RESUMEN
The objective of the present study was to investigate some commonly detected halogenated textile pollutants for their bioavailability and hazardous properties. Release into artificial sweat and skin absorption in vitro were examined as well as mutagenic effects by Ames test, and skin-sensitizing properties from a peptide reactivity assay combined with a cell test. All investigated compounds were shown to migrate from the textile into sweat and be absorbed by the skin, although to a different extent. The experimental values for migration were found to be up to 390 times higher compared to literature values. Two of the studied compounds, 2,5-dinitrochlorobenzene and 3,5-dinitrobromobenzene, both exhibited mutagenic effects in the Ames test, while both 2,5-dinitrochlorobenzene and 2,6-dichlorobenzene-1,4-diamine were classified as skin sensitizers. The allergenic reactivity of the latter was found to be due to an oxidized transformation product. Risks for the induction of skin allergy and other non-carcinogenic effects from dermal exposure to the individual compounds were found low, even when considering clothing with the highest reported levels. However, the complex mixtures of chemicals often present in garments may still constitute a health risk, especially when considering the many hours of daily exposure. It is important to further study the toxicity of other frequently occurring chemicals as well as the synergistic effects of chemicals that co-occur in clothing.
RESUMEN
The aim of this study was to assess the potential use of a selective small molecule MALT1 inhibitor in solid tumor treatment as an immunotherapy targeting regulatory T-cells (Tregs). In vitro, MALT1 inhibition suppressed the proteolytic cleavage of the MALT1-substrate HOIL1 and blocked IL-2 secretion in Jurkat cells. It selectively suppressed the proliferation of PBMC-derived Tregs, with no effect on conventional CD4+T-cells. In vivo, however, no evident anti-tumor effect was achieved by MALT1 inhibition monotherapy or in combination with anti-CTLA4 in the MB49 cancer model. Despite decreased Treg-frequencies in lymph nodes of tumor-bearing animals, intratumoral Treg depletion was not observed. We also showed that MALT1-inhibition caused a reduction of antigen-specific CD8+T-cells in an adoptive T-cell transfer model. Thus, selective targeting of Tregs would be required to improve the immunotherapeutic effect of MALT1-inhibition. Also, various dosing schedules and combination therapy strategies should be carefully designed and evaluated further.
Asunto(s)
Leucocitos Mononucleares , Neoplasias , Animales , Linfocitos T Reguladores , Linfocitos T CD8-positivos , ProteolisisRESUMEN
Deciphering transcription factor networks from microarray data remains difficult. This study presents a simple method to infer the regulation of transcription factors from microarray data based on well-characterized target genes. We generated a catalog containing transcription factors associated with 2720 target genes and 6401 experimentally validated regulations. When it was available, a distinction between transcriptional activation and inhibition was included for each regulation. Next, we built a tool (www.tfacts.org) that compares submitted gene lists with target genes in the catalog to detect regulated transcription factors. TFactS was validated with published lists of regulated genes in various models and compared to tools based on in silico promoter analysis. We next analyzed the NCI60 cancer microarray data set and showed the regulation of SOX10, MITF and JUN in melanomas. We then performed microarray experiments comparing gene expression response of human fibroblasts stimulated by different growth factors. TFactS predicted the specific activation of Signal transducer and activator of transcription factors by PDGF-BB, which was confirmed experimentally. Our results show that the expression levels of transcription factor target genes constitute a robust signature for transcription factor regulation, and can be efficiently used for microarray data mining.
Asunto(s)
Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Análisis de Secuencia por Matrices de Oligonucleótidos , Programas Informáticos , Factores de Transcripción/metabolismo , Becaplermina , Catálogos como Asunto , Línea Celular Tumoral , Células Cultivadas , Interpretación Estadística de Datos , Humanos , Factor de Crecimiento Derivado de Plaquetas/farmacología , Proteínas Proto-Oncogénicas c-sis , Reproducibilidad de los Resultados , Factores de Transcripción STAT/metabolismo , Interfaz Usuario-ComputadorRESUMEN
Growth factors of the PDGF and FGF families act through receptor tyrosine kinases. These receptors can be activated by chromosomal rearrangements in myeloid neoplasms associated with hypereosinophilia. We identified a new fusion gene between KANK1 and PDGFRbeta in a patient with thrombocythemia. We showed that such fusion oncoproteins derived from PDGF and FGF receptors escape the normal degradation pathways, leading to their accumulation in cells. This process amplifies signalling leading to cell proliferation. Using microarrays and bioinformatics, we showed that several transcription factors contribute to the control cell growth, including STATS, FOXO and SREBP.
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Neoplasias/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos/fisiología , Receptores del Factor de Crecimiento Derivado de Plaquetas/fisiología , Humanos , Neoplasias/patologíaRESUMEN
BACKGROUND: Chimeric oncogenes encoding constitutively active protein tyrosine kinases are associated with chronic myeloid neoplasms. TEL-PDGFRbeta (TPbeta, also called ETV6-PDGFRB) is a hybrid protein produced by the t(5;12) translocation, FIP1L1-PDGFRalpha (FPalpha) results from a deletion on chromosome 4q12 and ZNF198-FGFR1 is created by the t(8;13) translocation. These fusion proteins are found in patients with myeloid neoplasms associated with eosinophilia. Wild-type receptor tyrosine kinases are efficiently targeted for degradation upon activation, in a process that requires Cbl-mediated monoubiquitination of receptor lysines. Since protein degradation pathways have been identified as useful targets for cancer therapy, the aim of this study was to compare the degradation of hybrid and wild-type receptor tyrosine kinases. DESIGN AND METHODS: We used Ba/F3 as a model cell line, as well as leukocytes from two patients, to analyze hybrid protein degradation. RESULTS: In contrast to the corresponding wild-type receptors, which are quickly degraded upon activation, we observed that TPbeta, FPalpha and the ZNF198-FGFR1 hybrids escaped down-regulation in Ba/F3 cells. The high stability of TPbeta and FPalpha hybrid proteins was confirmed in leukocytes from leukemia patients. Ubiquitination of TPbeta and FPalpha was much reduced compared to that of wild-type receptors, despite marked Cbl phosphorylation in cells expressing hybrid receptors. The fusion of a destabilizing domain to TPbeta induced protein degradation. Instability was reverted by adding the destabilizing domain ligand, Shield1. The destabilization of this modified TPbeta reduced cell transformation and STAT5 activation. CONCLUSIONS: We have shown that chimeric receptor tyrosine kinases escape ubiquitination and down-regulation and that their stabilization is critical to efficient stimulation of cell proliferation.
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Proteínas de Fusión Oncogénica/metabolismo , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Ubiquitinación , Factores de Escisión y Poliadenilación de ARNm/metabolismo , Anciano , Animales , Becaplermina , Western Blotting , Línea Celular , Proliferación Celular , Células Cultivadas , Genotipo , Humanos , Síndrome Hipereosinofílico/sangre , Leucemia Mielomonocítica Crónica/sangre , Leucocitos/citología , Leucocitos/efectos de los fármacos , Leucocitos/metabolismo , Masculino , Persona de Mediana Edad , Proteínas de Fusión Oncogénica/genética , Fosforilación , Factor de Crecimiento Derivado de Plaquetas/farmacología , Proteínas Proto-Oncogénicas c-cbl/metabolismo , Proteínas Proto-Oncogénicas c-sis , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/genética , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/genética , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Transfección , Factores de Escisión y Poliadenilación de ARNm/genéticaRESUMEN
Highly potent and selective 4-amidofuran-3-one inhibitors of cathepsin S are described. The synthesis and structure-activity relationship of a series of inhibitors with a sulfonamide moiety in the P3 position is presented. Several members of the series show sub-nanomolar inhibition of the target enzyme as well as an excellent selectivity profile and good cellular potency. Molecular modeling of the most interesting inhibitors describes interactions in the extended S3 pocket and explains the observed selectivity towards cathepsin K.
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Catepsinas/antagonistas & inhibidores , Furanos/química , Inhibidores de Proteasas/química , Sulfonamidas/química , Catepsina K , Simulación por Computador , Furanos/síntesis química , Furanos/farmacología , Humanos , Modelos Moleculares , Inhibidores de Proteasas/síntesis química , Inhibidores de Proteasas/farmacología , Relación Estructura-Actividad , Sulfonamidas/síntesis química , Sulfonamidas/farmacologíaRESUMEN
Sterol-regulatory element binding proteins (SREBPs) control the expression of genes involved in fatty acid and cholesterol biosynthesis. Using microarrays, we observed that mature SREBP-1 also induced the expression of genes unrelated to lipid metabolism, such as heme oxygenase 1 (HMOX1), plasma glutathione peroxidase, the phosphatidylinositol-3 kinase regulatory subunit p55 gamma, synaptic vesicle glycoprotein 2A, and COTE1. The expression of these genes was repressed upon addition of sterols, which block endogenous SREBP cleavage, and was induced by the statin drug mevinolin. Stimulation of fibroblasts with platelet-derived growth factor, which activates SREBP-1, had a similar effect. Fasted mice that were refed with a high-carbohydrate diet presented an increased expression of HMOX1 and p55 gamma in the liver. Overall, the transcriptional signature of SREBP-1 in fibroblasts stimulated by growth factors was very similar to that described in liver cells. We analyzed the HMOX1 promoter and found one SREBP binding site of the E-box type, which was required for regulation by SREBP-1a and SREBP-1c but was insensitive to SREBP-2. In conclusion, our data suggest that SREBP-1 regulates the expression of stress response and signaling genes, which could contribute to the metabolic response to insulin and growth factors in various tissues.
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Regulación Enzimológica de la Expresión Génica , Hemo-Oxigenasa 1/biosíntesis , Fosfatidilinositol 3-Quinasas/biosíntesis , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/fisiología , Animales , Línea Celular Tumoral , Fibroblastos/metabolismo , Humanos , Hidroxicolesteroles/farmacología , Lovastatina/farmacología , Masculino , Ratones , Factor de Crecimiento Derivado de Plaquetas/farmacología , Regiones Promotoras Genéticas/fisiologíaRESUMEN
The majority of polycythemia vera (PV) patients harbor a unique somatic mutation (V617F) in the pseudokinase domain of JAK2, which leads to constitutive signaling. Here we show that the homologous mutations in JAK1 (V658F) and in Tyk2 (V678F) lead to constitutive activation of these kinases. Their expression induces autonomous growth of cytokine-dependent cells and constitutive activation of STAT5, STAT3, mitogen-activated protein kinase, and Akt signaling in Ba/F3 cells. The mutant JAKs exhibit constitutive signaling also when expressed in fibrosarcoma cells deficient in JAK proteins. Expression of the JAK2 V617F mutant renders Ba/F3 cells hypersensitive to insulin-like growth factor 1 (IGF1), which is a hallmark of PV erythroid progenitors. Upon selection of Ba/F3 cells for autonomous growth induced by the JAK2 V617F mutant, cells respond to IGF1 by activating STAT5, STAT3, Erk1/2, and Akt on top of the constitutive activation characteristic of autonomous cells. The synergic effect on proliferation and STAT activation appears specific to the JAK2 V617F mutant. Our results show that the homologous V617F mutation induces activation of JAK1 and Tyk2, suggesting a common mechanism of activation for the JAK1, JAK2, and Tyk2 mutants. JAK3 is not activated by the homologous mutation M592F, despite the presence of the conserved GVC preceding sequence. We suggest that mutations in the JAK1 and Tyk2 genes may be identified as initial molecular defects in human cancers and autoimmune diseases.
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Mutación , Policitemia Vera/enzimología , Policitemia Vera/genética , Proteínas Tirosina Quinasas/metabolismo , Receptor Cross-Talk , Receptor IGF Tipo 1/metabolismo , Línea Celular Tumoral , Activación Enzimática , Humanos , Janus Quinasa 1 , Ligandos , Transducción de Señal , TYK2 Quinasa , Factores de Transcripción/metabolismoRESUMEN
We analyzed the transcriptional program elicited by stimulation of normal human fibroblasts with platelet-derived growth factor (PDGF) using cDNA microarrays. 103 significantly regulated transcripts that had not been previously linked to PDGF signaling were identified. Among them, a cluster of genes involved in fatty acid and cholesterol biosynthesis, including stearoyl-CoA desaturase (SCD), fatty acid synthase, and hydroxymethylglutaryl-CoA synthase (HMGCS), was up-regulated by PDGF after 24 h of treatment, and their expression correlated with increased membrane lipid production. These genes are known to be controlled by sterol regulatory element-binding proteins (SREBP). PDGF increased the amount of mature SREBP-1 and regulated the promoters of SCD and HMGCS in an SREBP-dependent manner. In line with these results, blocking SREBP processing by addition of 25-hydroxycholesterol blunted the effects of PDGF on lipogenic enzymes. SREBP activation was dependent on the phosphatidylinositol 3-kinase (PI3K) pathway, as judged from the effects of the inhibitor LY294002 and mutation of the PDGFbeta receptor tyrosines that bind the PI3K adaptor subunit p85. Fibroblast growth factors (FGF-2 and FGF-4) and other growth factors mimicked the effects of PDGF on NIH3T3 and human fibroblasts. In conclusion, our results suggest that growth factors induce membrane lipid synthesis via the activation SREBP and PI3K.
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Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Lípidos de la Membrana/biosíntesis , Lípidos de la Membrana/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Factores de Transcripción/metabolismo , Células Cultivadas , Activación Enzimática/efectos de los fármacos , Humanos , Proteína 1 de Unión a los Elementos Reguladores de EsterolesRESUMEN
Gab1 is a scaffolding/docking protein that has been suggested to play a role in signal transduction downstream of certain plasma membrane receptors, including platelet-derived growth factor (PDGF) receptors. We found that PDGF induced a rapid Gab1 phosphorylation, which depended on the recruitment of Grb2, indicating that Grb2 acts as a bridge between Gab1 and the PDGF beta-receptor. PDGF also enhanced the binding of Gab1 to the phosphatase SHP-2, but not to p85. To further study the role of Gab1 in PDGF signaling, we transfected porcine aortic endothelial cells with a doxycycline-inducible Gab1 construct. Increased Gab1 expression enhanced the recruitment and activation of SHP-2, as well as the phosphorylation of the mitogen-activated protein kinases Erk and p38 by PDGF. Gab1 expression also enhanced the formation of lamellipodia and cellular protrusions. In Gab1-deficient mouse embryonic fibroblasts, the same phenotype was induced by restoring the expression of wild-type Gab1, but not a mutant Gab1 that was unable to associate with SHP-2. These effects of PDGF on the actin cytoskeleton were not altered by the inhibition of p38 or Erk, but could be blocked by a dominant-negative form of Rac (Asn(17)). Finally, Gab1-deficient fibroblasts showed a decreased chemotactic response toward gradients of PDGF as compared with wild-type cells. In conclusion, Gab1 plays a selective role in the regulation of the mitogen-activated protein kinases Erk and p38 downstream of the PDGF beta-receptor, and contributes to cytoskeletal reorganization and chemotaxis in response to PDGF.
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Quimiotaxis , Citoesqueleto/química , Fosfoproteínas/fisiología , Factor de Crecimiento Derivado de Plaquetas/química , Proteínas Adaptadoras Transductoras de Señales , Animales , Becaplermina , Western Blotting , Células COS , Células Cultivadas , Citoesqueleto/metabolismo , Proteínas de Unión al ADN/metabolismo , Relación Dosis-Respuesta a Droga , Activación Enzimática , Fibroblastos/metabolismo , Regulación de la Expresión Génica , Genes Dominantes , Glutatión Transferasa/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular , Ratones , Microscopía Fluorescente , Nitrofenoles/química , Compuestos Organofosforados/química , Fosforilación , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Pruebas de Precipitina , Unión Proteica , Proteína Tirosina Fosfatasa no Receptora Tipo 11 , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas Proto-Oncogénicas c-sis , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Factor de Transcripción STAT3 , Transducción de Señal , Porcinos , Factores de Tiempo , Transactivadores/metabolismo , TransfecciónRESUMEN
Proline-rich tyrosine kinase 2 (Pyk2), a non-receptor tyrosine kinase structurally related to focal adhesion kinase, has been implicated in the regulation of mitogen-activated protein kinase cascades and ion channels, the induction of apoptosis, and in the modulation of the cytoskeleton. In order to understand how Pyk2 signaling mediates these diverse cellular functions, we performed a yeast two-hybrid screening using the C-terminal part of Pyk2 that contains potential protein-protein interaction sites as bait. A prominent binder of Pyk2 identified by this method was the Arf-GTPase-activating protein ASAP1. Pyk2-ASAP1 interaction was confirmed in pull-down as well as in co-immunoprecipitation experiments, and contact sites were mapped to the proline-rich regions of Pyk2 and the SH3 domain of ASAP1. Pyk2 directly phosphorylates ASAP1 on tyrosine residues in vitro and increases ASAP1 tyrosine phosphorylation when co-expressed in HEK293T cells. Phosphorylation of tyrosine 308 and 782 affects the phosphoinositide binding profile of ASAP1, and fluorimetric Arf-GTPase assays with purified proteins revealed an inhibition of ASAP1 GTPase-activating protein activity by Pyk2-mediated tyrosine phosphorylation. We therefore provide evidence for a functional interaction between Pyk2 and ASAP1 and a regulation of ASAP1 and hence Arf1 activity by Pyk2-mediated tyrosine phosphorylation.
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Factor 1 de Ribosilacion-ADP/metabolismo , Proteínas Portadoras/metabolismo , Proteínas Tirosina Quinasas/fisiología , Animales , Línea Celular , ADN Complementario/metabolismo , Quinasa 2 de Adhesión Focal , Glutatión Transferasa/metabolismo , Humanos , Sistema de Señalización de MAP Quinasas , Mutagénesis Sitio-Dirigida , Células PC12 , Mapeo Peptídico , Fosforilación , Pruebas de Precipitina , Prolina/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Tirosina Quinasas/metabolismo , Ratas , Proteínas Recombinantes de Fusión/metabolismo , Factores de Tiempo , Transfección , Técnicas del Sistema de Dos Híbridos , Tirosina/química , Dominios Homologos srcRESUMEN
We have previously shown that the binding site for GTPase activating protein of Ras (RasGAP) in the PDGF beta-receptor, Tyr771, is phosphorylated to a much lower extent in the heterodimeric configuration of PDGF alpha- and beta-receptors, compared to the PDGF beta-receptor homodimer. The decreased recruitment of the RasGAP to the receptor leads to prolonged activation of the Ras/MAP kinase pathway, which could explain the increase in mitogenicity seen upon induction of heterodimers. The molecular mechanism underlying these differences was investigated. We could show that the loss of phosphorylation of Tyr771 was dependent on presence of intact binding sites for the protein tyrosine phosphatase SHP-2 on the PDGF beta-receptor. Thus, in PDGF receptor mutants in which binding of SHP-2 was lost, a higher degree of phosphorylation of Tyr771 was seen, while other phosphorylation sites in the receptor remained virtually unaffected. Thus, SHP-2 appears to play an important role in modulating phosphorylation of Y771, thereby controlling RasGAP recruitment and Ras/MAP kinase signaling in the heterodimeric configuration of the PDGF receptors.