inhibition of chemotactic peptide-induced rises in cytosolic Ca2+ concentration with a half-maximal effect at 480 nM. Gö 6850 (1 microM) inhibited PMA-induced lysozyme release by 55%. Gö 6976 had no effect on the parameters studied. Our data suggest the following: (1) n- Rather than c-PKC isoenzymes are involved in the regulation of various human neutrophil functions. (2) Different n-PKC isoenzymes may mediate activation of NADPH oxidase by various stimuli. (3) Different n-PKC isoenzymes may be involved in the mediation of the effects of PMA on various cell functions.

Asunto(s)

Neutrófilos/enzimología , Proteína Quinasa C/metabolismo , Carbazoles/farmacología , Activación Enzimática , Humanos , Técnicas In Vitro , Indoles/farmacología , Isoenzimas/metabolismo , Maleimidas/farmacología , Muramidasa/metabolismo , N-Formilmetionina Leucil-Fenilalanina/farmacología , Estallido Respiratorio/efectos de los fármacos , Superóxidos/metabolismo , Acetato de Tetradecanoilforbol/farmacología

RESUMEN

Suppression of human immunodeficiency virus-1 (HIV-1) reactivation in latently infected cells by protein kinase C (PKC) inhibitors has been described. Based on an initial finding with the indolocarbazole inhibitor Gö 6976 we have examined several members of this new class of potent and specific PKC inhibitors with respect to their ability to prevent the PKC-mediated induction of HIV-1 replication in the latently infected U1 cell line. Two of these compounds strongly inhibited not only PMA-induced release of p24-antigen and infectious virus particles into the supernatant (50% inhibition at 0.04-0.35 microM) but also TNF-alpha-mediated HIV-1 reactivation in the same concentration range. Significant lower toxicities compared to Gö 6976 were observed for the new compounds, with 50% cytotoxic concentrations at 5.2 microM for Gö 7775 and 3.4 microM for Gö 7716. This resulted in selectivity indices which were 10-20-times higher compared to the reference compound Gö 6976 and were comparable to those of registered anti-AIDS drugs. No anti-HIV-1 activity was observed for a closely related indolocarbazole analogue with no inhibitory activity in the PKC in vitro enzyme assay. This study demonstrates the important role of PKC in reactivation of HIV-1 in latently infected cells and points to the potential of indolocarbazoles to preserve the latent state of HIV-1 infection.

Asunto(s)

Antineoplásicos/farmacología , Carbazoles/farmacología , VIH-1/efectos de los fármacos , Indoles/farmacología , Proteína Quinasa C/antagonistas & inhibidores , Carbazoles/toxicidad , Línea Celular , Humanos , Indoles/toxicidad , Acetato de Tetradecanoilforbol/farmacología , Activación Viral/efectos de los fármacos , Latencia del Virus/efectos de los fármacos

RESUMEN

Cellular DNA from 25 Actinomyces naeslundii and Actinomyces viscosus strains belonging to the 7 taxonomic clusters of Fillery et al. (1978) and several unclustered strains was obtained by enzymatic and N-lauroylsarcosine/guanidine isothiocyanate treatment of whole cells, followed by extraction of the nucleic acid. The DNA samples were digested with restriction endonucleases BamHI or PvuII, and agarose gel electrophoresis was used to obtain DNA fingerprints. The DNA fragments were subjected to Southern blot hybridization with a digoxigenin-labeled cDNA probe transcribed from Escherichia coli 16S and 23S rRNA. The patterns of bands from genomic (DNA fingerprints) and rDNA fingerprints (ribotypes) were used for comparison between the taxonomic cluster strains and strains within clusters. Representative strains from each taxonomic cluster provided different BamHI DNA fingerprints and ribotype patterns with 3 to 9 distinct bands. Some strains within a cluster showed identical ribotype patterns with both endonucleases (A. naeslundii B120 and A. naeslundii B102 from cluster 3), while others showed the same pattern with BamHI but a different pattern with PvuII (A. naeslundii ATCC 12104 and 398A from cluster 5). A viscosus ATCC 15987 (cluster 7) and its parent strain T6 yielded identical fingerprint and ribotype patterns. The genomic diversity revealed by DNA fingerprinting and ribotyping demonstrates that these techniques, which do not require phenotypic expression, are suited for study of the oral ecology of the Actinomyces, and for epidemiological tracking of specific Actinomyces strains associated with caries lesions and sites of periodontal destruction.

Asunto(s)

Actinomyces/clasificación , Actinomyces/genética , Técnicas de Tipificación Bacteriana , ADN Bacteriano/análisis , Actinomyces viscosus/clasificación , Actinomyces viscosus/genética , Animales , Southern Blotting , Cricetinae , Dermatoglifia del ADN/métodos , Sondas de ADN , ADN Bacteriano/aislamiento & purificación , ADN Ribosómico/análisis , Caries Dental/microbiología , Placa Dental/microbiología , Desoxirribonucleasa BamHI , Desoxirribonucleasas de Localización Especificada Tipo II , Electroforesis en Gel de Agar , Humanos , Boca/microbiología , ARN Ribosómico/genética , Ratas

RESUMEN

Human immunodeficiency virus (HIV-1) infection is followed by a period of latency or a low-level-persistent (LLP) state that results in an asymptomatic infection of the host. Productive viral expression may be triggered by a variety of activators including mitogens, antigens, and cytokines. Protein kinase C (PKC) has been shown to be important in the intracellular cascade of signals induced by such activators. With U1 and ACH-2 cell lines representative of an HIV-1 postintegration state, the effect of Gö 6976, a synthetic inhibitor of PKC was tested. Gö 6976 is a nonglycosidic indolocarbazole found to potently inhibit HIV-1 induction by Bryostatin 1, tumor necrosis factor alpha, and interleukin 6. Gö 6976 effectively blocks viral transcription induced by Bryostatin 1 or tumor necrosis factor alpha that leads to the inhibition of intracellular viral protein synthesis and viral shedding. Gö 6976 also blocks interleukin 6-mediated posttranscriptional induction of viral proteins. The IC50 of Gö 6976 shows a 12- to 60-fold more potent effect than for H-7, another PKC inhibitor with a similar mechanism. The inhibitory effect is reduced when Gö 6976 is not added before or within 1 hr of induction by the potent PKC activator Bryostatin 1. However, U1 cells can be grown for long periods in a nontoxic concentration of Gö 6976 (300 nM), which confers virtual inhibition of HIV-1 induction without the development of resistance. Results indicate that inhibition of HIV-1 proviral induction from latent/low-level-producing infectious states with potent PKC inhibitors like Gö 6976 may represent an additional and promising antiviral approach.

Asunto(s)

Carbazoles/farmacología , VIH-1/crecimiento & desarrollo , Indoles/farmacología , Proteína Quinasa C/antagonistas & inhibidores , Sulfonamidas , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina , Brioestatinas , Inducción Enzimática/efectos de los fármacos , VIH-1/efectos de los fármacos , Humanos , Técnicas In Vitro , Isoquinolinas/farmacología , Lactonas/farmacología , Macrólidos , Piperazinas/farmacología , ARN Viral/biosíntesis , ADN Polimerasa Dirigida por ARN/biosíntesis , Proteínas de los Retroviridae/biosíntesis , Transcripción Genética/efectos de los fármacos , Células Tumorales Cultivadas , Replicación Viral/efectos de los fármacos