RESUMO
Septins, a family of GTP-binding proteins that assemble into higher order structures, interface with the membrane, actin filaments and microtubules, and are thus important regulators of cytoarchitecture. Septin 9 (SEPT9), which is frequently overexpressed in tumors and mutated in hereditary neuralgic amyotrophy (HNA), mediates the binding of septins to microtubules, but the molecular determinants of this interaction remained uncertain. We demonstrate that a short microtubule-associated protein (MAP)-like motif unique to SEPT9 isoform 1 (SEPT9_i1) drives septin octamer-microtubule interaction in cells and in vitro reconstitutions. Septin-microtubule association requires polymerizable septin octamers harboring SEPT9_i1. Although outside of the MAP-like motif, HNA mutations abrogate this association, identifying a putative regulatory domain. Removal of this domain from SEPT9_i1 sequesters septins on microtubules, promotes microtubule stability and alters actomyosin fiber distribution and tension. Thus, we identify key molecular determinants and potential regulatory roles of septin-microtubule interaction, paving the way to deciphering the mechanisms underlying septin-associated pathologies. This article has an associated First Person interview with the first author of the paper.
Assuntos
Septinas , Fibras de Estresse , Humanos , Proteínas Associadas aos Microtúbulos , Microtúbulos/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Septinas/genética , Septinas/metabolismo , Fibras de Estresse/metabolismoRESUMO
iASPP is a protein mostly known as an inhibitor of p53 pro-apoptotic activity and a predicted regulatory subunit of the PP1 phosphatase, which is often overexpressed in tumors. We report that iASPP associates with the microtubule plus-end binding protein EB1, a central regulator of microtubule dynamics, via an SxIP motif. iASPP silencing or mutation of the SxIP motif led to defective microtubule capture at the cortex of mitotic cells, leading to abnormal positioning of the mitotic spindle. These effects were recapitulated by the knockdown of the membrane-to-cortex linker Myosin-Ic (Myo1c), which we identified as a novel partner of iASPP. Moreover, iASPP or Myo1c knockdown cells failed to round up upon mitosis because of defective cortical stiffness. We propose that by increasing cortical rigidity, iASPP helps cancer cells maintain a spherical geometry suitable for proper mitotic spindle positioning and chromosome partitioning.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Mitose , Proteínas Repressoras/metabolismo , Fuso Acromático/metabolismo , Motivos de Aminoácidos , Células HEK293 , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Miosina Tipo I/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Ligação Proteica , Proteínas Repressoras/químicaRESUMO
Focal adhesion (FA) turnover depends on microtubules and actin. Microtubule ends are captured at FAs, where they induce rapid FA disassembly. However, actin's roles are less clear. Here, we use polarization-resolved microscopy, FRAP, live cell imaging, and a mutant of Adenomatous polyposis coli (APC-m4) defective in actin nucleation to investigate the role of actin assembly in FA turnover. We show that APC-mediated actin assembly is critical for maintaining normal F-actin levels, organization, and dynamics at FAs, along with organization of FA components. In WT cells, microtubules are captured repeatedly at FAs as they mature, but once a FA reaches peak maturity, the next microtubule capture event leads to delivery of an autophagosome, triggering FA disassembly. In APC-m4 cells, microtubule capture frequency and duration are altered, and there are long delays between autophagosome delivery and FA disassembly. Thus, APC-mediated actin assembly is required for normal feedback between microtubules and FAs, and maintaining FAs in a state "primed" for microtubule-induced turnover.
Assuntos
Actinas/metabolismo , Proteína da Polipose Adenomatosa do Colo/metabolismo , Adesões Focais/metabolismo , Microtúbulos/metabolismo , Humanos , Células Tumorais CultivadasRESUMO
Oral-facial-digital (OFD) syndromes are rare heterogeneous disorders characterized by the association of abnormalities of the face, the oral cavity and the extremities, some due to mutations in proteins of the transition zone of the primary cilia or the closely associated distal end of centrioles. These two structures are essential for the formation of functional cilia, and for signaling events during development. We report here causal compound heterozygous mutations of KIAA0753/OFIP in a patient with an OFD VI syndrome. We show that the KIAA0753/OFIP protein, whose sequence is conserved in ciliated species, associates with centrosome/centriole and pericentriolar satellites in human cells and forms a complex with FOR20 and OFD1. The decreased expression of any component of this ternary complex in RPE1 cells causes a defective recruitment onto centrosomes and satellites. The OFD KIAA0753/OFIP mutant loses its capacity to interact with FOR20 and OFD1, which may be the molecular basis of the defect. We also show that KIAA0753/OFIP has microtubule-stabilizing activity. OFD1 and FOR20 are known to regulate the integrity of the centriole distal end, confirming that this structural element is a target of importance for pathogenic mutations in ciliopathies.
Assuntos
Centríolos/metabolismo , Centrossomo/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Síndromes Orofaciodigitais/metabolismo , Proteínas/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Centríolos/ultraestrutura , Centrossomo/ultraestrutura , Cílios/genética , Cílios/metabolismo , Cílios/patologia , Sequência Conservada , Feminino , Expressão Gênica , Heterozigoto , Humanos , Recém-Nascido , Proteínas Associadas aos Microtúbulos/genética , Dados de Sequência Molecular , Mutação , Síndromes Orofaciodigitais/genética , Síndromes Orofaciodigitais/patologia , Ligação Proteica , Proteínas/genética , Alinhamento de SequênciaRESUMO
Aberrant glycosylation or overexpression of cell-surface glycosylated tumor-associated Ags (TAA) distinguish neoplastic from normal cells. Interactions of TAA MUC1 and HER2/neu with dendritic cells (DC) preclude efficient processing, which impairs immune responses. It is thus important to define the mechanisms of interactions between DC and glycosylated TAA and their trafficking and processing for further T cell activation. In this work, we study interactions between DC and the oncofetal fucose-rich glycovariants of bile salt-dependent lipase (BSDL), expressed in pancreatic cancer tissues and referred to as pathological BSDL carrying the fucosylated J28 glycotope (pBSDL-J28) because it is characterized by the mAb J28. The expression of pBSDL-J28 was assessed by immunohistochemistry and quantified by confocal microscopy. Nontumoral pancreatic tissues and cells do not express pBSDL-J28. Using multidisciplinary approaches and functional studies, we provide the first evidence, to our knowledge, that this tumoral glycoprotein is rapidly internalized by human DC through macropinocytosis and endocytosis via mannose receptors and then transported to late endosomes for processing. Interestingly, pBSDL-J28 per se induced DC maturation with increased expression of costimulatory and CD83 molecules associated with cytokine secretion (IL-8 and IL-6). Surprisingly, DC retained their full ability to internalize Ags, making this maturation atypical. Finally, the allogeneic pBSDL-J28-treated DC stimulated lymphocyte proliferation. Besides, pulsing DC with pBSDL-J28 C-terminal glycopolypeptide and maturation with CD40L triggered CD4(+) and CD8(+) T cell proliferation. Therefore, interactions of pBSDL-J28, expressed on tumoral pancreatic tissue, with DC may lead to adequate Ag trafficking and processing and result in T cell activation.
Assuntos
Antígenos de Neoplasias/metabolismo , Biomarcadores Tumorais/metabolismo , Diferenciação Celular/imunologia , Células Dendríticas/imunologia , Endocitose/imunologia , Neoplasias Pancreáticas/imunologia , Esterol Esterase/metabolismo , Apresentação de Antígeno/imunologia , Antígenos de Neoplasias/fisiologia , Biomarcadores Tumorais/fisiologia , Técnicas de Cocultura , Células Dendríticas/metabolismo , Células Dendríticas/patologia , Células HEK293 , Humanos , Lectinas Tipo C/metabolismo , Ativação Linfocitária/imunologia , Receptor de Manose , Lectinas de Ligação a Manose/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Transporte Proteico/imunologia , Receptores de Superfície Celular/metabolismo , Esterol Esterase/fisiologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linfócitos T/patologiaRESUMO
Actin assembly at the cell front drives membrane protrusion and initiates the cell migration cycle. Microtubules (MTs) extend within forward protrusions to sustain cell polarity and promote adhesion site turnover. Memo is an effector of the ErbB2 receptor tyrosine kinase involved in breast carcinoma cell migration. However, its mechanism of action remained unknown. We report in this study that Memo controls ErbB2-regulated MT dynamics by altering the transition frequency between MT growth and shortening phases. Moreover, although Memo-depleted cells can assemble the Rac1-dependent actin meshwork and form lamellipodia, they show defective localization of lamellipodial markers such as alpha-actinin-1 and a reduced number of short-lived adhesion sites underlying the advancing edge of migrating cells. Finally, we demonstrate that Memo is required for the localization of the RhoA guanosine triphosphatase and its effector mDia1 to the plasma membrane and that Memo-RhoA-mDia1 signaling coordinates the organization of the lamellipodial actin network, adhesion site formation, and MT outgrowth within the cell leading edge to sustain cell motility.
Assuntos
Actinas/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Adesão Celular/fisiologia , Movimento Celular/fisiologia , Microtúbulos/fisiologia , Ferroproteínas não Heme/fisiologia , Proteína rhoA de Ligação ao GTP/fisiologia , Neoplasias da Mama , Linhagem Celular Tumoral , Membrana Celular/fisiologia , Citosol/fisiologia , Forminas , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Ferroproteínas não Heme/deficiência , Ferroproteínas não Heme/genética , Pseudópodes/fisiologiaRESUMO
Members of the LAP protein family, LET-413 in Caenorhabditis elegans, Scribble in Drosophila melanogaster, and Erbin, Lano, Densin-180 and hScrib in mammals, have conserved structural features. LET-413 and Scribble are junctional proteins involved in establishing and maintaining epithelial cell polarity. scribble also behaves as a neoplastic tumor suppressor gene. We show here that, in epithelial cells, hScrib is recruited at cell-cell junctions in an E-cadherin-dependent manner as shown by calcium switch assays in MDCK cells, re-expression of E-cadherin in MDA-231 cells treated by 5-Aza-2'-deoxycytidine (5Aza), and siRNA experiments. hScrib is restricted at the basolateral membrane of epithelial cells by its LRR domain, and is enriched in Triton X-100-insoluble fractions. In breast cancers, most lobular tumors did not express hScrib and E-cadherin while ductal tumors had a less frequent downregulation of hScrib. Our data provide additional insights on the modalities of recruitment of hScrib at the cell-cell junctions, and establish a potential link between the E-cadherin and hScrib tumor suppressors.
Assuntos
Caderinas/metabolismo , Junções Intercelulares/metabolismo , Proteínas de Membrana/metabolismo , Animais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Caderinas/genética , Cálcio/farmacologia , Linhagem Celular , Membrana Celular/metabolismo , Colo/citologia , Colo/metabolismo , Proteínas do Citoesqueleto/metabolismo , Cães , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Humanos , Junções Intercelulares/genética , Leucina/metabolismo , Proteínas de Membrana/genética , Microscopia Eletrônica , Ligação Proteica , Sequências Repetitivas de Aminoácidos , Transativadores/metabolismo , Proteínas Supressoras de Tumor , alfa Catenina , beta CateninaRESUMO
Tec is the prototypical member of the Tec tyrosine kinases family, which plays an important role in T cell signaling. We show in this study that Tec translocates to the immunological synapse when a T cell contacts a dendritic cell. Surprisingly, the presence of the pleckstrin homology (PH) domain of Tec is not required for this accumulation, and despite a strong activation of 3'-phosphorylated phosphoinositide lipids synthesis during the synapse formation, the Tec PH domain is not redistributed to the T cell plasma membrane. In contrast, we demonstrate that an active Src homology 3 domain is absolutely required, underlining the essential role played by this part of the molecule in the recruitment and/or stabilization of Tec at the immunological synapse. Our results nevertheless suggest that the PH domain controls the kinase activity of the molecule in vivo. We finally demonstrate that the two domains are necessary to trigger transcriptional events following Ag presentation. These data support a model in which the plasma membrane recruitment of the PH-containing protein Tec is not dependent on the production of 3'-phosphorylated phosphoinositide lipids by the PI3K, but rather on an intact Src homology 3 domain.
Assuntos
Células Apresentadoras de Antígenos/metabolismo , Proteínas Tirosina Quinases/metabolismo , Linfócitos T/metabolismo , Sequência de Aminoácidos , Células Apresentadoras de Antígenos/enzimologia , Células Apresentadoras de Antígenos/imunologia , Humanos , Mutação , Estrutura Terciária de Proteína , Proteínas Tirosina Quinases/genética , Homologia de Sequência , Linfócitos T/enzimologia , Linfócitos T/imunologiaRESUMO
Plasmacytoid dendritic cells (PDCs) are crucial effectors in innate immunity. In this study, we show that imatinib, a potent inhibitor of BCR/ABL tyrosine kinase activity, in the presence of Flt3-Ligand, could induce CD34+ progenitors from chronic myeloid leukemia (CML) to give rise in vitro to typical BDCA-2+ type I interferon-producing PDCs. The effect of imatinib on PDC generation was related to up-regulation of Flt3 on leukemic CD34+ progenitors. Moreover, patients with chronic myeloid leukemia (CML) who were in complete cytogenetic or molecular response after imatinib treatment restored their blood PDCs both quantitatively and functionally comparable to healthy donors, in contrast to patients not responding to imatinib, further confirming that disease response to imatinib is accompanied by restoration of PDC function in vivo. These findings provide evidence that response to imatinib is capable to restore some DC-related immune functions in CML that might be beneficial for long-term disease control.
Assuntos
Células Dendríticas/fisiologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/imunologia , Piperazinas/uso terapêutico , Pirimidinas/uso terapêutico , Antineoplásicos/uso terapêutico , Benzamidas , Células Dendríticas/efeitos dos fármacos , Citometria de Fluxo , Humanos , Mesilato de Imatinib , Leucemia Mielogênica Crônica BCR-ABL Positiva/sangue , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologiaRESUMO
The three human TACC (transforming acidic coiled-coil) genes encode a family of proteins with poorly defined functions that are suspected to play a role in oncogenesis. A Xenopus TACC homolog called Maskin is involved in translational control, while Drosophila D-TACC interacts with the microtubule-associated protein MSPS (Mini SPindleS) to ensure proper dynamics of spindle pole microtubules during cell division. We have delineated here the interactions of TACC1 with four proteins, namely the microtubule-associated chTOG (colonic and hepatic tumor-overexpressed gene) protein (ortholog of Drosophila MSPS), the adaptor protein TRAP (tudor repeat associator with PCTAIRE2), the mitotic serine/threonine kinase Aurora A and the mRNA regulator LSM7 (Like-Sm protein 7). To measure the relevance of the TACC1-associated complex in human cancer we have examined the expression of the three TACC, chTOG and Aurora A in breast cancer using immunohistochemistry on tissue microarrays. We show that expressions of TACC1, TACC2, TACC3 and Aurora A are significantly correlated and downregulated in a subset of breast tumors. Using siRNAs, we further show that depletion of chTOG and, to a lesser extent of TACC1, perturbates cell division. We propose that TACC proteins, which we also named 'Taxins', control mRNA translation and cell division in conjunction with microtubule organization and in association with chTOG and Aurora A, and that these complexes and cell processes may be affected during mammary gland oncogenesis.
Assuntos
Neoplasias da Mama/enzimologia , Proteínas Fetais/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Quinases/metabolismo , Aurora Quinase A , Aurora Quinases , Neoplasias da Mama/metabolismo , Células CACO-2 , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular , Feminino , Células HeLa , Humanos , Filogenia , Mapeamento de Interação de Proteínas , Proteínas Serina-Treonina Quinases , Proteínas de XenopusRESUMO
IFN-alpha is an important cytokine for the generation of a protective T cell-mediated immune response to viruses. In this study, we asked whether IFN-alpha can regulate the functional properties of dendritic cells (DCs). We show that monocytes cultured in the presence of GM-CSF and IFN-alpha can differentiate into DCs (IFN-alpha-derived DCs (IFN-DCs)). When compared with DCs generated in the presence of GM-CSF and IL-4 (IL-4-derived DCs), IFN-DCs exhibited a typical DC morphology and expressed, in addition to DC markers CD1a and blood DC Ag 4, a similar level of costimulatory and class II MHC molecules, but a significantly higher level of MHC class I molecules. After maturation with CD40 ligand, IFN-DCs up-regulated costimulatory, class I and II MHC molecules and expressed mature DC markers such as CD83 and DC-lysosome-associated membrane protein. IFN-DCs were endowed with potent functional activities. IFN-DCs secreted large amounts of the inflammatory cytokines IL-6, IL-10, TNF-alpha, IL-1beta, and IL-18, and promoted a Th1 response that was independent of IL-12p70 and IL-18, but substantially inhibited by IFN-alpha neutralization. Furthermore, immature IFN-DCs induced a potent autologous Ag-specific immune response, as evaluated by IFN-gamma secretion and expansion of CD8(+) T cells specific for CMV. Also, IFN-DCs expressed a large number of Toll-like receptors (TLRs), including acquisition of TLR7, which is classically found on the natural type I IFN-producing plasmacytoid DCs. Like plasmacytoid DCs, IFN-DCs could secrete IFN-alpha following viral stimulation or TLR7-specific stimulation. Taken together, these results illustrate the critical role of IFN-alpha at the early steps of immune response to pathogens or in autoimmune diseases.
Assuntos
Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Interferon-alfa/fisiologia , Glicoproteínas de Membrana/biossíntese , Monócitos/citologia , Monócitos/imunologia , Receptores de Superfície Celular/biossíntese , Adulto , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/imunologia , Diferenciação Celular/imunologia , Células Cultivadas , Técnicas de Cocultura , Citocinas/biossíntese , Células Dendríticas/citologia , Células Dendríticas/virologia , Epitopos de Linfócito T/biossíntese , Humanos , Interferon-alfa/metabolismo , Interferon gama/biossíntese , Interleucina-12/fisiologia , Interleucina-18/fisiologia , Células L , Ativação Linfocitária , Camundongos , Monócitos/metabolismo , Subunidades Proteicas/fisiologia , Simplexvirus/imunologia , Receptor 7 Toll-Like , Receptores Toll-LikeRESUMO
All-trans retinoic acid (ATRA) and retinoid derivatives are essential agents for multiple biological processes. Numerous immune system dysfunctions can occur in the case of retinoid deficiency. Because of the central role of dendritic cells (DCs) in controlling immunity and the wide effects of retinoids on the immune system homeostasis, we investigated the ability of ATRA to influence the differentiation of DCs from circulating peripheral blood monocytes. Human peripheral blood monocytes were cultured with granulocyte-macrophage colony-stimulating factor (GM-CSF) and various concentrations of ATRA. Differentiated cells were assayed for their morphology, phenotype, antigen uptake, allostimulatory capacity and cytokine secretion profile. ATRA (10(-12) mol/l) and GM-CSF drove the differentiation of monocytes into dendritic-like cells (ATRA-DC). ATRA-DCs exhibited DC morphology, had a phenotype of immature DCs, with the expression of CD1a, and upregulation of adhesion and co-stimulatory molecules. ATRA-DCs could induce a proliferative response in naive CD4+ T cells. Although ATRA-DCs retained their antigen-capture capacity, they secreted interleukin (IL)-12p70 without the need for any maturation agent. In addition, ATRA-DCs could drive T cells towards an IL-12-dependent T-helper cell type 1 response with secretion of interferon-gamma. DCs appear to be potential targets for ATRA, giving new insights into the immunomodulatory function of retinoids, with implications potentially related to immunotherapy.
Assuntos
Antineoplásicos/farmacologia , Leucócitos Mononucleares/citologia , Tretinoína/farmacologia , Linfócitos T CD4-Positivos/imunologia , Diferenciação Celular/efeitos dos fármacos , Divisão Celular , Células Cultivadas , Células Dendríticas/imunologia , Citometria de Fluxo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Humanos , Interferon gama/metabolismo , Interleucina-12/metabolismo , Interleucina-4/metabolismo , Leucócitos Mononucleares/efeitos dos fármacos , Teste de Cultura Mista de Linfócitos , Células Th1/imunologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The TP53INP1 gene encodes two protein isoforms, TP53INP1alpha and TP53INP1beta, located into the nucleus. Their synthesis is increased during cellular stress by p53-mediated activation of transcription. Overexpression of these isoforms induces apoptosis, suggesting an involvement of TP53INP1s in p53-mediated cell death. It was recently shown that p53-dependent apoptosis is promoted by homeodomain-interacting protein kinase-2 (HIPK2), which is known to bind p53 and induce its phosphorylation in promyelocytic leukemia protein nuclear bodies (PML-NBs). In this work we show that TP53INP1s localize with p53, PML-IV, and HIPK2 into the PML-NBs. In addition, we show that TP53INP1s interact physically with HIPK2 and p53. In agreement with these results we demonstrate that TP53INP1s, in association with HIPK2, regulate p53 transcriptional activity on p21, mdm2, pig3, and bax promoters. Furthermore, TP53INP1s overexpression induces G1 arrest and increases p53-mediated apoptosis. Although a TP53INP1s and HIPK2 additive effect was observed on apoptosis, G1 arrest was weaker when HIPK2 was transfected together with TP53INP1. These results indicate that TP53INP1s and HIPK2 could be partners in regulating p53 activity.
Assuntos
Proteínas de Transporte/fisiologia , Proteínas de Choque Térmico , Proteínas Nucleares/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Proteína Supressora de Tumor p53/fisiologia , Apoptose , Proteínas de Transporte/análise , Ciclo Celular , Células Cultivadas , Humanos , Proteínas Nucleares/análise , Proteínas Serina-Treonina Quinases/análise , Transcrição GênicaRESUMO
The localization of caspases and their substrates in different cellular compartments may be one way to regulate apoptosis. Caspase-3-dependent proteolysis of inhibitor caspase-activated deoxyribonuclease (ICAD) activates caspase-activated deoxyribonuclease (CAD), which induces apoptotic internucleosomal DNA degradation. The nuclear localization of ICAD, pro- and active-caspase-3 molecules remains a controversial issue. Using a combination of immunodetection of endogenous molecules and confocal microscopy, we analysed the kinetics of the procaspase-3 and CAD activation induced by FAS triggering in Jurkat cells. Through a semi-quantitative image analysis, we showed a constitutive nuclear localization of pro-caspase 3 and ICAD in non-apoptotic cells. FAS stimulation induced 7A6 apoptotic antigen expression, which could be related to three different sequential patterns of nuclear chromatin organization. Active-caspase-3 first appeared in the cytoplasm and was next observed in the nucleus. Simultaneously, the amount of ICAD located in the nucleus decreased, whereas the amount of ICAD located in the cytoplasm remained unchanged. Thus, our experiments using in situ immunodetection of endogenous molecules show that the ICAD cleavage by the active-caspase-3 probably takes place in the nucleus. These results provide new perspectives about the subcellular compartmentation and traffic of caspases during the apoptotic process.
Assuntos
Apoptose/fisiologia , Caspases/metabolismo , Núcleo Celular/enzimologia , Citoplasma/enzimologia , Proteínas Reguladoras de Apoptose , Caspase 3 , Desoxirribonucleases/antagonistas & inibidores , Precursores Enzimáticos/metabolismo , Humanos , Células Jurkat , Microscopia Confocal , Proteínas/metabolismo , Receptor fas/metabolismoRESUMO
Thalidomide (Thal) was shown to be a potent immunomodulating agent. Because of their central role in controlling immunity, we investigated the effects of Thal on monocyte-derived dendritic cells (Mo-DC). The addition of 10 micro g/ml or 20 micro g/ml Thal from the beginning of monocyte culture with granulocyte macrophage-colony stimulating factor and interleukin (IL)-4 did not block Mo-DC differentiation. Moreover, Thal alone could not induce Mo-DC maturation. However, Thal exerted a modulation of Mo-DC functional properties. At 10 micro g/ml, Thal modified the allostimulatory capacity of DC little, whereas a dose of 20 micro g/ml up-regulated this capacity (P=.05) and increased IL-12p70 production in a dose-dependent manner between 10 and 20 micro g/ml (P=.001). Mo-DC generated with 10 micro g/ml Thal were poor stimulators of T helper cell type 1 (Th1) responses (P=.01), but 20 micro g/ml was able to strengthen Th1 responses (P=.03). Also, Thal induced a significant reduction of IL-10 production in response to the maturation-inducing stimulus CD40L. Similarly, tumor necrosis factor alpha production was significantly decreased when Mo-DC were exposed to 10 micro g/ml Thal, and a dose of 20 micro g/ml did not induce any significant changes. The effects of Thal in vitro on the secretion of IL-12p70 and strengthening of Th1 responses might contribute to the antitumor effects of Thal. Thus, DC appear to be potential targets for the immunomodulatory capacity of Thal, defining a new mechanism of action of this drug.
Assuntos
Adjuvantes Imunológicos/farmacologia , Células Dendríticas/imunologia , Talidomida/farmacologia , Animais , Antígenos CD/análise , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Citocinas/biossíntese , Células Dendríticas/efeitos dos fármacos , Relação Dose-Resposta a Droga , Citometria de Fluxo , Humanos , Teste de Cultura Mista de Linfócitos , Camundongos , Monócitos/efeitos dos fármacos , Monócitos/imunologiaRESUMO
Myeloid and plasmacytoid dendritic cells (MDC, PDC) play a key role in the initiation of immune responses. We found a reduction of DC subsets among 20 chronic myeloid leukaemia (CML) patients in chronic phase (MDC, mean, 0.10% +/- 0.10, P = 0.02; PDC, mean, 0.11% +/- 0.08, P = 0.006 versus controls). The maintenance of blood DC correlated with the presence of high percentages of circulating CD34+/CD38- progenitors that were able to give rise in vitro to CD1a+ DC. The reduced DC numbers may contribute to leukaemia escape from immune control and restoration of DC may be a goal for CML immunotherapy.
Assuntos
ADP-Ribosil Ciclase/imunologia , Antígenos CD34/imunologia , Antígenos CD/imunologia , Células Dendríticas/imunologia , Células-Tronco Hematopoéticas/imunologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/sangue , ADP-Ribosil Ciclase 1 , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos CD1/imunologia , Feminino , Humanos , Masculino , Glicoproteínas de Membrana , Pessoa de Meia-IdadeRESUMO
Through a genomic survey of the Caenorhabditis elegans genome for genes encoding tyrosine kinase receptors (RTK) we identified a family of four RTKs which are structurally related to vascular endothelial growth factor receptors (VEGFRs). We named this family the ver gene family (for Vascular Endothelial growth factor receptor Related). It was intriguing to find this type of RTK in an animal devoid of a vascular system. The common sites of expression of the ver genes are specialized cells of neural origin: ver-1 (T17A3.1) is expressed in the support (glial) cells of amphid and phasmid neurons, ver-2 (T17A3.8) in ADL, a pair of chemosensorial neurons, and ver-3 (F59F3.1) in the ALA neuron. In mammals, the VEGFRs are associated with angiogenesis and neurogenesis. We provide here the first observation that these molecules may be primarily and solely involved in neurogenesis in a living organism.
Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Genoma , Neurônios/fisiologia , Receptores de Fatores de Crescimento do Endotélio Vascular/genética , Animais , Células Quimiorreceptoras/fisiologia , Expressão Gênica/fisiologia , Mamíferos , Neovascularização Fisiológica/fisiologia , Especificidade da EspécieRESUMO
The three human TACC genes encode a family of proteins that are suspected to play a role in carcinogenesis. Their function is not precisely known; a Xenopus TACC protein called Maskin is involved in translational control, while the Drosophila D-TACC associates with microtubules and centrosomes. We have characterized the human TACC1 gene and its products. The TACC1 gene is located in region p12 of chromosome 8; its mRNA is ubiquitously expressed and encodes a protein with an apparent molecular mass of 125 kDa, which is cytoplasmic and mainly perinuclear. We show that TACC1 mRNA gene expression is down-regulated in various types of tumors. Using immunohistochemistry of tumor tissue-microarrays and sections, we confirm that the level of TACC1 protein is down-regulated in breast cancer. Finally, using the two-hybrid screen in yeast, GST pull-downs and co-immunoprecipitations, we identified two potential binding partners for TACC1, LSM7 and SmG. They constitute a conserved subfamily of Sm-like small proteins that associate with U6 snRNPs and play a role in several aspects of mRNA processing. We speculate that down-regulation of TACC1 may alter the control of mRNA homeostasis in polarized cells and participates in the oncogenic processes.
Assuntos
Neoplasias da Mama/genética , Proteínas Fetais , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias Ductais, Lobulares e Medulares/genética , Proteínas Nucleares , RNA Mensageiro/metabolismo , Northern Blotting , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Primers do DNA/química , Regulação para Baixo , Feminino , Imunofluorescência , Glutationa Transferase/metabolismo , Humanos , Immunoblotting , Proteínas de Membrana/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Pessoa de Meia-Idade , Proteínas de Neoplasias/genética , Neoplasias Ductais, Lobulares e Medulares/metabolismo , Neoplasias Ductais, Lobulares e Medulares/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Fragmentos de Peptídeos/imunologia , Reação em Cadeia da Polimerase , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Saccharomyces cerevisiae , Frações Subcelulares , Células Tumorais Cultivadas/citologia , Técnicas do Sistema de Duplo-HíbridoRESUMO
Dendritic cells (DC) are a group of potent antigen-presenting cells (APC) specialized for initiating T cell immune responses. They originate from the bone marrow and upon stimulation with bacterial products, cytokines or CD40 ligation they acquire the ability to migrate to the secondary lymphoid organs. In vitro DC can be generated from human CD34(+) bone marrow cells and CD14(+) peripheral blood monocytes after culture with different cytokine combinations. Since most leukemic cells and tumors in general are devoid of APC capacities, various strategies have been used to increase their recognition and confer the capacity of antigen presentation on them. Because of our interest in the design of vaccine immunotherapy protocols for the adjuvant treatment of patients with lymphoid malignancies (LM), we chose to explore the capacity of human acute lymphoblastic leukemia, chronic lymphocytic leukemia and plasma cell leukemia to differentiate into cells with APC and DC features. Our results among a sample of 10 patients demonstrate that such approach is feasible. Leukemic cells could be induced in the presence of IL-4 and CD40L to exhibit a DC morphology with a phenotype of mature DC-like cells. They could also induce a potent proliferative response in naive CD4(+) T cells. In addition, they expressed chemokine receptor CCR7 and CD62L, and could drive T cells towards a T(h)1 response with secretion of IFN-gamma. Our strategy leading to increased LM cell immunogenicity may have potential clinical applications and LM appear to be attracting candidates for adjuvant vaccination and adoptive immunotherapy.