RESUMEN
The human T-cell lymphotropic virus type 1 (HTLV-1) is the cause of adult T-cell leukemia and inflammatory diseases including HTLV-1-associated myelopathy/tropical spastic paraparesis. HTLV-1 can be transmitted through sexual contact, mother-to-child transmission, and exposure to contaminated blood. Microbicides are agents that interfere with microbial infectivity at mucous membranes, and candidates are under development for use against sexually transmitted viruses such as human immunodeficiency virus type 1. We previously demonstrated that cell surface polyanionic heparan sulfate proteoglycans bind the HTLV-1 envelope glycoprotein surface subunit gp46, facilitating cell-cell and cell-free virus spread in vitro. We now show, using assays for Env-receptor binding inhibition, Env-induced cell-cell fusion, cell-cell virus spread, and pseudotype HTLV-1 infectivity, that the soluble polyanions PRO 2000 and dextran sulfate are potent inhibitors of HTLV-1 spread in vitro, with PRO 2000 being the more promising candidate. The results of these studies suggest that candidate topical microbicides may be of use in reducing HTLV-1 sexual transmission.
Asunto(s)
Antivirales/farmacología , Infecciones por HTLV-I/prevención & control , Virus Linfotrópico T Tipo 1 Humano/efectos de los fármacos , Naftalenosulfonatos/farmacología , Polímeros/farmacología , Antivirales/química , Fusión Celular , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Productos del Gen env/efectos de los fármacos , Infecciones por HTLV-I/transmisión , Células HeLa , Humanos , Naftalenosulfonatos/química , Péptidos/farmacología , Polímeros/química , Proteínas Oncogénicas de Retroviridae/efectos de los fármacos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Virión/efectos de los fármacosRESUMEN
The protein-modifying agent arsenite stimulates glucose uptake in 3T3-L1 adipocytes. In the current study we have analysed the signalling pathways that contribute to this response. By subcellular fractionation we observed that arsenite, like insulin, induces translocation of the GLUT1 and GLUT4 glucose transporters from the low-density membrane fraction to the plasma membrane. Arsenite did not activate early steps of the insulin receptor (IR)-signalling pathway and the response was insensitive to inhibition of phosphatidylinositol-3'-kinase (PI-3') kinase by wortmannin. These findings indicate that the 'classical' IR-IR substrate-PI-3' kinase pathway, that is essential for insulin-induced GLUT4 translocation, is not activated by arsenite. However, arsenite-treatment did induce tyrosine-phosphorylation of c-Cbl. Furthermore, treatment of the cells with the tyrosine kinase inhibitor, tyrphostin A25, abolished arsenite-induced glucose uptake, suggesting that the induction of a tyrosine kinase by arsenite is essential for glucose uptake. Both arsenite and insulin-induced glucose uptake were inhibited partially by the p38 MAP kinase inhibitor, SB203580. This compound had no effect on the magnitude of translocation of glucose transporters indicating that the level of glucose transport is determined by additional factors. Arsenite- and insulin-induced glucose uptake responded in a remarkably similar dose-dependent fashion to a range of pharmacological- and peptide-inhibitors for atypical PKC-lambda, a downstream target of PI-3' kinase signalling in insulin-induced glucose uptake. These data show that in 3T3-L1 adipocytes both arsenite- and insulin-induced signalling pathways project towards a similar cellular response, namely GLUT1 and GLUT4 translocation and glucose uptake. This response to arsenite is not functionally linked to early steps of the IR-IRS-PI-3' kinase pathway, but does coincide with c-Cbl phosphorylation, basal levels of PKC-lambda activity and p38 MAPK activation.
Asunto(s)
Adipocitos/metabolismo , Arsenitos/farmacología , Glucosa/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas de Transporte de Monosacáridos/metabolismo , Proteínas Musculares , Células 3T3 , Adipocitos/efectos de los fármacos , Animales , Transporte Biológico/efectos de los fármacos , Caveolina 1 , Caveolinas/efectos de los fármacos , Caveolinas/metabolismo , Células Cultivadas , Desoxiglucosa/farmacocinética , Inhibidores Enzimáticos/farmacología , Transportador de Glucosa de Tipo 1 , Transportador de Glucosa de Tipo 4 , Imidazoles/farmacología , Insulina/metabolismo , Insulina/farmacología , Ratones , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/efectos de los fármacos , Proteínas de Transporte de Monosacáridos/efectos de los fármacos , Proteína Oncogénica v-cbl , Péptidos/química , Péptidos/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación/efectos de los fármacos , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/metabolismo , Piridinas/farmacología , Receptor de Insulina/efectos de los fármacos , Receptor de Insulina/metabolismo , Proteínas Oncogénicas de Retroviridae/efectos de los fármacos , Proteínas Oncogénicas de Retroviridae/metabolismo , Transducción de Señal , Tirosina/metabolismo , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
Primary macrophages isolated from hck(-/-)fgr(-/-) mice display altered morphology and F-actin cytoskeletal structures and reduced migration. The ability of phorbol myristyl acetate (PMA), a protein kinase C activator that has been reported to increase macrophage spreading and carcinoma cell motility, to rescue these hck(-/-)fgr(-/-) defects was tested. Although PMA-treated wild-type and hck(-/-)fgr(-/-) macrophages exhibited a similar flattened, spread phenotype, PMA did not rescue the hck(-/-)fgr(-/-) macrophage migration defect. Instead, both PMA-treated wild type and hck(-/-)fgr(-/-) macrophages were defective in spontaneous and chemotactic migration and tyrosine phosphorylation of the Cbl protooncoprotein was decreased in both. Moreover, c-cbl(-/-) macrophages displayed the same impairment of motility as hck(-/-)fgr(-/-) macrophages and a similar morphology with less polarization and more dorsal ruffling than wild-type macrophages. As Hck and Fgr expression and activity were not decreased in c-cbl(-/-) macrophages, these results suggest that Cbl is likely to be an important downstream mediator of the Src family kinase-regulated macrophage motility pathway.
Asunto(s)
Actinas/metabolismo , Quimiotaxis de Leucocito/fisiología , Macrófagos/metabolismo , Proteínas Oncogénicas de Retroviridae/deficiencia , Actinas/efectos de los fármacos , Animales , Tamaño de la Célula/efectos de los fármacos , Tamaño de la Célula/fisiología , Células Cultivadas , Quimiotaxis de Leucocito/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Macrófagos/citología , Macrófagos/efectos de los fármacos , Ratones , Ratones Noqueados , Proteína Oncogénica v-cbl , Fosforilación/efectos de los fármacos , Proteínas Tirosina Quinasas/deficiencia , Proteínas Tirosina Quinasas/genética , Proteínas Proto-Oncogénicas/deficiencia , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-hck , Proteínas Oncogénicas de Retroviridae/efectos de los fármacos , Proteínas Oncogénicas de Retroviridae/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Acetato de Tetradecanoilforbol/farmacología , Tirosina/metabolismo , Familia-src Quinasas/efectos de los fármacos , Familia-src Quinasas/metabolismoRESUMEN
We have investigated the relationship between the molecular chaperone heat shock protein-90 (Hsp90) and the signal transducing capacity of the Src-family kinase Hck. Inhibition of Hsp90 with geldanamycin suppressed the ability of bacterial lipopolysaccharide to enhance the cell adhesion properties of macrophages, a phenomenon most likely explained by the reduced expression and activity of Hck in macrophages lacking Hsp90 function. The contribution of Hsp90 to signal transduction by Hck was biochemically dissected further by examining its role in the de novo folding and maintenance of wild-type Hck and its constitutively active counterpart, Hck499F. The folding of nascent wild-type Hck and Hck499F into catalytically active conformations, and their accumulation in cells was found to be dependent on Hsp90 function. Notably, mature Hck499F had a greater requirement for on-going support from Hsp90 than did mature wild-type Hck. This particular finding might have important implications for our understanding of the evolution of oncogenic protein kinases.
Asunto(s)
Adhesión Celular/fisiología , Proteínas HSP90 de Choque Térmico/metabolismo , Receptores de Lipopolisacáridos/metabolismo , Activación de Macrófagos/fisiología , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Transducción de Señal/fisiología , Animales , Benzoquinonas , Dominio Catalítico/efectos de los fármacos , Dominio Catalítico/fisiología , Células Cultivadas/citología , Células Cultivadas/efectos de los fármacos , Células Cultivadas/metabolismo , Inhibidores Enzimáticos/farmacología , Quinasa 2 de Adhesión Focal , Lactamas Macrocíclicas , Lipopolisacáridos/farmacología , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Proteína Oncogénica v-cbl , Fosforilación/efectos de los fármacos , Pliegue de Proteína , Estructura Terciaria de Proteína/efectos de los fármacos , Estructura Terciaria de Proteína/fisiología , Proteínas Tirosina Quinasas/efectos de los fármacos , Proteínas Proto-Oncogénicas/efectos de los fármacos , Proteínas Proto-Oncogénicas c-hck , Quinonas/farmacología , Proteínas Oncogénicas de Retroviridae/efectos de los fármacos , Proteínas Oncogénicas de Retroviridae/metabolismo , Tirosina/efectos de los fármacos , Tirosina/metabolismo , Familia-src Quinasas/efectos de los fármacos , Familia-src Quinasas/metabolismoRESUMEN
Our previous data demonstrated that cellular and nuclear tyrosine-phosphorylated Vav associate with phosphoinositide 3-kinase during all-trans-retinoic acid-dependent granulocytic differentiation of HL-60 cells. In this study, aimed to analyze the mechanism by which Vav is recruited and activated, we report that the Src homology 2 domain of Vav interacts with tyrosine-phosphorylated proteins in a differentiation-dependent manner. Two adaptor proteins, Cbl and SLP-76, were identified, showing a discrete distribution inside the cells, with Cbl absent from the nuclei and SLP-76 particularly abundant in the nuclear compartment. Of note, Vav interacts with the tyrosine kinase Syk, which is also present in the nuclear compartment and may phosphorylate Vav in vitro when cells differentiate. Inhibition of Syk activity by piceatannol prevents both in vitro and in vivo Vav tyrosine phosphorylation, its association with the regulatory subunit of phosphoinositide 3-kinase, and the nuclear modifications typically observed during granulocytic differentiation of this cell line. These findings suggest that tyrosine-phosphorylated Vav and its association with phosphoinositide 3-kinase play a crucial role in all-trans-retinoic acid-induced reorganization of the nucleoskeleton, which is responsible for the changes in nuclear morphology observed during granulocytic differentiation of HL-60 cells.
Asunto(s)
Proteínas de Ciclo Celular , Diferenciación Celular/fisiología , Granulocitos/metabolismo , Células Madre Hematopoyéticas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Compartimento Celular/efectos de los fármacos , Compartimento Celular/genética , Diferenciación Celular/efectos de los fármacos , Núcleo Celular/efectos de los fármacos , Núcleo Celular/genética , Núcleo Celular/metabolismo , Inhibidores Enzimáticos/farmacología , Precursores Enzimáticos/efectos de los fármacos , Precursores Enzimáticos/genética , Precursores Enzimáticos/metabolismo , Granulocitos/citología , Granulocitos/efectos de los fármacos , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/efectos de los fármacos , Humanos , Péptidos y Proteínas de Señalización Intracelular , Proteína Oncogénica v-cbl , Fosfatidilinositol 3-Quinasas/efectos de los fármacos , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfoproteínas/efectos de los fármacos , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilación/efectos de los fármacos , Estructura Terciaria de Proteína/efectos de los fármacos , Estructura Terciaria de Proteína/genética , Proteínas Tirosina Quinasas/efectos de los fármacos , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/efectos de los fármacos , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-vav , Proteínas Oncogénicas de Retroviridae/efectos de los fármacos , Proteínas Oncogénicas de Retroviridae/genética , Proteínas Oncogénicas de Retroviridae/metabolismo , Estilbenos/farmacología , Quinasa Syk , Tretinoina/farmacología , Células Tumorales Cultivadas , Tirosina/efectos de los fármacos , Tirosina/metabolismoRESUMEN
The 120 kD product of the c-cbl oncogene is rapidly tyrosine phosphorylated and recruited to the EGF receptor following ligand binding. Cbl's oncogenic potential is activated by a large carboxy-terminal truncation that generated v-cbl and removes the Ring finger and proline-rich SH3-binding domains. Here we show that this truncation reveals a novel and highly conserved domain that can interact directly with the EGF receptor in a phosphorylation dependent manner. Furthermore we demonstrate that the v-cbl domain is not utilized by c-cbl for recruitment to the receptor since this binding property is not evident in c-cbl constructs with proline domain deletions, and it is only revealed following deletion of the Ring finger. We also analyse a loss-of-function mutation from the C. elegans homologue, sli-1, and show that the corresponding mutation in v-cbl ablates transformation and EGF receptor association. Thus our findings provide further evidence that v-cbl possesses a novel and evolutionarily conserved phosphotyrosine binding domain and that the dual capability of EGF receptor binding by cbl involves two distinct mechanisms. In addition these findings raise the possibility that v-cbl may transform by competing with c-cbl for phosphorylated binding sites on activated receptor complexes.
Asunto(s)
Proteínas de Caenorhabditis elegans , Transformación Celular Neoplásica/genética , Receptores ErbB/metabolismo , Proteínas del Helminto/genética , Mutación , Proteínas Oncogénicas de Retroviridae/genética , Ubiquitina-Proteína Ligasas , Células 3T3/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Caenorhabditis elegans/genética , Secuencia Conservada , Factor de Crecimiento Epidérmico/farmacología , Ratones , Proteína Oncogénica v-cbl , Fosforilación/efectos de los fármacos , Prolina/química , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-cbl , Conejos , Proteínas Oncogénicas de Retroviridae/efectos de los fármacos , Proteínas Oncogénicas de Retroviridae/metabolismo , Tirosina/metabolismoRESUMEN
The NF-kappa B/Rel family of transcription factors participates in the activation of a diverse range of genes involved in inflammation, immune response, lymphoid differentiation, growth control and development. The present review provides a brief overview of NF-kappa B/Rel activation and a detailed analysis of important biological and biochemical inhibitors of the NF-kappa B/Rel pathway. Given the pleiotropic role of NF-kappa B in controlling cytokines and other immunoregulatory genes, the inhibition of NF-kappa B activation by steroid hormones, antioxidants, protease inhibitors and other compounds may provide a pharmacological basis for interfering with pathological inflammatory conditions, cancer and AIDS.
Asunto(s)
Citocinas/biosíntesis , FN-kappa B/antagonistas & inhibidores , FN-kappa B/metabolismo , Proteínas Oncogénicas de Retroviridae/efectos de los fármacos , Proteínas Oncogénicas de Retroviridae/metabolismo , Factores de Transcripción , Animales , Antioxidantes/farmacología , Aspirina/farmacología , Ciclosporina/farmacología , Citocinas/efectos de los fármacos , Dinoprostona/farmacología , Predicción , Glucocorticoides/farmacología , Humanos , Inmunosupresores/farmacología , Mutación , Óxido Nítrico/metabolismo , Proteínas Oncogénicas v-rel , Fosforilación/efectos de los fármacos , Inhibidores de Proteasas/farmacología , Proteínas Proto-Oncogénicas/efectos de los fármacos , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Tacrolimus/farmacología , Factor de Transcripción ReIBRESUMEN
P120cbl, the product of the c-cbl proto-oncogene, has previously been shown to become tyrosine phosphorylated following EGF stimulation of cells, and to bind constitutively to the SH3 domain of the adaptor protein Grb2. Here we show that another adaptor protein, Crk, binds through its SH2 domain to tyrosine phosphorylated p120cbl. In addition, Crk becomes phosphorylated on tyrosine and serine following EGF treatment of PC12 and other cell lines. In unstimulated cells, while Grb2 is not bound to any tyrosine phosphoprotein, Crk is bound via its SH2 domain to tyrosine phosphorylated p130cas, the Crk-associated v-Src substrate. Following EGF treatment, Crk dissociates from p130cas, possibly due to a higher affinity of Crk SH2 for p120cbl compared with p130cas. Interaction between Grb2 and p120cbl increases threefold following EGF treatment of cells; in vitro, this induction of Grb2 association with unphosphorylated p120cbl can be mimicked by the addition of tyrosine phosphorylated Shc, suggesting a transfer of information between the SH2 and SH3 domains of Grb2. These data indicate that adaptor proteins can exchange binding partners in response to stimuli, and that different adaptor proteins can bind to the same partners by different mechanisms.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Adaptadoras del Transporte Vesicular , Moléculas de Adhesión Celular/metabolismo , Proteínas Oncogénicas/metabolismo , Fosfoproteínas/metabolismo , Proteínas/metabolismo , Proteínas Oncogénicas de Retroviridae/metabolismo , Transducción de Señal , Animales , Sitios de Unión , Cateninas , Proteína Sustrato Asociada a CrK , Factor de Crecimiento Epidérmico/metabolismo , Factor de Crecimiento Epidérmico/farmacología , Proteína Adaptadora GRB2 , Proteína Oncogénica v-crk , Células PC12/efectos de los fármacos , Fosforilación , Pruebas de Precipitina , Proteínas/efectos de los fármacos , Ratas , Proteína p130 Similar a la del Retinoblastoma , Proteínas Oncogénicas de Retroviridae/efectos de los fármacos , Proteínas Adaptadoras de la Señalización Shc , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src , Tirosina/metabolismo , Dominios Homologos src , Catenina deltaRESUMEN
Highly immunogenic ("xenogenized") tumor variants appear after treatment of murine lymphoma L5178Y with the triazene derivative DTIC, this phenomenon being associated with the appearance of structurally abnormal gp70 env proteins in the cell variants. In the present study, we have isolated and sequenced several PCR-amplified gp70 cDNA genes from L5178Y cells. One of the resulting clones was used as a probe in Southern and Northern analysis of parental and xenogenized cells. The results indicated that xenogenization of tumor cells is associated with changes in retroviral env sequences detectable at the genomic level.
Asunto(s)
Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Genes Virales/efectos de los fármacos , Proteínas Oncogénicas de Retroviridae/genética , Proteínas Oncogénicas de Retroviridae/inmunología , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/inmunología , Animales , Secuencia de Bases , Southern Blotting , Clonación Molecular , Dacarbazina/farmacología , Femenino , Genes Virales/inmunología , Leucemia L5178/genética , Leucemia L5178/inmunología , Masculino , Ratones , Ratones Endogámicos DBA , Datos de Secuencia Molecular , Polimorfismo de Longitud del Fragmento de Restricción , Proteínas Oncogénicas de Retroviridae/efectos de los fármacos , Transcripción Genética/efectos de los fármacos , Trasplante Heterólogo , Proteínas del Envoltorio Viral/efectos de los fármacosRESUMEN
Metallothionein has been implicated in resistance to anticancer drugs. We examined whether transient induction of metallothionein by dexamethasone causes resistance to cis-diamminedichloroplatinum(II) (cis-DDP) in malignant and nonmalignant cells. Normal rat kidney cells (6m2) were infected with a modified v-mos oncogene construct in which expression of v-mos and consequently transformation was temperature-sensitive occurring at the permissive temperature of less than 33 degrees C and not at the nonpermissive temperature of 37 degrees C. Temperature-sensitive oncogenic transformation by v-mos attenuated induction of metallothionein by dexamethasone. No induction of metallothionein was observed in a revertant 6m2 cell line (54-5A4), which expressed v-mos and was transformed at 37 degrees C. Only nontransformed 6m2 cells displayed resistance to cis-DDP after dexamethasone pretreatment for 24 h. Dexamethasone pretreatment did not cause marked resistance to doxorubicin or melphalan in nontransformed 6m2 cells. When 6m2 cells (37 degrees C) were pretreated with dexamethasone (0.5 microM) for 24 h and then incubated in dexamethasone-free medium for 24 h, both metallothionein levels and resistance to cis-DDP decreased significantly. Thus, transient resistance to cis-DDP can be produced by a nonmetal inducer of metallothionein in nontransformed cells. Glucocorticoid-induced protection is suppressed in cells expressing v-mos and this might form the basis of future strategies to improve the therapeutic index of cis-DDP.
Asunto(s)
Transformación Celular Neoplásica/genética , Dexametasona/antagonistas & inhibidores , Riñón/metabolismo , Metalotioneína/biosíntesis , Compuestos Organoplatinos/metabolismo , Proteínas Oncogénicas de Retroviridae/genética , Animales , Transformación Celular Neoplásica/inducido químicamente , Transformación Celular Neoplásica/metabolismo , Resistencia a Medicamentos , Riñón/efectos de los fármacos , Proteínas Oncogénicas v-mos , Compuestos Organoplatinos/antagonistas & inhibidores , Ratas , Proteínas Oncogénicas de Retroviridae/efectos de los fármacosRESUMEN
Vitamin E succinate inhibited proliferation of C4#1 cells, an established avian retrovirus [reticuloendotheliosis virus (REV)]-transformed immature lymphoid tumor cell line, in a dose-dependent manner. The cytostatic effects of vitamin E succinate were reversible in that treated cells regained their ability to divide after vitamin E succinate removal. Possible mechanism(s) for the antiproliferative actions of vitamin E succinate were investigated. Analyses of C4#1 cell surface membrane antigen profiles and morphology indicated that vitamin E succinate was not inducing differentiation of the tumor cells to a more mature, differentiated, nonproliferative state. Five antioxidants, including a synthetic analogue of vitamin E, Trolox, as well as the active vitamin form, DL-alpha-tocopherol, were incapable of inhibiting C4#1 tumor cell growth, indicating that a mechanism of action other than or in addition to functions as an antioxidant may be operating. Cell cycle analyses suggested that C4#1 tumor cells treated with vitamin E succinate were blocked in the G0G1/early S phases of the cell cycle. Tumor growth arrested by vitamin E succinate did not affect the expression of the REV-encoded oncogene, v-rel, at either the RNA or protein level. These studies demonstrated that vitamin E, in the form of vitamin E succinate, inhibited the growth of retrovirus-transformed tumor cells in vitro and suggested that the antiproliferative effects of vitamin E succinate did not involve antioxidant properties but rather, as yet, unidentified mechanisms leading to cell cycle blockage.