RESUMEN
OBJECTIVE: Cannabinoids are able to reduce tumor growth in xenograft models, but their therapeutic potential as anti-cancer drugs in humans is unclear yet. In vitro studies of the effect of cannabinoids on cancer cells are often carried out in absence of serum or in low serum concentration (i.e. 0.5% serum), conditions that limit cellular growth and therefore can increase the response of cells to additional challenges such as the presence of cannabinoids. However, the tumor microenvironment can be teaming with growth factors. In this study we assessed the viability and proliferation of cancer cells treated with cannabidiol in presence of a serum concentration that commonly sustains cell growth (10% serum). RESULTS: The results show that cannabidiol exerts a markedly different effect on the viability of the human HT-29 cancer cell line when cultured in presence of 0.5% serum in comparison to 10% serum, displaying a cytotoxic effect only in the former situation. In presence of 10% serum, no inhibitory effect of cannabidiol on DNA replication of HT-29 cells was detected, and a weak inhibition was observed for other cancer cell lines. These results indicate that the effect of cannabidiol is cell context-dependent being modulated by the presence of growth factors.
Asunto(s)
Antineoplásicos , Cannabidiol , Cannabinoides , Neoplasias , Antineoplásicos/farmacología , Cannabidiol/farmacología , Cannabinoides/farmacología , Línea Celular Tumoral , Proliferación Celular , Humanos , Neoplasias/tratamiento farmacológicoRESUMEN
The lymphocyte function-associated antigen-1 (LFA-1) (also known as CD11a/CD18 and αLß2), is just one of many integrins in the human body, but its significance is derived from its exclusive presence in leukocytes. In this review, we summarize the studies relating LFA-1 and its major ligand ICAM-1 (or CD54) with cancer, through the function of lymphocytes and myeloid cells on tumor cells. We consider how LFA-1 mediates the interaction of leukocytes with tumors and the role of ICAM-1 in tumor dynamics, which can be independent of its interaction with LFA-1. We also offer a more detailed examination of the role of LFA-1 within B-cell chronic lymphocytic leukemia. Finally, we discuss the role that exosomes harboring LFA-1 play in tumor growth and metastasis.
RESUMEN
Ataxia-telangiectasia mutated (ATM) is a cell cycle checkpoint kinase that upon activation by DNA damage leads to cell cycle arrest and DNA repair or apoptosis. The absence of Atm or the occurrence of loss-of-function mutations in Atm predisposes to tumorigenesis. MAPK7 has been implicated in numerous types of cancer with pro-survival and pro-growth roles in tumor cells, but its functional relation with tumor suppressors is not clear. In this study, we show that absence of MAPK7 delays death due to spontaneous tumor development in Atm-/- mice. Compared with Atm-/- thymocytes, Mapk7-/-Atm-/- thymocytes exhibited an improved response to DNA damage (increased phosphorylation of H2AX) and a restored apoptotic response after treatment of mice with ionizing radiation. These findings define an antagonistic function of ATM and MAPK7 in the thymocyte response to DNA damage, and suggest that the lack of MAPK7 inhibits thymic lymphoma growth in Atm-/- mice by partially restoring the DNA damage response in thymocytes.
Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada/genética , Transformación Celular Neoplásica/genética , Proteína Quinasa 7 Activada por Mitógenos/genética , Animales , Proteínas de la Ataxia Telangiectasia Mutada/deficiencia , Linfocitos B/metabolismo , Linfocitos B/patología , Ciclo Celular/genética , Transformación Celular Neoplásica/metabolismo , Daño del ADN/genética , Daño del ADN/efectos de la radiación , Eliminación de Gen , Expresión Génica , Hematopoyesis/genética , Histonas/metabolismo , Ratones , Ratones Noqueados , Proteína Quinasa 7 Activada por Mitógenos/metabolismo , Mutación , Fosforilación , Radiación Ionizante , Transducción de Señal , Timocitos/metabolismo , Timocitos/patologíaRESUMEN
Regulation of the levels of the TCR/CD3 complex at the cell surface is critical to proper T cell development and mature T cell activation. We provide evidence that the MAPK ERK5 regulates the surface expression of the TCR/CD3 complex by controlling the degradation of the CD3ζ chain and the recovery of the complex after anti-CD3ε stimulation. ERK5 knockdown led to TCR/CD3 up-regulation at the cell surface and increased amounts of the CD3ζ chain. Inhibition of the MEK5-dependent phosphorylation status of the kinase domain of ERK5 in human T CD4(+) cells reduced CD3ζ ubiquitination and degradation, limiting TCR/CD3 down-regulation in anti-CD3-stimulated cells. Moreover, TCR/CD3 recovery at the cell surface, after anti-CD3ε treatment, is impaired by ERK5 knockdown or pharmacological inhibition of autophosphorylation in the ERK5 C-terminal region. ERK5 loss in thymocytes augmented cellular CD3ζ and increased cell surface levels of TCR/CD3 on CD4(+)CD8(+) thymocytes. This correlated with enhanced generation of CD4(+)CD8(-)CD25(+) thymocytes. Our findings define ERK5 as a novel kinase that modulates the levels of TCR/CD3 at the cell surface by promoting CD3ζ degradation and TCR/CD3 recovery after TCR stimulation.
Asunto(s)
Regulación de la Expresión Génica , Proteína Quinasa 7 Activada por Mitógenos/metabolismo , Receptores de Antígenos de Linfocitos T/genética , Linfocitos T/metabolismo , Animales , Anticuerpos Monoclonales/farmacología , Complejo CD3/inmunología , Complejo CD3/metabolismo , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Línea Celular , Membrana Celular/metabolismo , Regulación hacia Abajo , Técnicas de Silenciamiento del Gen , Humanos , Células Jurkat , Ratones , Ratones Noqueados , Ratones Transgénicos , Proteína Quinasa 7 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 7 Activada por Mitógenos/genética , Proteolisis , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Timocitos/efectos de los fármacos , Timocitos/inmunología , Timocitos/metabolismo , UbiquitinaciónRESUMEN
An adequate supply of nucleotides is essential for accurate DNA replication, and inappropriate deoxyribonucleotide triphosphate (dNTP) concentrations can lead to replication stress, a common source of DNA damage, genomic instability and tumourigenesis. Here, we provide evidence that Erk5 is necessary for correct nucleotide supply during erythroid development. Mice with Erk5 knockout in the haematopoietic lineage showed impaired erythroid development in bone marrow, accompanied by altered dNTP levels and increased DNA mutagenesis in erythroid progenitors as detected by exome sequencing. Moreover, Erk5-depleted leukemic Jurkat cells presented a marked sensitivity to thymidine-induced S phase stalling, as evidenced by increased H2AX phosphorylation and apoptosis. The increase in thymidine sensitivity correlated with a higher dTTP/dCTP ratio. These results indicate that Erk5 is necessary to maintain the balance of nucleotide levels, thus preventing dNTP misincorporation and DNA damage in proliferative erythroid progenitors and leukemic Jurkat T cells.
Asunto(s)
Eritropoyesis/fisiología , Proteína Quinasa 7 Activada por Mitógenos/metabolismo , Timidina/farmacología , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Western Blotting , Ciclo Celular/efectos de los fármacos , Ciclo Celular/genética , Línea Celular Tumoral , Daño del ADN/efectos de los fármacos , Daño del ADN/genética , Desoxirribonucleósidos/metabolismo , Células Eritroides/efectos de los fármacos , Células Eritroides/metabolismo , Eritropoyesis/genética , Células HL-60 , Humanos , Células Jurkat/efectos de los fármacos , Células Jurkat/metabolismo , Ratones , Ratones Noqueados , Proteína Quinasa 7 Activada por Mitógenos/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Syndecans are cell membrane proteoglycans that can modulate the activity and dynamics of some growth factor receptors and integrins. Here, we show the down-regulation of integrin lymphocyte function-associated antigen-1 (LFA-1) and inhibition of adhesion of Jurkat T cells transfected with syndecan-2. The PDZ-binding domain in the cytoplasmic region of syndecan-2 was necessary to block the LFA-1 high-affinity conformation, and to reduce cellular adhesion. A second cytoplasmic motif comprising tyrosines 179 and 191, and serines 187 and 188 contributed also to reduce LFA-1 function and cellular adhesion. Inhibition of the LFA-1 high-affinity conformation by syndecan-2 was independent of the expression of the talin head domain and RhoA, Rac1 and Cdc42 GTPases. These results demonstrate the importance of PDZ-binding domain of syndecan-2 for controlling LFA-1 affinity and cell adhesion.
Asunto(s)
Adhesión Celular/fisiología , Antígeno-1 Asociado a Función de Linfocito/biosíntesis , Dominios PDZ/genética , Sindecano-2/metabolismo , Linfocitos B/metabolismo , Línea Celular Tumoral , Membrana Celular/metabolismo , Movimiento Celular , Regulación hacia Abajo , Endotelio/citología , Endotelio/metabolismo , Proteínas Activadoras de GTPasa/biosíntesis , Humanos , Células Jurkat , Antígeno-1 Asociado a Función de Linfocito/metabolismo , Fosfoproteínas/biosíntesis , Unión Proteica , Sindecano-2/genética , Linfocitos T/metabolismo , Talina/biosíntesis , Transfección , Proteína de Unión al GTP rac1/biosíntesis , Proteína de Unión al GTP rhoA/biosíntesisRESUMEN
T cells express the heparan sulphate proteoglycans syndecan-2 and syndecan-4. Syndecan-4 plays a T-cell inhibitory role; however, the function of syndecan-2 is unknown. In an attempt to examine this function, syndecan-2 was expressed constitutively in Jurkat T cells. Interestingly, the expression of syndecan-2 decreased the surface levels of T-cell receptor (TCR)/CD3 complex, concomitant with intracellular retention of CD3ε and partial degradation of the TCR-ζ chain. Immunofluorescence microscopy revealed that intracellular CD3ε co-located with Rab-4 endosomes. However, the intracellular pool of CD3ε did not recycle to the cell surface. The lower TCR/CD3 surface levels caused by syndecan-2 led to reduced TCR/CD3 responsiveness. We show that the cytosolic PDZ-binding domain of syndecan-2 is not necessary to elicit TCR/CD3 down-regulation. These results identify a previously unrecognized means of controlling surface TCR/CD3 expression by syndecan-2.
Asunto(s)
Complejo CD3/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Sindecano-2/inmunología , Regulación hacia Abajo , Humanos , Células JurkatRESUMEN
PURPOSE: The goal of this study was to characterize and compare mesenchymal stem cells from adult human adipose tissue (ADS cells) with progenitor cell lines from the human corneoscleral limbus and to analyze their potential for the expression of epithelial markers. METHODS: Stem cell markers (CD34, CD90, p63, and ABCG2) and epithelial cell markers (CK3/76, CK12, CK76, CK19, and CK1/5/10/14) were analyzed by immunostaining, flow cytometry, Western blot analysis, and PCR methods. The authors assayed adhesion and proliferation on different extracellular matrix proteins. RESULTS: ADS cells expressed a set of progenitor cell markers, including p63 and ABCG2. CK12 expression in ADS cell cultures increased spontaneously and progressively by differential adhesion, which demonstrates the cells' potential and capability to acquire epithelial-like cell characteristics. The authors observed an increase in the adhesion and proliferation of ADS cells seeded onto different basement membrane extracellular matrix proteins. Laminin substrates reduced the proliferative state of ADS cells. CONCLUSIONS: The expression of putative stem cell markers (CD90, ABCG2, and p63) and cytokeratins (CK12 and CK76) supports the hypothesis that ADS cells have self-renewal capacity and intrinsic plasticity that enables them to acquire some epithelial-like characteristics. Therefore, adult ADS cells could be a potential source for cell therapy in ocular surface regeneration.
Asunto(s)
Adipocitos/metabolismo , Tejido Adiposo/citología , Biomarcadores/metabolismo , Células Epiteliales/metabolismo , Limbo de la Córnea/metabolismo , Células del Estroma/citología , Tejido Adiposo/metabolismo , Adulto , Animales , Western Blotting , Diferenciación Celular , Células Cultivadas , Modelos Animales de Enfermedad , Células Epiteliales/citología , Citometría de Flujo , Humanos , Limbo de la Córnea/citología , Ratones , Microscopía de Contraste de Fase , Reacción en Cadena de la Polimerasa , ARN/análisis , Células Madre/metabolismoRESUMEN
Syndecans bind to cell adhesion molecules, growth factors and cytokines, and can act as coreceptors, and in this way modulate leukocyte cell function. Here, expression of the syndecans on primary human CD4 T cells was examined. Cell stimulation dramatically increased the amount of syndecan-4, and in a lower extent that of syndecan-2. Expression of syndecan-2 and -4 show different induction kinetics. Whereas syndecan-4 expression is fast and significant, that of syndecan-2 is more delayed and short-lived decreasing its mRNA expression at day 4. Both CD45RA+ naive and CD45RA- memory CD4 T cells express syndecan-2 and -4 upon activation. When incubated with human peripheral blood lymphocytes in a mixed leukocyte reaction, anti-syndecan-4 but not anti-syndecan-2 antibodies, decreased T cell proliferation. However, cross-linking of cell-bound syndecan-2 or syndecan-4 via immobilized antibodies blocked proliferation and decreased TNF production of T cells in the presence of optimal levels of anti-CD3. These findings suggest that syndecan-2 and -4 act as inhibitors of T cell activation. We also investigated the role that MAPK signalling pathways play in control of syndecan expression in T cells. We show that production of syndecan-2 but not syndecan-4 requires signaling via p38 MAP kinase alpha/beta in T CD4 cells. As mechanisms that confer syndecan-2 expression are unknown, we analyse the chromatin hypersensitivity of syndecan-2 promoter proximal region in Jurkat T cells and endothelial cells. The analysis reveals a chromatin accessible site in the +3.5kb intronic region, concomitant with a region showing high evolutionary conservation. We isolate and analyse 5'-flanking regions of human syndecan-2 gene, by transfection assays. The +3.5kb hypersensitive site in the intronic region demonstrates basal promoter activity in Jurkat. This study provides evidence for the up-regulation of syndecan-2 and -4 in human primary CD4 T cells during in vitro activation and suggest an inhibitory role for these syndecans in CD4 T cells.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Activación de Linfocitos/inmunología , Sindecano-2/inmunología , Sindecano-4/inmunología , Complejo CD3/inmunología , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/enzimología , Células Cultivadas , Cromatina/química , Desoxirribonucleasa I/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Humanos , Memoria Inmunológica/efectos de los fármacos , Intrones/genética , Proteínas Quinasas JNK Activadas por Mitógenos/antagonistas & inhibidores , Células Jurkat , Activación de Linfocitos/efectos de los fármacos , Prueba de Cultivo Mixto de Linfocitos , Regiones Promotoras Genéticas/genética , Inhibidores de Proteínas Quinasas/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Sindecano-2/genética , Sindecano-4/genética , Regulación hacia Arriba/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidoresAsunto(s)
Western Blotting/métodos , Reactivos de Enlaces Cruzados/química , Proteínas de Unión al ARN/análisis , Rayos Ultravioleta , Reactivos de Enlaces Cruzados/efectos de la radiación , Electroforesis en Gel de Poliacrilamida , Ensayo de Cambio de Movilidad Electroforética/métodos , Filtración , Proteínas/química , ARN/química , Proteínas de Unión al ARN/químicaRESUMEN
Posttranscriptional regulatory mechanisms control TNFalpha expression through AU-rich elements in the 3'UTR of its mRNA. This is mediated through Erk and p38 MAP kinase signaling, although the mechanisms involved remain poorly understood. Here, we show that the MAP kinase signal-integrating kinases (Mnks), which are activated by both these pathways, regulate TNFalpha expression in T cells via the 3'UTR. A selective Mnk inhibitor or siRNA-mediated knockdown of Mnk1 inhibits TNFalpha production in T cells, whereas Mnk1 overexpression enhances expression of a reporter construct containing the TNFalpha 3'UTR. We identify ARE binding proteins that are Mnk substrates, such as hnRNP A1, which they phosphorylate at two sites in vitro. hnRNP A1 is phosphorylated in response to T cell activation, and this is blocked by Mnk inhibition. Moreover, Mnk-mediated phosphorylation decreases binding of hnRNP A1 to TNFalpha-ARE in vitro or TNFalpha-mRNA in vivo. Therefore, Mnks are novel players in cytokine regulation and potential new targets for anti-inflammatory therapy.
Asunto(s)
Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/metabolismo , Sistema de Señalización de MAP Quinasas/fisiología , Factor de Necrosis Tumoral alfa/biosíntesis , Regiones no Traducidas 3'/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Genes Reporteros , Ribonucleoproteína Nuclear Heterogénea A1 , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/antagonistas & inhibidores , Humanos , Células Jurkat , Fosforilación , Unión Proteica/genética , Unión Proteica/inmunología , Biosíntesis de Proteínas/fisiología , ARN Mensajero/metabolismo , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/genética , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
The gene encoding the human TNF alpha receptor (TNFR) 2 contains polymorphisms in the 3' untranslated region (UTR). Previous studies have shown that some variant alleles in this region are associated with obesity and insulin resistance. However, the effect of these polymorphisms on the expression of TNFR2 has not been studied to date. To examine the role played by different haplotypes in the control of TNFR2 expression (haplotypes A1-A5, referring to nucleotides 1663 G/A, 1668 T/G, and 1690 T/C), we introduced these sequences into the 3'-UTR of a heterologous reporter gene and expressed the corresponding constructs in a human T-cell line. We demonstrate that a 485-nt fragment of the TNFR2 3'-UTR that contains a U-rich region decreases reporter expression and that haplotypes A1-A4 exert a stronger effect than A5. Furthermore, time-course assays of mRNA stability using actinomycin D revealed that haplotypes A1-A4 destabilize the mRNA. The proximal TNFR2 3'-UTR, independently of haplotype differences, responded to T-cell activation by increasing mRNA decay. Electromobility shift analysis demonstrated that protein(s) found in T-cell extracts bind to the 485-nt fragment. We suggest that an increased rate of TNFR2 mRNA decay protects cells from unrestrained TNF alpha effects and that this protection is weakened in A5 subjects. These findings may explain the association of this haplotype with obesity and increased leptin levels.
Asunto(s)
Regiones no Traducidas 3'/genética , Regulación de la Expresión Génica , Genes Reporteros/genética , Polimorfismo Genético/genética , Receptores Tipo II del Factor de Necrosis Tumoral/genética , Regiones no Traducidas 3'/química , Regiones no Traducidas 3'/metabolismo , Secuencia de Bases , Citomegalovirus/genética , Dactinomicina/farmacología , Estabilidad de Medicamentos , Citometría de Flujo , Expresión Génica/genética , Proteínas Fluorescentes Verdes/genética , Haplotipos , Humanos , Células Jurkat , Cinética , Leptina/sangre , Activación de Linfocitos , Datos de Secuencia Molecular , Obesidad/genética , Regiones Promotoras Genéticas/genética , Estructura Secundaria de Proteína , ARN Mensajero/análisis , Receptores Tipo II del Factor de Necrosis Tumoral/química , Receptores Tipo II del Factor de Necrosis Tumoral/metabolismo , Linfocitos T , TransfecciónRESUMEN
Adenosine- and uridine-rich elements (AREs) located in 3'-untranslated regions are the best-known determinants of RNA instability. These elements have also been shown to control translation in certain mRNAs, including mRNAs for prominent pro-inflammatory and tumor growth-related proteins, and physiological anti-inflammatory processes that target ARE-controlled translation of mRNAs coding for pro-inflammatory proteins have been described. A major research effort is now being made to understand the mechanisms by which the translation of these mRNAs is controlled and the signalling pathways involved. This review focuses on the role of ARE-containing gene translation in inflammation, and the disease models that have improved our understanding of ARE-mediated translational control.
Asunto(s)
Regiones no Traducidas 3' , Adenosina/química , Biosíntesis de Proteínas , ARN Mensajero/química , Uridina/química , Adenosina/metabolismo , Transducción de Señal , Linfocitos T/inmunología , Uridina/metabolismoRESUMEN
Patients with antibody deficiency disorders are highly susceptible to microbial infections. Intravenous (i.v.) immunoglobulin concentrates were originally developed as replacement therapy for such patients. The present study assesses the measles virus neutralizing antibody titers and the antibody-dependent cell-mediated cytotoxicity (ADCC) capacities against Epstein-Barr virus (EBV)-infected cells of immunoglobulin G (IgG) preparations produced for i.v. use (i.v. IgG). The level of neutralizing antibodies against measles virus was determined by a syncytium neutralization test with Vero cells as targets. The measles virus neutralizing antibody titers of the i.v. IgG preparations were >3 x 10(2) and were an average of 1.0 log higher than the titers in pooled plasma from healthy subjects. The two IgG preparations tested showed similar ADCC activities against EBV-infected Raji cells, being active at concentrations of 3 mg/ml or higher. i.v. IgG bound to Raji cells but not to the EBV-negative Ramos cells, as evaluated by flow cytometry. Our in vitro findings may provide further support for the use of i.v. IgG for the prevention and treatment of infections caused by specific viral pathogens.
Asunto(s)
Anticuerpos Antivirales/inmunología , Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , Inmunoglobulina G/administración & dosificación , Virus del Sarampión/inmunología , Animales , Anticuerpos Antivirales/sangre , Linfocitos B , Línea Celular Transformada , Chlorocebus aethiops , Pruebas Inmunológicas de Citotoxicidad , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Herpesvirus Humano 4/inmunología , Humanos , Inyecciones Intravenosas , Pruebas de Neutralización , Células VeroRESUMEN
Tumor necrosis factor alpha (TNF-alpha), a proinflammatory cytokine, is produced abundantly by monocytes and macrophages. We have compared LPS-stimulated TNF-alpha production and regulation in freshly isolated human monocytes and macrophages differentiated in vitro. A significant increase in LPS-induced TNF-alpha protein secretion was observed in macrophages over freshly isolated monocytes without comparable differences in TNF-alpha mRNA induction. Polysome gradient analysis showed polysome-mRNA distribution did not change, whereas TNF-alpha mRNA stability increased in macrophages. Tristetraprolin mRNA expression was constitutive and decreased with differentiation-linked kinetics. Blockable LPS-inducible MAP kinase activity (p38, ERK) affected TNF-alpha biosynthesis differentially at the transcriptional and post-transcriptional level throughout the culture period. We suggest that the increase in TNF-alpha secretion in macrophages relates to changes in post-transcriptional processing, which is regulated indirectly by the expression of RNA-binding proteins. Changes in gene expression throughout monocytic differentiation equip the cell to act as a more potent producer of this proinflammatory cytokine.