RESUMEN
Glia cells are involved in upper motor neuron degeneration in amyotrophic lateral sclerosis (ALS). Protease activated receptor 1 (PAR1) pathway is related to brain pathologies. Brain PAR1 is located on peri-synaptic astrocytes, adjacent to pyramidal motor neurons, suggesting possible involvement in ALS. Brain thrombin activity in superoxide dismutase 1 (SOD1) mice was measured using a fluorometric assay, and PAR1 levels by western blot. PAR1 was localized using immunohistochemistry staining. Treatment targeted PAR1 pathway on three levels; thrombin inhibitor TLCK (N-Tosyl-Lys-chloromethylketone), PAR1 antagonist SCH-79797 and the Ras intracellular inhibitor FTS (S-trans-trans-farnesylthiosalicylic acid). Mice were weighed and assessed for motor function and survival. SOD1 brain thrombin activity was increased (p < 0.001) particularly in the posterior frontal lobe (p = 0.027) and hindbrain (p < 0.01). PAR1 levels were decreased (p < 0.001, brain, spinal cord, p < 0.05). PAR1 and glial fibrillary acidic protein (GFAP) staining decreased in the cerebellum and cortex. SOD1 mice lost weight (≥17 weeks, p = 0.047), and showed shorter rotarod time (≥14 weeks, p < 0.01). FTS 40mg/kg significantly improved rotarod scores (p < 0.001). Survival improved with all treatments (p < 0.01 for all treatments). PAR1 antagonism was the most efficient, with a median survival improvement of 10 days (p < 0.0001). Our results support PAR1 pathway involvement in ALS.
Asunto(s)
Esclerosis Amiotrófica Lateral/metabolismo , Encéfalo/metabolismo , Modelos Animales de Enfermedad , Receptor PAR-1/metabolismo , Superóxido Dismutasa-1/metabolismo , Esclerosis Amiotrófica Lateral/genética , Animales , Astrocitos/metabolismo , Peso Corporal/efectos de los fármacos , Farnesol/análogos & derivados , Farnesol/farmacología , Proteína Ácida Fibrilar de la Glía/metabolismo , Humanos , Ratones , Ratones Transgénicos , Neuronas Motoras/metabolismo , Mutación , Pirroles/farmacología , Quinazolinas/farmacología , Salicilatos/farmacología , Transducción de Señal/efectos de los fármacos , Superóxido Dismutasa-1/genética , Análisis de Supervivencia , Clorometilcetona Tosilisina/farmacologíaRESUMEN
In some organisms, small RNA pathways can act nonautonomously, with responses spreading from cell to cell. Dedicated intercellular RNA delivery pathways have not yet been characterized in mammals, although secretory compartments have been found to contain RNA. Here we show that, upon cell contact, T cells acquire from B cells small RNAs that can impact the expression of target genes in the recipient T cells. Synthetic microRNA (miRNA) mimetics, viral miRNAs expressed by infected B cells, and endogenous miRNAs could all be transferred into T cells. These mechanisms may allow small RNA-mediated communication between immune cells. The documented transfer of viral miRNAs raises the possible exploitation of these pathways for viral manipulation of the host immune response.
Asunto(s)
Linfocitos B/metabolismo , Comunicación Celular , Regulación de la Expresión Génica , MicroARNs/metabolismo , ARN Viral/metabolismo , Linfocitos T/metabolismo , Células Cultivadas , Humanos , Células JurkatRESUMEN
Alternative splicing is a key cellular mechanism for generating distinct isoforms, whose relative abundances regulate critical cellular processes. It is therefore essential that inclusion levels of alternative exons be tightly regulated. However, how the precision of inclusion levels among individual cells is governed is poorly understood. Using single-cell gene expression, we show that the precision of inclusion levels of alternative exons is determined by the degree of evolutionary conservation at their flanking intronic regions. Moreover, the inclusion levels of alternative exons, as well as the expression levels of the transcripts harboring them, also contribute to this precision. We further show that alternative exons whose inclusion levels are considerably changed during stem cell differentiation are also subject to this regulation. Our results imply that alternative splicing is coordinately regulated to achieve accuracy in relative isoform abundances and that such accuracy may be important in determining cell fate.
Asunto(s)
Empalme Alternativo , Regulación de la Expresión Génica , ARN Mensajero/metabolismo , Diferenciación Celular , Evolución Molecular , Exones , Perfilación de la Expresión Génica/métodos , Genoma Humano , Células HEK293 , Humanos , Células MCF-7 , Análisis de la Célula Individual , Células Madre/citologíaRESUMEN
The Ras oncogene transmits signals, which regulate various cellular processes including cell motility, differentiation, growth and death. Since Ras signalling is abnormally activated in more than 30% of human cancers, Ras and its downstream signalling pathways are considered good targets for therapeutic interference. Ras is post-translationally modified by the addition of a farnesyl group, which permits its attachment to the plasma membrane. Exploiting this knowledge, a synthetic Ras inhibitor, S-trans, trans-farnesylthiosalicylic acid (FTS; Salirasib), was developed. FTS resembles the farnesylcysteine group of Ras, and acts as an effective Ras antagonist. In the present review, the effect of FTS in combination with various other drugs, as tested in vitro and in vivo, and its therapeutic potential are discussed. As reviewed, FTS cooperates with diverse therapeutic agents, which significantly improves treatment outcome. Therefore, combinations of FTS with other agents have a potential to serve as anti-cancer or anti-inflammatory therapies.
Asunto(s)
Antineoplásicos/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Farnesol/análogos & derivados , Neoplasias/tratamiento farmacológico , Salicilatos/farmacología , Transducción de Señal/efectos de los fármacos , Animales , Antiinflamatorios/farmacología , Farnesol/farmacología , HumanosRESUMEN
The ETS transcription factor ERG plays a central role in definitive hematopoiesis, and its overexpression in acute myeloid leukemia (AML) is associated with a stem cell signature and poor prognosis. Yet how ERG causes leukemia is unclear. Here we show that pan-hematopoietic ERG expression induces an early progenitor myeloid leukemia in transgenic mice. Integrated genome-scale analysis of gene expression and ERG binding profiles revealed that ERG activates a transcriptional program similar to human AML stem/progenitor cells and to human AML with high ERG expression. This transcriptional program was associated with activation of RAS that was required for leukemia cells growth in vitro and in vivo. We further show that ERG induces expression of the Pim1 kinase oncogene through a novel hematopoietic enhancer validated in transgenic mice and human CD34(+) normal and leukemic cells. Pim1 inhibition disrupts growth and induces apoptosis of ERG-expressing leukemic cells. The importance of the ERG/PIM1 axis is further underscored by the poorer prognosis of AML highly expressing ERG and PIM1. Thus, integrative genomic analysis demonstrates that ERG causes myeloid progenitor leukemia characterized by an induction of leukemia stem cell transcriptional programs. Pim1 and the RAS pathway are potential therapeutic targets of these high-risk leukemias.
Asunto(s)
Regulación Leucémica de la Expresión Génica/fisiología , Leucemia Mieloide Aguda/genética , Proteínas Proto-Oncogénicas c-pim-1/metabolismo , Transactivadores/genética , Factores de Transcripción/metabolismo , Animales , Antineoplásicos , Elementos de Facilitación Genéticos/genética , Genómica , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones SCID , Ratones Transgénicos , Células Progenitoras Mieloides/fisiología , Trasplante de Neoplasias , Transcripción Genética/fisiología , Regulador Transcripcional ERGRESUMEN
Many cases of heritable environmental responses have been documented but the underlying mechanisms are largely unknown. Recently, inherited RNA interference has been shown to act as a multigenerational genome surveillance apparatus. We suggest that inheritance of regulatory RNAs is at the root of many other epigenetic phenomena, the trigger that induces other epigenetic mechanisms, such as the depositing of histone modifications and DNA methylation. In addition, we explore the possibility that interacting organisms influence each other's transcriptomes by exchanging heterologous non-coding RNAs.
Asunto(s)
Evolución Biológica , Epigénesis Genética/fisiología , Células Procariotas/fisiología , ARN no Traducido , Animales , PlantasRESUMEN
Lymphocytes establish dynamic cell-cell interactions with the cells they scan. Previous studies show that upon cell contact, various membrane-associated proteins, such as Ras-family proteins, transfer from B to T and NK lymphocytes. Mutations in RAS genes that encode constitutively active, GTP-bound, oncoproteins are rather common in human cancers; for instance, melanoma. Cancer immunoediting has been postulated to contribute to the elimination of malignant melanoma. Thus, we asked whether Ras oncoproteins can transfer from melanoma to T cells, including tumor-infiltrating lymphocytes (TILs), and subsequently induce functional effects in the adopting T cells. To explore this issue, we genetically engineered an HLA-A2(+) melanoma cell line, MEL526, to express GFP or GFP-tagged H-Ras mutants stably. In this study, we show by an in vitro coculture system that GFP-tagged H-Ras, but not GFP, transfers from MEL526 to T cells and localizes to the inner aspect of their plasma membrane. This cell-contact-dependent process was increased by TCR stimulation and did not require strict Ag specificity. Importantly, we found a positive correlation between the levels of the acquired constitutively active H-RasG12V and ERK1/2 phosphorylation within the adopting TILs. We also show a significant increase in IFN-γ production and cytotoxic activity in TILs that acquired H-RasG12V compared to TILs that acquired a different H-Ras mutant. In conclusion, our findings demonstrate a hitherto unknown phenomenon of intercellular transfer of Ras oncoproteins from melanoma to TILs that consequently augments their effector functions.
Asunto(s)
Linfocitos Infiltrantes de Tumor/inmunología , Melanoma/inmunología , Melanoma/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/fisiología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Línea Celular , Técnicas de Cocultivo , Humanos , Linfocitos Infiltrantes de Tumor/metabolismo , Linfocitos Infiltrantes de Tumor/patología , Melanoma/patología , Cultivo Primario de Células , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Linfocitos T/patología , Transfección , Células Tumorales CultivadasRESUMEN
BACKGROUND: Hepatic stellate cells (HSCs) have a key role in the formation of hepatic fibrosis. The active form of vitamin D, 1,25(OH)2D3, has been found to have antiproliferative and antifibrotic effects in various tissues including liver. Farnesylthiosalicylic acid (FTS), a novel Ras antagonist, was also found to inhibit hepatic fibrosis. AIMS: The purpose of this study was to examine the antiproliferative and antifibrotic effects of the combined treatment of 1,25(OH)2D3 and FTS on primary cultured HSCs. METHODS: Primary HSCs, isolated from rat's livers, were treated with 1,25(OH)2D3, FTS or a combination of both. Proliferation was assessed by bromodeoxyuridine. Expression of p-ERK, ERK, Ras-GTP, total-Ras, CyclinD1 and fibrotic markers was measured by western blotting analysis and real-time PCR. Cytotoxicity was assessed by lactate dehydrogenase method. RESULTS: The combined treatment inhibited HSCs proliferation by threefold. The effect was synergistic and non-cytotoxic. In concordance, the combined treatment suppressed CyclinD1 expression by ~2-fold, whereas 1,25(OH)2D3 or FTS alone showed a significantly lower inhibitory effect. The effect of the combined treatment on CyclinD1 expression was mediated via Ras-GTP and p-ERK signal transduction pathway. The effect on fibrotic markers showed that 1,25(OH)2D3 decreased collagen Iα1 expression by ~40%, FTS by ~50% and the combined treatment by ~60%. 1,25(OH)2D3 inhibited tissue inhibitor of metalloproteinases-1 (TIMP-1) expression by 20%. FTS alone or 1,25(OH)2D3 + FTS inhibited TIMP-1 expression by 60%. FTS inhibited transforming growth factor-ß (TGF-ß) expression by 25%, while 1,25(OH)2D3 had no effect. CONCLUSION: Although the combination of 1,25(OH)2D3 and FTS did not demonstrate an additive antifibrotic effect, it showed a synergistic antiproliferative effect on primary HSCs. Therefore, the combined treatment may have a potential therapeutic value in the initiation of fibrotic process.
Asunto(s)
Calcitriol/farmacología , Farnesol/análogos & derivados , Células Estrelladas Hepáticas/efectos de los fármacos , Salicilatos/farmacología , Animales , Biomarcadores , Calcitriol/administración & dosificación , Proliferación Celular , Ciclina D1 , Sinergismo Farmacológico , Quinasas MAP Reguladas por Señal Extracelular/genética , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Farnesol/administración & dosificación , Farnesol/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Células Estrelladas Hepáticas/fisiología , Masculino , Ratas , Ratas Wistar , Salicilatos/administración & dosificación , Proteínas ras/genética , Proteínas ras/metabolismoRESUMEN
Non-cell-autonomous proteins are incorporated into cells that form tight contacts or are invaded by bacteria, but identifying the full repertoire of transferred proteins has been a challenge. Here we introduce a quantitative proteomics approach to sort out non-cell-autonomous proteins synthesized by other cells or intracellular pathogens. Our approach combines stable-isotope labeling of amino acids in cell culture (SILAC), high-purity cell sorting and bioinformatics analysis to identify the repertoire of relevant non-cell-autonomous proteins. This 'trans-SILAC' method allowed us to discover many proteins transferred from human B to natural killer cells and to measure biosynthesis rates of Salmonella enterica proteins in infected human cells. Trans-SILAC should be a useful method to examine protein exchange between different cells of multicellular organisms or pathogen and host.
Asunto(s)
Marcaje Isotópico/métodos , Transporte de Proteínas , Proteoma , Antígeno B7-2/metabolismo , Proteínas Bacterianas/biosíntesis , Comunicación Celular , Células Cultivadas , Humanos , Biosíntesis de ProteínasRESUMEN
The small GTPase proteins, Ras and Rheb, serve as molecular switches regulating cell proliferation, differentiation and apoptosis. Ras also regulates Rheb by inactivating the tuberous sclerosis complex (TSC), which includes products of the TSC1 and TSC2 genes encoding hamartin (TSC1) and tuberin (TSC2), respectively, and acts as a Rheb-specific GTPase-activating protein. Loss of function of TSC1 or TSC2 results in an increase in active Rheb.GTP with the consequent translational abnormalities and excessive cell proliferation characteristic of the genetic disorders, tuberous sclerosis and lymphangioleiomyomatosis (LAM). To determine whether inactivation of Rheb, Ras or both might be a potential treatment for LAM, we used TSC2-null ELT3 cells as a LAM model. The cells were treated with the Ras inhibitor S-trans,trans-farnesylthiosalicylic acid (FTS; salirasib), which mimics the C-terminal S-farnesyl cysteine common to Ras and Rheb. This C-terminus is critical for their attachment to cellular membranes and for their biological activities. Untreated, the ELT3 cells expressed significant amounts of Rheb but little Ras.GTP, and this phenotype was reversed by TSC2 reexpression. Treatment with FTS decreased Ras.GTP only slightly in the TSC2-null cells, but reduced their overactive Rheb as well as their proliferation, migration and tumor growth. Notably, TSC2 reexpression in these ELT3 cells rescued them from the inhibitory effect of FTS. Evidently, therefore, FTS blocks active Rheb in TSC2-null ELT3 cells and may have therapeutic potential for LAM.
Asunto(s)
Farnesol/análogos & derivados , Linfangioleiomiomatosis/tratamiento farmacológico , Proteínas de Unión al GTP Monoméricas/antagonistas & inhibidores , Neuropéptidos/antagonistas & inhibidores , Salicilatos/farmacología , Proteínas Supresoras de Tumor/deficiencia , Animales , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Cisteína/análogos & derivados , Cisteína/metabolismo , Farnesol/farmacología , Linfangioleiomiomatosis/genética , Linfangioleiomiomatosis/metabolismo , Linfangioleiomiomatosis/patología , Linfocitos Nulos , Ratones , Ratones Desnudos , Proteínas de Unión al GTP Monoméricas/genética , Proteínas de Unión al GTP Monoméricas/metabolismo , Mutación/genética , Neuropéptidos/genética , Neuropéptidos/metabolismo , Fosforilación/efectos de los fármacos , Proteína Homóloga de Ras Enriquecida en el Cerebro , Ratas , Proteínas Quinasas S6 Ribosómicas/metabolismo , Proteína 2 del Complejo de la Esclerosis Tuberosa , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Proteínas ras/genética , Proteínas ras/metabolismoRESUMEN
Emerging data suggest that regulatory T cell (Treg) dysfunction and consequent breakdown of immunological self-tolerance in autoimmunity can be mediated by factors that are not Treg-intrinsic (e.g., cytokines). Indeed, recent studies show that in rheumatoid arthritis the proinflammatory cytokine TNF reduces the suppressive function of Tregs, whereas in vivo TNF blockade restores this function and accordingly self-tolerance. However, until now a coherent mechanism by which TNF regulates the Treg has not been described. In this paper, we show that TNF induces preferential and significant activation of the canonical NF-kappaB pathway in human Tregs as compared with CD25(-) conventional T cells. Furthermore, TNF induced primarily in CD45RA(-) Tregs a transcription program highly enriched for typical NF-kappaB target genes, such as the cytokines lymphotoxin-alpha and TNF, the TNFR superfamily members FAS, 4-1BB, and OX-40, various antiapoptotic genes, and other important immune-response genes. FACS analysis revealed that TNF also induced upregulation of cell surface expression of 4-1BB and OX40 specifically in CD45RA(-)FOXP3(+) Tregs. In contrast, TNF had only a minimal effect on the Treg's core transcriptional signature or on the intracellular levels of the FOXP3 protein in Tregs. Importantly, TNF treatment modulated the capacity of Tregs to suppress the proliferation and IFN-gamma secretion by conventional T cells, an effect that was fully reversed by cotreatment with anti-TNFR2 mAbs. Our findings thus provide new mechanistic insight into the role of TNF and TNFR2 in the pathogenesis of autoimmunity.
Asunto(s)
Regulación de la Expresión Génica/inmunología , Tolerancia Inmunológica/inmunología , FN-kappa B/inmunología , Subgrupos de Linfocitos T/inmunología , Linfocitos T Reguladores/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Autoinmunidad/inmunología , Separación Celular , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Factores de Transcripción Forkhead/biosíntesis , Factores de Transcripción Forkhead/inmunología , Expresión Génica , Humanos , Interferón gamma/biosíntesis , Interferón gamma/inmunología , Antígenos Comunes de Leucocito/biosíntesis , Antígenos Comunes de Leucocito/inmunología , Activación de Linfocitos/inmunología , FN-kappa B/metabolismo , Receptores Tipo II del Factor de Necrosis Tumoral/biosíntesis , Receptores Tipo II del Factor de Necrosis Tumoral/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/inmunología , Subgrupos de Linfocitos T/metabolismo , Linfocitos T Reguladores/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
BACKGROUND: Ras proteins are crucial for cell differentiation and proliferation. Targeting Ras with farnesylthiosalicylic acid (FTS), a Ras antagonist, has been suggested as a therapeutic strategy in proliferative and inflammatory diseases. AIMS: To examine the role of Ras and the therapeutic potential of FTS in experimental colitis. METHODS: Colitis was induced in 26 mice by adding 2.5% dextran sodium sulfate to their drinking water for 7 days during which 12 study mice were treated with FTS and 14 control mice were given normal saline. Two additional controls included 10 naïve mice treated with FTS and 7 naïve non-treated mice. The animals were followed clinically and sacrificed after 7 days. Their colons were isolated for histological assessment and for measurement of myeloperoxidase activity (MPO), tumor necrosis factor-α(TNF-α), and interleukin-1ß(Il-1ß) levels. Ras and activated Ras expression was determined by immunoblotting assays. T cell populations in the colon and spleen were analyzed by flow-cytometry. RESULTS: FTS induced a 2.1-fold reduction in activated Ras levels (P < 0.004). FTS-treated mice had lower disease activity scores (3.9 ± 1.7 vs. 7.5 ± 2.3, P < 0.001), and lower levels of MPO activity (1.65 ± 0.6 vs. 2.6 ± 0.8 units/g, P < 0.007), Il-1ß (2.4 ± 3.6 vs. 24.3 ± 17.5 pg/mg, P < 0.01) and TNF-α (0.63 ± 0.5 vs. 1.9 ± 1 pg/mg, P < 0.04). FTS increased regulatory T cell population in the spleen (1.9 ± 0.4-fold, P < 0.04), and decreased effector T cell populations in the colon and spleen by 24 ± 3% (P < 0.03) and 27 ± 1% (P < 0.02), respectively. FTS had no remarkable side effects. CONCLUSIONS: Ras is involved in the inflammatory processes of induced colitis in mice and its inhibition by FTS ameliorates the severity of the inflammation.
Asunto(s)
Colitis/prevención & control , Colitis/fisiopatología , Inhibidores Enzimáticos/uso terapéutico , Farnesol/análogos & derivados , Salicilatos/uso terapéutico , Proteínas ras/antagonistas & inhibidores , Proteínas ras/fisiología , Animales , Western Blotting , Colon/patología , Inhibidores Enzimáticos/farmacología , Farnesol/uso terapéutico , Femenino , Citometría de Flujo , Interleucina-1beta/análisis , Ratones , Ratones Endogámicos BALB C , Técnicas de Cultivo de Órganos , Factor de Necrosis Tumoral alfa/análisisRESUMEN
Nasal epithelial cells secret mucins and are exposed in vivo to airflow-induced mechanophysical stresses, including wall shear stress (WSS), temperature, and humidity. In this work, human nasal epithelial cells cultured under air-liquid interface conditions were subjected to fields of airflow-induced oscillatory WSS at different temperature and humidity conditions. Changes in mucin secretion due to WSS were measured and the role of the cytoskeleton in mucin secretion was explored. Mucin secretion significantly increased in response to WSS in a magnitude-dependent manner with respect to static cultures and independently of the airflow temperature and humidity. In static cultures, mucin secretion decreased at high humidity with or without elevation of the temperature with respect to cultures at a comfortable climate. In cultures exposed to WSS, mucin secretion increased at high temperature with respect to cultures at comfortable climate conditions. The polymerization of actin microfilaments was shown to increase mucin secretion under WSS, whereas the dynamics of microtubule polymerization did not affect secretion. In conclusion, the data in this study show that mucin secretion is sensitive to oscillatory WSS as well as high temperature and humidity conditions.
Asunto(s)
Células Epiteliales/metabolismo , Mucinas/metabolismo , Mucosa Nasal/citología , Mucosa Nasal/metabolismo , Estrés Mecánico , Citoesqueleto de Actina/efectos de los fármacos , Citoesqueleto de Actina/metabolismo , Adulto , Fenómenos Biomecánicos/efectos de los fármacos , Células Cultivadas , Colchicina/farmacología , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Humanos , Tubulina (Proteína)/metabolismoRESUMEN
Histone deacetylase (HDAC) inhibitors, such as valproic acid (VPA), constitute a novel class of anticancer agents that cause an increase in acetylated histones and thus restore the expression of dormant tumor-suppressor and other genes related to cell differentiation, cell-cycle arrest or apoptosis of tumor cells. The Ras inhibitor farnesylthiosalicylic acid (FTS, salirasib) attenuates cancer cell proliferation in vitro and in vivo and, under certain circumstances, induces cell death. FTS by itself does not induce differentiation or complete growth arrest. The abovementioned activity of VPA as a differentiation agent suggested that it might be worth investigating its possible therapeutic potential in synergistic combination with FTS. Here, we examined whether the combined application of VPA and FTS could synergistically inhibit the proliferation of cancer cells that express oncogenic K-Ras (A549 nonsmall-cell lung carcinoma cells), DLD1 (colon carcinoma cells) or chronically active wild-type K-Ras and constitutively active B-Raf (ARO, thyroid carcinoma cells). The results showed that combined treatment with VPA and FTS synergistically reduces proliferation in all of these cancer cell lines by downregulating Ras and blocking the expression of Survivin and Aurora A. These alterations, which were most pronounced following the combined treatment, led to a mitotic crisis, as reflected by mislocalization of the chromosomal passenger complex. Our findings thus demonstrate that combination therapy with VPA and FTS might offer a promising therapeutic approach to the treatment of epithelial tumors.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Proteínas Asociadas a Microtúbulos/genética , Proteínas Serina-Treonina Quinasas/genética , Antineoplásicos/uso terapéutico , Aurora Quinasas , Western Blotting , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Células Pequeñas/patología , División Celular , Línea Celular Tumoral , Neoplasias del Colon/patología , Regulación hacia Abajo/efectos de los fármacos , Farnesol/análogos & derivados , Farnesol/uso terapéutico , Citometría de Flujo , Inhibidores de Histona Desacetilasas/uso terapéutico , Histona Desacetilasas/genética , Histona Desacetilasas/metabolismo , Humanos , Proteínas Inhibidoras de la Apoptosis , Neoplasias Pulmonares/patología , ARN Neoplásico/genética , ARN Neoplásico/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Salicilatos/uso terapéutico , Survivin , Ácido Valproico/uso terapéuticoRESUMEN
K-Ras functions as a critical node in the mitogen-activated protein kinase (MAPK) pathway that regulates key cellular functions including proliferation, differentiation, and apoptosis. Following growth factor receptor activation K-Ras.GTP forms nanoclusters on the plasma membrane through interaction with the scaffold protein galectin-3. The generation of nanoclusters is essential for high fidelity signal transduction via the MAPK pathway. To explore the mechanisms underlying K-Ras.GTP nanocluster formation, we developed a mathematical model of K-Ras-galectin-3 interactions. We designed a computational method to calculate protein collision rates based on experimentally determined protein diffusion rates and diffusion mechanisms and used a genetic algorithm to search the values of key model parameters. The optimal estimated model parameters were validated using experimental data. The resulting model accurately replicates critical features of K-Ras nanoclustering, including a fixed ratio of clustered K-Ras.GTP to monomeric K-Ras.GTP that is independent of the concentration of K-Ras.GTP. The model reproduces experimental results showing that the cytosolic level of galectin-3 determines the magnitude of the K-Ras.GTP clustered fraction and illustrates that nanoclustering is regulated by key nonequilibrium processes. Our kinetic model identifies a potential biophysical mechanism for K-Ras nanoclustering and suggests general principles that may be relevant for other plasma-membrane-localized proteins.
Asunto(s)
Membrana Celular/enzimología , Modelos Biológicos , Nanoestructuras/química , Proteínas ras/metabolismo , Simulación por Computador , Activación Enzimática , Galectina 3/química , Galectina 3/metabolismo , Guanosina Trifosfato/metabolismo , Estructura Cuaternaria de Proteína , Proteínas ras/químicaRESUMEN
A miniaturized, bead-based protein-protein-interaction assay was developed to study the interaction of Rho GTPases with regulatory proteins. The setup, which uses only minute amounts of sample, was used to analyze small molecules that inhibit the interaction between Rho GTPases and RhoGDI alpha. Prenylcysteine analogues and the replacement of GDP by non-hydrolysable GTP analogues prevented the formation of Rho GTPase-RhoGDI alpha complexes in a concentration-dependent manner.
Asunto(s)
Inhibidores de Disociación de Guanina Nucleótido/análisis , Proteómica/métodos , Proteínas de Unión al GTP rho/análisis , Activación Enzimática , Inhibidores de Disociación de Guanina Nucleótido/metabolismo , Unión Proteica , Proteínas de Unión al GTP rho/antagonistas & inhibidores , Proteínas de Unión al GTP rho/metabolismo , Inhibidores de la Disociación del Nucleótido Guanina rho-EspecíficoRESUMEN
Ras proteins regulate cell growth, differentiation, and apoptosis from various cellular platforms. We have recently identified a novel potential signaling platform, the rasosome, which moves rapidly near the plasma membrane (PM) and in the cytosol, carrying multiple copies of palmitoylated Ras proteins. In the present study we demonstrate that rasosomes are unique entities distinct from PM nanoclusters or from endocytotic compartments. In addition, we examine whether rasosomes can act as regulated Ras signaling platforms. We show that a single rasosome simultaneously carries different types of Ras molecules in their active and inactive state, suggesting that rasosomes can upload and download Ras signals. Total internal reflection fluorescence (TIRF) microscopy combined with fast time-lapse and a new spatial analysis algorithm demonstrate that rasosome movement near the PM is restricted to distinctive areas, rasosomal 'hotspots', localized between actin filament cages. In addition, Ras-binding domain of Raf-1 (RBD) is recruited to Ras in rasosomal hotspots as revealed by bimolecular fluorescence complementation experiments. Interestingly, epidermal growth factor stimulates H/NRas activation on rasosomes and the subsequent recruitment of RBD to rasosomes. Moreover, we show that rasosomes are loaded with Ras downstream effectors and modulators. These findings establish that physiological stimulation originating from PM hotspots is transduced to rasosomes, which appear to serve as robust Ras signaling platforms that spread signals across the cell.
Asunto(s)
Membrana Celular/metabolismo , Modelos Biológicos , Complejos Multienzimáticos/metabolismo , Transducción de Señal/fisiología , Proteínas ras/metabolismo , Animales , Células COS , Membrana Celular/genética , Chlorocebus aethiops , Activación Enzimática/fisiología , Lipoilación/fisiología , Complejos Multienzimáticos/genética , Proteínas ras/genéticaRESUMEN
In neuroblastoma LAN-1 cells harboring an amplified MycN gene, disruption of cooperation between Ras and MycN proteins by the Ras inhibitor farnesylthiosalicylic acid (FTS, Salirasib) reportedly arrests cell growth. Our aim was to establish whether this is a general phenomenon. We examined the effects of FTS on gene-expression profiles, growth and death of NCIH929 myeloma cells and K562 leukemia cells, which-like LAN-1 cells-exhibit Myc gene amplification and harbor active Ras. Under specified conditions, FTS reduced Ras and Myc and induced cell growth arrest and death in all Myc-amplified cell lines but not in SHEP, a neuroblastoma cell line without Myc gene amplification. Gene-expression analysis revealed a common pattern of FTS-induced endoplasmic reticulum (ER) stress, known as the unfolded protein response (UPR), in Myc-amplified cells, but not in SHEP. Thus, Ras negatively regulates ER stress in cells with amplified Myc. ER stress was also inducible by dominant-negative (DN)-Ras or shRNA to Ras isoforms, all of which induced an increase in BIP (the master regulator of ER stress) and its downstream targets Nrf2 and eIF2alpha, both regulated by active p-PERK. FTS also induced an increase in p-PERK, while small interfering RNA to PERK reduced Nrf2 and ATF4 and rescued cells from FTS-induced death. BIP and its downstream targets were also increased by inhibitors of MAPK p38 and MEK. Ras, acting through MAPK p38 and MEK, negatively regulates the ER stress cascades BIP/PERK/Nrf2 and eIF2alpha/ATF4/ATF3. These findings can explain the Ras-dependent protection of Myc-amplified cells from ER stress-associated death.
Asunto(s)
Antineoplásicos/farmacología , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Inhibidores Enzimáticos/farmacología , Farnesol/análogos & derivados , Amplificación de Genes , Proteínas Nucleares/genética , Proteínas Oncogénicas/genética , Salicilatos/farmacología , Respuesta de Proteína Desplegada , Proteínas ras/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Retículo Endoplásmico/genética , Farnesol/farmacología , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Células K562 , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Proteína Proto-Oncogénica N-Myc , Factor 2 Relacionado con NF-E2/metabolismo , Neuroblastoma , ARN Interferente Pequeño/metabolismo , Factores de Transcripción/metabolismo , Respuesta de Proteína Desplegada/efectos de los fármacos , Respuesta de Proteína Desplegada/genética , eIF-2 Quinasa/metabolismo , Proteínas ras/antagonistas & inhibidoresRESUMEN
Neurofibromatosis type 1 (NF1) is a genetic disorder characterized inter alia by cognitive and motor dysfunction and appearance of high-signal foci on T2-weighted images in the brain. Nf1(+/-) mice are useful models for studying aspects of NF1, including cognitive deficits. Here we assessed their motor performance and used quantitative transverse T2 relaxation MRI to identify structural abnormalities in their brains. Nf1(+/-) mice exhibited both enhanced and reduced T2 signals in distinct brain regions compared to wild-type mice, and their motor performance was impaired. As in NF1 patients, enhanced T2 signals in Nf1(+/-) mice were observed in the thalamus and basal ganglia. Reduced T2 signals were seen in motor-associated regions along the motor pathway, predominantly in the white matter of the cerebral peduncle and the optic tract. Correlation analysis between T2 signals and motor performance suggested that the motor deficits are associated with impairments in the cerebral peduncle and the amygdala.
Asunto(s)
Modelos Animales de Enfermedad , Imagen por Resonancia Magnética/métodos , Actividad Motora/fisiología , Neurofibromatosis 1/fisiopatología , Análisis de Varianza , Animales , Conducta Animal , Encéfalo/anomalías , Encéfalo/fisiopatología , Ratones , Ratones Endogámicos C57BL , Modelos Estadísticos , Neurofibromina 1/deficiencia , Neurofibromina 1/metabolismoRESUMEN
Neurofibromin contains several domains, most notably a GAP-related domain (GRD), that down-regulates Ras pathways. The functions of the non-GRD neurofibromin domains are largely known. Here we show that the pre-GRD region of neurofibromin alters the expression of genes involved in cell adhesion and migration and acts as a negative regulator of the Rac1/Pak1/LIMK1/cofilin pathway. Thus, neurofibromin-deficient glioblastoma and mouse fibroblasts are enriched in Rac1-GTP, p-Pak1, p-LIMK1 and p-cofilin, with all proteins exhibiting decreased expression upon expression of NF1(1-1163) polypeptide. Concomitantly, actin stress fibers and focal adhesion were disassembled and cell migration was halted. These effects were independent of the Ras signaling pathways. It seems that NF1(1-1163), through negative regulation of Rac-1, shifts the balance from a state of inactive phospho-cofilin to active unphosphorylated cofilin, resulting in severing of F-actin. Impairment of these cellular functions of neurofibromin provides novel insights into the invasiveness/progression of NF1-associated tumors.