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1.
Elife ; 132024 Mar 06.
Artículo en Inglés | MEDLINE | ID: mdl-38446032

RESUMEN

Cell motility processes highly depend on the membrane distribution of Phosphoinositides, giving rise to cytoskeleton reshaping and membrane trafficking events. Membrane contact sites serve as platforms for direct lipid exchange and calcium fluxes between two organelles. Here, we show that VAPA, an ER transmembrane contact site tether, plays a crucial role during cell motility. CaCo2 adenocarcinoma epithelial cells depleted for VAPA exhibit several collective and individual motility defects, disorganized actin cytoskeleton and altered protrusive activity. During migration, VAPA is required for the maintenance of PI(4)P and PI(4,5)P2 levels at the plasma membrane, but not for PI(4)P homeostasis in the Golgi and endosomal compartments. Importantly, we show that VAPA regulates the dynamics of focal adhesions (FA) through its MSP domain, is essential to stabilize and anchor ventral ER-PM contact sites to FA, and mediates microtubule-dependent FA disassembly. To conclude, our results reveal unknown functions for VAPA-mediated membrane contact sites during cell motility and provide a dynamic picture of ER-PM contact sites connection with FA mediated by VAPA.


Asunto(s)
Adhesiones Focales , Aparato de Golgi , Humanos , Células CACO-2 , Citoesqueleto de Actina , Movimiento Celular , Proteínas de Transporte Vesicular
2.
Nat Commun ; 15(1): 2235, 2024 Mar 12.
Artículo en Inglés | MEDLINE | ID: mdl-38472173

RESUMEN

Intracellular pathogens develop elaborate mechanisms to survive within the hostile environments of host cells. Theileria parasites infect bovine leukocytes and cause devastating diseases in cattle in developing countries. Theileria spp. have evolved sophisticated strategies to hijack host leukocytes, inducing proliferative and invasive phenotypes characteristic of cell transformation. Intracellular Theileria parasites secrete proteins into the host cell and recruit host proteins to induce oncogenic signaling for parasite survival. It is unknown how Theileria parasites evade host cell defense mechanisms, such as autophagy, to survive within host cells. Here, we show that Theileria annulata parasites sequester the host eIF5A protein to their surface to escape elimination by autophagic processes. We identified a small-molecule compound that reduces parasite load by inducing autophagic flux in host leukocytes, thereby uncoupling Theileria parasite survival from host cell survival. We took a chemical genetics approach to show that this compound induced host autophagy mechanisms and the formation of autophagic structures via AMPK activation and the release of the host protein eIF5A which is sequestered at the parasite surface. The sequestration of host eIF5A to the parasite surface offers a strategy to escape elimination by autophagic mechanisms. These results show how intracellular pathogens can avoid host defense mechanisms and identify a new anti-Theileria drug that induces autophagy to target parasite removal.


Asunto(s)
Parásitos , Theileria , Theileriosis , Animales , Bovinos , Theileria/genética , Theileriosis/parasitología , Interacciones Huésped-Parásitos/fisiología , Transducción de Señal
3.
Front Oncol ; 12: 958155, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-36387192

RESUMEN

Human TRIAP1 (TP53-regulated inhibitor of apoptosis 1; also known as p53CSV for p53-inducible cell survival factor) is the homolog of yeast Mdm35, a well-known chaperone that interacts with the Ups/PRELI family proteins and participates in the intramitochondrial transfer of lipids for the synthesis of cardiolipin (CL) and phosphatidylethanolamine. Although recent reports indicate that TRIAP1 is a prosurvival factor abnormally overexpressed in various types of cancer, knowledge about its molecular and metabolic function in human cells is still elusive. It is therefore critical to understand the metabolic and proliferative advantages that TRIAP1 expression provides to cancer cells. Here, in a colorectal cancer cell model, we report that the expression of TRIAP1 supports cancer cell proliferation and tumorigenesis. Depletion of TRIAP1 perturbed the mitochondrial ultrastructure, without a major impact on CL levels and mitochondrial activity. TRIAP1 depletion caused extramitochondrial perturbations resulting in changes in the endoplasmic reticulum-dependent lipid homeostasis and induction of a p53-mediated stress response. Furthermore, we observed that TRIAP1 depletion conferred a robust p53-mediated resistance to the metabolic stress caused by glutamine deprivation. These findings highlight the importance of TRIAP1 in tumorigenesis and indicate that the loss of TRIAP1 has extramitochondrial consequences that could impact on the metabolic plasticity of cancer cells and their response to conditions of nutrient deprivation.

4.
Elife ; 112022 03 23.
Artículo en Inglés | MEDLINE | ID: mdl-35319462

RESUMEN

Centrioles are formed by microtubule triplets in a ninefold symmetric arrangement. In flagellated protists and animal multiciliated cells, accessory structures tethered to specific triplets render the centrioles rotationally asymmetric, a property that is key to cytoskeletal and cellular organization in these contexts. In contrast, centrioles within the centrosome of animal cells display no conspicuous rotational asymmetry. Here, we uncover rotationally asymmetric molecular features in human centrioles. Using ultrastructure expansion microscopy, we show that LRRCC1, the ortholog of a protein originally characterized in flagellate green algae, associates preferentially to two consecutive triplets in the distal lumen of human centrioles. LRRCC1 partially co-localizes and affects the recruitment of another distal component, C2CD3, which also has an asymmetric localization pattern in the centriole lumen. Together, LRRCC1 and C2CD3 delineate a structure reminiscent of a filamentous density observed by electron microscopy in flagellates, termed the 'acorn.' Functionally, the depletion of LRRCC1 in human cells induced defects in centriole structure, ciliary assembly, and ciliary signaling, supporting that LRRCC1 cooperates with C2CD3 to organizing the distal region of centrioles. Since a mutation in the LRRCC1 gene has been identified in Joubert syndrome patients, this finding is relevant in the context of human ciliopathies. Taken together, our results demonstrate that rotational asymmetry is an ancient property of centrioles that is broadly conserved in human cells. Our work also reveals that asymmetrically localized proteins are key for primary ciliogenesis and ciliary signaling in human cells.


Asunto(s)
Proteínas de Ciclo Celular , Centriolos , Ciliopatías , Proteínas Asociadas a Microtúbulos , Animales , Proteínas de Ciclo Celular/genética , Centriolos/metabolismo , Centrosoma/metabolismo , Cilios/metabolismo , Humanos , Proteínas Asociadas a Microtúbulos/genética , Microtúbulos/metabolismo
5.
Toxins (Basel) ; 12(5)2020 05 11.
Artículo en Inglés | MEDLINE | ID: mdl-32403276

RESUMEN

Shiga toxins (Stxs) expressed by the enterohaemorrhagic Escherichia coli and enteric Shigella dysenteriae 1 pathogens are protein synthesis inhibitors. Stxs have been shown to induce apoptosis via the activation of extrinsic and intrinsic pathways in many cell types (epithelial, endothelial, and B cells) but the link between the protein synthesis inhibition and caspase activation is still unclear. Endoplasmic reticulum (ER) stress induced by the inhibition of protein synthesis may be this missing link. Here, we show that the treatment of Burkitt lymphoma (BL) cells with verotoxin-1 (VT-1 or Stx1) consistently induced the ER stress response by activation of IRE1 and ATF6-two ER stress sensors-followed by increased expression of the transcription factor C/REB homologous protein (CHOP). However, our data suggest that, although ER stress is systematically induced by VT-1 in BL cells, its role in cell death appears to be cell specific and can be the opposite: ER stress may enhance VT-1-induced apoptosis through CHOP or play a protective role through ER-phagy, depending on the cell line. Several engineered Stxs are currently under investigation as potential anti-cancer agents. Our results suggest that a better understanding of the signaling pathways induced by Stxs is needed before using them in the clinic.


Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Linfoma de Burkitt/tratamiento farmacológico , Estrés del Retículo Endoplásmico/efectos de los fármacos , Toxina Shiga I/farmacología , Factor de Transcripción Activador 6/genética , Factor de Transcripción Activador 6/metabolismo , Linfoma de Burkitt/genética , Linfoma de Burkitt/metabolismo , Linfoma de Burkitt/patología , Línea Celular Tumoral , Endorribonucleasas/genética , Endorribonucleasas/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Factor de Transcripción CHOP/genética , Factor de Transcripción CHOP/metabolismo
6.
Int J Pharm ; 532(2): 748-756, 2017 Nov 05.
Artículo en Inglés | MEDLINE | ID: mdl-28546071

RESUMEN

Oxazaphosphorines including cyclophosphamide, trofosfamide and ifosfamide (IFO) belong to the alkylating agent class and are indicated in the treatment of numerous cancers. However, IFO is subject to limiting side-effects in high-dose protocols. To circumvent IFO drawbacks in clinical practices, preactivated IFO analogs were designed to by-pass the toxic metabolic pathway. Among these IFO analogs, some of them showed the ability to self-assemble due to the use of a poly-isoprenyloxy chain as preactivating moiety. We present here, the in vitro activity of the nanoassembly formulations of preactivated IFO derivatives with a C-4 geranyloxy, farnesyloxy and squalenoxy substituent on a large panel of tumor cell lines. The chemical and colloidal stabilities of the geranyloxy-IFO (G-IFO), farnesyloxy-IFO (F-IFO) and squalenoxy-IFO (SQ-IFO) NAs were further evaluated in comparison to their free formulation. Finally, pharmacokinetic parameters and maximal tolerated dose of the most potent preactivated IFO analog (G-IFO) were determined and compared to IFO, paving the way to in vivo studies.


Asunto(s)
Antineoplásicos Alquilantes/administración & dosificación , Ifosfamida/análogos & derivados , Ifosfamida/administración & dosificación , Nanoestructuras/administración & dosificación , Animales , Antineoplásicos Alquilantes/farmacocinética , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Humanos , Ifosfamida/química , Ifosfamida/farmacocinética , Masculino , Dosis Máxima Tolerada , Ratones Desnudos , Nanoestructuras/química , Prenilación
7.
Haematologica ; 101(12): 1469-1478, 2016 12.
Artículo en Inglés | MEDLINE | ID: mdl-27515249

RESUMEN

Megakaryocytes are naturally polyploid cells that increase their ploidy by endomitosis. However, very little is known regarding the mechanism by which they escape the tetraploid checkpoint to become polyploid. Recently, it has been shown that the tetraploid checkpoint was regulated by the Hippo-p53 pathway in response to a downregulation of Rho activity. We therefore analyzed the role of Hippo-p53 pathway in the regulation of human megakaryocyte polyploidy. Our results revealed that Hippo-p53 signaling pathway proteins are present and are functional in megakaryocytes. Although this pathway responds to the genotoxic stress agent etoposide, it is not activated in tetraploid or polyploid megakaryocytes. Furthermore, Hippo pathway was observed to be uncoupled from Rho activity. Additionally, polyploid megakaryocytes showed increased expression of YAP target genes when compared to diploid and tetraploid megakaryocytes. Although p53 knockdown increased both modal ploidy and proplatelet formation in megakaryocytes, YAP knockdown caused no significant change in ploidy while moderately affecting proplatelet formation. Interestingly, YAP knockdown reduced the mitochondrial mass in polyploid megakaryocytes and decreased expression of PGC1α, an important mitochondrial biogenesis regulator. Thus, the Hippo pathway is functional in megakaryocytes, but is not induced by tetraploidy. Additionally, YAP regulates the mitochondrial mass in polyploid megakaryocytes.


Asunto(s)
Diferenciación Celular , Megacariocitos/citología , Megacariocitos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Tetraploidía , Proteínas de Unión al GTP rho/metabolismo , Biomarcadores , Plaquetas/citología , Plaquetas/metabolismo , Proteínas de Ciclo Celular , Diferenciación Celular/genética , Expresión Génica , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Vía de Señalización Hippo , Humanos , Modelos Biológicos , Proteínas Nucleares/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/genética , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Poliploidía , Proteínas Serina-Treonina Quinasas/genética , Trombopoyesis/genética , Factores de Transcripción/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteínas de Unión al GTP rho/genética
8.
Nanoscale ; 8(22): 11588-94, 2016 Jun 02.
Artículo en Inglés | MEDLINE | ID: mdl-27216436

RESUMEN

Light and Transmission Electron Microscopies (LM and TEM) hold potential in bioimaging owing to the advantages of fast imaging of multiple cells with LM and ultrastructure resolution offered by TEM. Integrated or correlated LM and TEM are the current approaches to combine the advantages of both techniques. Here we propose an alternative in which the electron beam of a scanning TEM (STEM) is used to excite concomitantly the luminescence of nanoparticle labels (a process known as cathodoluminescence, CL), and image the cell ultrastructure. This CL-STEM imaging allows obtaining luminescence spectra and imaging ultrastructure simultaneously. We present a proof of principle experiment, showing the potential of this technique in image cytometry of cell vesicular components. To label the vesicles we used fluorescent diamond nanocrystals (nanodiamonds, NDs) of size ≈150 nm coated with different cationic polymers, known to trigger different internalization pathways. Each polymer was associated with a type of ND with a different emission spectrum. With CL-STEM, for each individual vesicle, we were able to measure (i) their size with nanometric resolution, (ii) their content in different ND labels, and realize intracellular component cytometry. In contrast to the recently reported organelle flow cytometry technique that requires cell sonication, CL-STEM-based image cytometry preserves the cell integrity and provides a much higher resolution in size. Although this novel approach is still limited by a low throughput, the automatization of data acquisition and image analysis, combined with improved intracellular targeting, should facilitate applications in cell biology at the subcellular level.

9.
Cell Mol Life Sci ; 73(19): 3745-60, 2016 10.
Artículo en Inglés | MEDLINE | ID: mdl-27076215

RESUMEN

Opposing views have been proposed regarding the role of tau, the principal microtubule-associated protein in axons. On the one hand, tau forms cross-bridges at the interface between microtubules and induces microtubule bundling in neurons. On the other hand, tau is also considered a polymer brush which efficiently separates microtubules. In mature axons, microtubules are indeed arranged in parallel arrays and are well separated from each other. To reconcile these views, we developed a mechanistic model based on in vitro and cellular approaches combined to analytical and numerical analyses. The results indicate that tau forms long-range cross-bridges between microtubules under macromolecular crowding conditions. Tau cross-bridges prevent the redistribution of tau away from the interface between microtubules, which would have occurred in the polymer brush model. Consequently, the short-range attractive force between microtubules induced by macromolecular crowding is avoided and thus microtubules remain well separated from each other. Interestingly, in this unified model, tau diffusion on microtubules enables to keep microtubules evenly distributed in axonal sections at low tau levels.


Asunto(s)
Axones/metabolismo , Microtúbulos/metabolismo , Proteínas tau/metabolismo , Animales , Corteza Cerebral/metabolismo , Simulación por Computador , Difusión , Fluorescencia , Sustancias Macromoleculares , Ratones , Dominios Proteicos , Tubulina (Proteína)/metabolismo , Proteínas tau/química
10.
Autophagy ; 11(12): 2275-87, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-26565591

RESUMEN

The Epstein-Barr virus (EBV) is associated with various lymphoproliferative disorders and lymphomas. We have previously demonstrated that treating wild-type TP53-expressing B cell lines with the TP53 pathway activator nutlin-3 induced apoptosis in EBV-negative and EBV-positive latency I cells whereas EBV-positive latency III cells remained much more apoptosis-resistant. Here, we report a constitutively high level of autophagy in these resistant cells which express high levels of the proautophagic protein BECN1/Beclin 1 based, at least in part, on the activation of the NFKB signaling pathway by the viral protein LMP1. Following treatment with nutlin-3, several autophagy-stimulating genes were upregulated both in EBV-negative and EBV-positive latency III cells. However the process of autophagy was only triggered in the latter and was associated with an upregulation of SESN1/sestrin 1 and inhibition of MTOR more rapid than in EBV-negative cells. A treatment with chloroquine, an inhibitor of autophagy, potentiated the apoptotic effect of nutlin-3, particularly in those EBV-positive cells which were resistant to apoptosis induced by nutlin-3 alone, thereby showing that autophagy participates in this resistant phenotype. Finally, using immunohistochemical staining, clinical samples from various B cell lymphoproliferations with the EBV-positive latency II or III phenotype were found to harbor a constitutively active autophagy.


Asunto(s)
Apoptosis/efectos de los fármacos , Autofagia , Linfocitos B/citología , Linfocitos B/virología , Herpesvirus Humano 4 , Linfoma/patología , Autofagia/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Humanos , Imidazoles/farmacología , Linfoma/virología , Piperazinas/farmacología , Proteína p53 Supresora de Tumor/metabolismo
11.
Biomaterials ; 45: 93-8, 2015 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-25662499

RESUMEN

The expression of a defective gene can lead to major cell dysfunctions among which cell proliferation and tumor formation. One promising therapeutic strategy consists in silencing the defective gene using small interfering RNA (siRNA). In previous publications we showed that diamond nanocrystals (ND) of primary size 35 nm, rendered cationic by polyethyleneimine-coating, can efficiently deliver siRNA into cell, which further block the expression of EWS/FLI-1 oncogene in a Ewing sarcoma disease model. However, a therapeutic application of such nanodiamonds requires their elimination by the organism, particularly in urine, which is impossible for 35 nm particles. Here, we report that hydrogenated cationic nanodiamonds of primary size 7 nm (ND-H) have also a high affinity for siRNA and are capable of delivering them in cells. With siRNA/ND-H complexes, we measured a high inhibition efficacy of EWS/FLI-1 gene expression in Ewing sarcoma cell line. Electron microscopy investigations showed ND-H in endocytosis compartments, and especially in macropinosomes from which they can escape before siRNA degradation occurred. In addition, the association of EWS/FLI-1 silencing by the siRNA/ND-H complex with a vincristine treatment yielded a potentiation of the toxic effect of this chemotherapeutic drug. Therefore ND-H appears as a promising delivery agent in anti-tumoral gene therapy.


Asunto(s)
Técnicas de Transferencia de Gen , Nanodiamantes/química , Proteínas de Fusión Oncogénica/genética , Gases em Plasma/química , Proteína Proto-Oncogénica c-fli-1/genética , ARN Interferente Pequeño/metabolismo , Proteína EWS de Unión a ARN/genética , Sarcoma de Ewing/metabolismo , Cationes , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Endocitosis/efectos de los fármacos , Fluorescencia , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Hidrogenación , Nanodiamantes/ultraestructura , Proteínas de Fusión Oncogénica/metabolismo , Proteína Proto-Oncogénica c-fli-1/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteína EWS de Unión a ARN/metabolismo , Sarcoma de Ewing/genética , Sarcoma de Ewing/ultraestructura , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/metabolismo , Vincristina/farmacología
12.
J Med Chem ; 58(2): 705-17, 2015 Jan 22.
Artículo en Inglés | MEDLINE | ID: mdl-25494842

RESUMEN

Oxazaphosphorines are alkylating agents used in routine clinical practices for treatment of cancer for many years. They are antitumor prodrugs that require cytochrome P450 bioactivation leading to 4-hydroxy derivatives. In the case of ifosfamide (IFO), the bioactivation produces two toxic metabolites: acrolein, a urotoxic compound, concomitantly generated with the isophosphoramide mustard; and chloroacetaldehyde, a neurotoxic and nephrotoxic compound, arising from the oxidation of the side chains. To improve the therapeutic index of IFO, we have designed preactivated IFO derivatives with the covalent binding of several O- and S-alkyl moieties including polyisoprenoid groups at the C-4 position of the oxazaphosphorine ring to avoid cytochrome bioactivation favoring the release of the active entity and limiting the chloroacetaldehyde release. Thanks to the grafted terpene moieties, some of these new conjugates demonstrated spontaneous self-assembling properties into nanoassemblies when dispersed in water. The cytotoxic activities on a panel of human tumor cell lines of these novel oxazaphosphorines, in bulk form or as nanoassemblies, and the release of 4-hydroxy-IFO from these preactivated IFO analogues in plasma are reported.


Asunto(s)
Antineoplásicos Alquilantes/síntesis química , Ifosfamida/análogos & derivados , Mostazas de Fosforamida/metabolismo , Profármacos/síntesis química , Antineoplásicos Alquilantes/metabolismo , Antineoplásicos Alquilantes/farmacología , Línea Celular Tumoral , Diseño de Fármacos , Humanos , Ifosfamida/metabolismo , Profármacos/metabolismo , Profármacos/farmacología
13.
Nucleic Acids Res ; 42(13): 8678-91, 2014 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-25013173

RESUMEN

The sequence of events leading to stress granule assembly in stressed cells remains elusive. We show here, using isotope labeling and ion microprobe, that proportionally more RNA than proteins are present in stress granules than in surrounding cytoplasm. We further demonstrate that the delivery of single strand polynucleotides, mRNA and ssDNA, to the cytoplasm can trigger stress granule assembly. On the other hand, increasing the cytoplasmic level of mRNA-binding proteins like YB-1 can directly prevent the aggregation of mRNA by forming isolated mRNPs, as evidenced by atomic force microscopy. Interestingly, we also discovered that enucleated cells do form stress granules, demonstrating that the translocation to the cytoplasm of nuclear prion-like RNA-binding proteins like TIA-1 is dispensable for stress granule assembly. The results lead to an alternative view on stress granule formation based on the following sequence of events: after the massive dissociation of polysomes during stress, mRNA-stabilizing proteins like YB-1 are outnumbered by the burst of nonpolysomal mRNA. mRNA freed of ribosomes thus becomes accessible to mRNA-binding aggregation-prone proteins or misfolded proteins, which induces stress granule formation. Within the frame of this model, the shuttling of nuclear mRNA-stabilizing proteins to the cytoplasm could dissociate stress granules or prevent their assembly.


Asunto(s)
Gránulos Citoplasmáticos/metabolismo , Polirribosomas/metabolismo , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Estrés Fisiológico , Animales , Células Cultivadas , Citoplasma/química , Gránulos Citoplasmáticos/química , ADN de Cadena Simple/metabolismo , Proteínas HSP70 de Choque Térmico/antagonistas & inhibidores , Inhibidores de Proteasoma/farmacología , Multimerización de Proteína , Transporte de Proteínas , Proteínas/análisis , Puromicina/farmacología , ARN/análisis , ARN Mensajero/fisiología , Ratas
14.
Cancer Res ; 73(7): 2247-58, 2013 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-23358685

RESUMEN

The spliceosome is a large ribonucleoprotein complex that guides pre-mRNA splicing in eukaryotic cells. Here, we determine whether the spliceosome could constitute an attractive therapeutic target in cancer. Analysis of gene expression arrays from lung, breast, and ovarian cancers datasets revealed that several genes encoding components of the core spliceosome composed of a heteroheptameric Sm complex were overexpressed in malignant disease as compared with benign lesions and could also define a subset of highly aggressive breast cancers. siRNA-mediated depletion of SmE (SNRPE) or SmD1 (SNRPD1) led to a marked reduction of cell viability in breast, lung, and melanoma cancer cell lines, whereas it had little effect on the survival of the nonmalignant MCF-10A breast epithelial cells. SNRPE or SNRPD1 depletion did not lead to apoptotic cell death but autophagy, another form of cell death. Indeed, induction of autophagy was revealed by cytoplasmic accumulation of autophagic vacuoles and by an increase in both LC3 (MAP1LC3A) protein conversion and the amount of acidic autophagic vacuoles. Knockdown of SNRPE dramatically decreased mTOR mRNA and protein levels and was accompanied by a deregulation of the mTOR pathway, which, in part, explains the SNRPE-dependent induction of autophagy. These findings provide a rational to develop new therapeutic agents targeting spliceosome core components in oncology.


Asunto(s)
Autofagia , Neoplasias de la Mama/patología , Neoplasias Pulmonares/patología , Melanoma/patología , Empalmosomas/genética , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Proteínas Nucleares snRNP/antagonistas & inhibidores , Apoptosis , Western Blotting , Mama/efectos de los fármacos , Mama/metabolismo , Mama/patología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Proliferación Celular , Células Cultivadas , Femenino , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/metabolismo , Melanoma/tratamiento farmacológico , Melanoma/metabolismo , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo , Proteínas Nucleares snRNP/genética , Proteínas Nucleares snRNP/metabolismo
15.
PLoS One ; 6(7): e22567, 2011.
Artículo en Inglés | MEDLINE | ID: mdl-21811634

RESUMEN

BACKGROUND: A relationship between the increased density of tumor-associated macrophages (TAMs) and decreased survival was recently reported in thyroid cancer patients. Among these tumors, anaplastic thyroid cancer (ATC) is one of the most aggressive solid tumors in humans. TAMs (type M2) have been recognized as promoting tumor growth. The purpose of our study was to analyze with immunohistochemistry the presence of TAMs in a series of 27 ATC. METHODOLOGY/PRINCIPAL FINDINGS: Several macrophages markers such as NADPH oxidase complex NOX2-p22phox, CD163 and CD 68 were used. Immunostainings showed that TAMs represent more than 50% of nucleated cells in all ATCs. Moreover, these markers allowed the identification of elongated thin ramified cytoplasmic extensions, bestowing a "microglia-like" appearance on these cells which we termed "Ramified TAMs" (RTAMs). In contrast, cancer cells were totally negative. Cellular stroma was highly simplified since apart from cancer cells and blood vessels, RTAMs were the only other cellular component. RTAMs were evenly distributed and intermingled with cancer cells, and were in direct contact with other RTAMs via their ramifications. Moreover, RTAMs displayed strong immunostaining for connexin Cx43. Long chains of interconnected RTAMs arose from perivascular clusters and were dispersed within the tumor parenchyma. When expressed, the glucose transporter Glut1 was found in RTAMs and blood vessels, but rarely in cancer cells. CONCLUSION: ATCs display a very dense network of interconnected RTAMs in direct contact with intermingled cancer cells. To our knowledge this is the first time that such a network is described in a malignant tumor. This network was found in all our studied cases and appeared specific to ATC, since it was not found in differentiated thyroid cancers specimens. Taken together, these results suggest that RTAMs network is directly related to the aggressiveness of the disease via metabolic and trophic functions which remain to be determined.


Asunto(s)
Macrófagos/patología , Neoplasias de la Tiroides/patología , Anciano , Anciano de 80 o más Años , Vasos Sanguíneos/metabolismo , Vasos Sanguíneos/patología , Complejo CD3/metabolismo , Línea Celular Tumoral , Forma de la Célula , Conexina 43/metabolismo , Uniones Comunicantes/metabolismo , Uniones Comunicantes/ultraestructura , Transportador de Glucosa de Tipo 1/metabolismo , Humanos , Inmunohistoquímica , Antígeno Ki-67/metabolismo , Macrófagos/metabolismo , Macrófagos/ultraestructura , Persona de Mediana Edad , NADPH Oxidasas/metabolismo , Proteínas de Neoplasias/metabolismo , Tomografía de Emisión de Positrones , Coloración y Etiquetado , Fracciones Subcelulares/metabolismo , Carcinoma Anaplásico de Tiroides , Neoplasias de la Tiroides/diagnóstico por imagen , Neoplasias de la Tiroides/ultraestructura , Tomografía Computarizada por Rayos X
16.
Blood ; 113(9): 1957-66, 2009 Feb 26.
Artículo en Inglés | MEDLINE | ID: mdl-19005181

RESUMEN

Epstein-Barr virus (EBV)-associated nasopharyngeal carcinoma (NPC) is the third most frequent virus-associated human malignancy. How this tumor escapes immune recognition despite the expression of several viral antigens has remained poorly understood. Our previous in vitro studies have shown that NPC cells release exosomes containing high amounts of galectin-9, a ligand of the membrane receptor Tim-3, which is able to induce apoptosis in mature Th1 lymphocytes. Here, we sought to determine whether galectin-9-carrying exosomes were produced in NPC patients and whether such exosomes might play a role in the immune evasion of NPC cells. We report that galectin-9-containing exosomes are selectively detected in plasma samples from NPC patients and mice xenografted with NPC tumors. The incorporation into exosomes protects galectin-9 against proteolytic cleavage but retains its Tim-3-binding capacity. Importantly, NPC exosomes induce massive apoptosis in EBV-specific CD4(+) cells used as a model of target T cells. This effect is inhibited by both anti-Tim-3 and antigalectin-9 blocking antibodies. These results indicate that blocking galectin-9/Tim-3 interaction in vivo might alleviate the Th1-suppressive effect of NPC exosomes and sustain antitumoral T-cell responses and thereby improve clinical efficacy of immunotherapeutic approaches against NPC.


Asunto(s)
Carcinoma/inmunología , Infecciones por Virus de Epstein-Barr/inmunología , Exosomas/metabolismo , Galectinas/metabolismo , Neoplasias Nasofaríngeas/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Escape del Tumor , Animales , Anticuerpos/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/inmunología , Vasos Sanguíneos/metabolismo , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/patología , Carcinoma/sangre , Carcinoma/complicaciones , Carcinoma/metabolismo , Línea Celular Tumoral , Difusión , Infecciones por Virus de Epstein-Barr/sangre , Infecciones por Virus de Epstein-Barr/complicaciones , Infecciones por Virus de Epstein-Barr/metabolismo , Exosomas/patología , Galectinas/antagonistas & inhibidores , Galectinas/sangre , Galectinas/inmunología , Células HeLa , Receptor 2 Celular del Virus de la Hepatitis A , Humanos , Ratones , Neoplasias Nasofaríngeas/sangre , Neoplasias Nasofaríngeas/complicaciones , Neoplasias Nasofaríngeas/metabolismo , Unión Proteica/efectos de los fármacos , Receptores Virales/antagonistas & inhibidores , Receptores Virales/inmunología , Receptores Virales/metabolismo , Linfocitos T Colaboradores-Inductores/patología , Trasplante Heterólogo , Escape del Tumor/inmunología
17.
Neoplasia ; 10(11): 1183-94, 2008 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-18953427

RESUMEN

The oncogenic process leading to nasopharyngeal carcinoma (NPC) requires the combination of genetic and epigenetic alterations, latent infection by the Epstein-Barr virus and local inflammation. A transcriptome analysis of NPC xenografts identified the gene encoding the cellular inhibitor of apoptosis protein 2 (c-IAP2) among the top five most intensely expressed. Consistently, the very high levels of the c-IAP2 protein were detected in 11 of 13 NPC biopsies. RMT 5265, a structural analog of second mitochondria-derived activator of caspase (SMAC), induced the rapid degradation of c-IAP2 in nasopharyngeal epithelial cells, whether malignant or not, but blocked clonal cell growth in NPC cells only. In short-term experiments, RMT 5265 induced apoptosis in a fraction of NPC cells, and this apoptosis was dramatically enhanced when RMT 5265 was combined with Toll-like receptor 3 (TLR3) stimulation. By contrast, the cooperative effect with tumor necrosis factor alpha was only marginal. The apoptosis induced by the combination of RMT 5265 and TLR3 stimulation was mediated by caspase-8 and associated with a decrease in the cellular content of the long isoform of FLICE-like inhibitory protein. Similar caspase-8 activation was obtained when siRNA knockdown of c-IAP2 was combined with TLR3 stimulation. In conclusion, c-IAP2 has a specific protective function in NPC cells challenged by TLR3 agonists. This protective function is probably important to make NPC cells tolerant to their own production of small viral RNAs, which are potential agonists of TLR3. Our data will help to design a rational use of IAP inhibitors in NPC patients.


Asunto(s)
Apoptosis , Infecciones por Virus de Epstein-Barr , Proteínas Inhibidoras de la Apoptosis/metabolismo , Neoplasias Nasofaríngeas/metabolismo , ARN Viral/metabolismo , Receptor Toll-Like 3/metabolismo , Análisis de Varianza , Animales , Proteína 3 que Contiene Repeticiones IAP de Baculovirus , Caspasa 8/metabolismo , Supervivencia Celular , Activación Enzimática , Infecciones por Virus de Epstein-Barr/genética , Infecciones por Virus de Epstein-Barr/metabolismo , Expresión Génica , Perfilación de la Expresión Génica , Humanos , Hibridación Fluorescente in Situ , Proteínas Inhibidoras de la Apoptosis/antagonistas & inhibidores , Proteínas Inhibidoras de la Apoptosis/genética , Ratones , Neoplasias Nasofaríngeas/patología , Trasplante de Neoplasias , Complejo de la Endopetidasa Proteasomal/metabolismo , ARN Interferente Pequeño , ARN Viral/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Receptor Toll-Like 3/genética , Trasplante Heterólogo , Ubiquitina-Proteína Ligasas
19.
BMC Cancer ; 6: 283, 2006 Dec 08.
Artículo en Inglés | MEDLINE | ID: mdl-17156439

RESUMEN

BACKGROUND: Nasopharyngeal carcinomas (NPC) are consistently associated with the Epstein-Barr virus (EBV). Their malignant epithelial cells contain the viral genome and express several antigenic viral proteins. However, the mechanisms of immune escape in NPCs are still poorly understood. EBV-transformed B-cells have been reported to release exosomes carrying the EBV-encoded latent membrane protein 1 (LMP1) which has T-cell inhibitory activity. Although this report suggested that NPC cells could also produce exosomes carrying immunosuppressive proteins, this hypothesis has remained so far untested. METHODS: Malignant epithelial cells derived from NPC xenografts--LMP1-positive (C15) or negative (C17)--were used to prepare conditioned culture medium. Various microparticles and vesicles released in the culture medium were collected and fractionated by differential centrifugation. Exosomes collected in the last centrifugation step were further purified by immunomagnetic capture on beads carrying antibody directed to HLA class II molecules. Purified exosomes were visualized by electron microscopy and analysed by western blotting. The T-cell inhibitory activities of recombinant LMP1 and galectin 9 were assessed on peripheral blood mononuclear cells activated by CD3/CD28 cross-linking. RESULTS: HLA-class II-positive exosomes purified from C15 and C17 cell supernatants were containing either LMP1 and galectin 9 (C15) or galectin 9 only (C17). Recombinant LMP1 induced a strong inhibition of T-cell proliferation (IC50 = 0.17 nM). In contrast recombinant galectin 9 had a weaker inhibitory effect (IC50 = 46 nM) with no synergy with LMP1. CONCLUSION: This study provides the proof of concept that NPC cells can release HLA class-II positive exosomes containing galectin 9 and/or LMP1. It confirms that the LMP1 molecule has intrinsic T-cell inhibitory activity. These findings will encourage investigations of tumor exosomes in the blood of NPC patients and assessment of their effects on various types of target cells.


Asunto(s)
Galectinas/metabolismo , Neoplasias Nasofaríngeas/virología , Vesículas Transportadoras/metabolismo , Proteínas de la Matriz Viral/metabolismo , Animales , Línea Celular Tumoral , Medios de Cultivo Condicionados , Exocitosis , Galectinas/genética , Galectinas/farmacología , Humanos , Leucocitos Mononucleares/efectos de los fármacos , Ratones , Neoplasias Nasofaríngeas/inmunología , Neoplasias Nasofaríngeas/ultraestructura , Proteínas Recombinantes/farmacología , Proteínas de la Matriz Viral/genética , Proteínas de la Matriz Viral/farmacología
20.
J Virol ; 79(21): 13326-37, 2005 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-16227255

RESUMEN

Nasopharyngeal carcinomas (NPC) are etiologically related to the Epstein-Barr virus (EBV), and malignant NPC cells have consistent although heterogeneous expression of the EBV latent membrane protein 1 (LMP1). LMP1 trafficking and signaling require its incorporation into membrane rafts. Conversely, raft environment is likely to modulate LMP1 activity. In order to investigate NPC-specific raft partners of LMP1, rafts derived from the C15 NPC xenograft were submitted to preparative immunoprecipitation of LMP1 combined with mass spectrometry analysis of coimmunoprecipitated proteins. Through this procedure, galectin 9, a beta-galactoside binding lectin and Hodgkin tumor antigen, was identified as a novel LMP1 partner. LMP1 interaction with galectin 9 was confirmed by coimmunoprecipitation and Western blotting in whole-cell extracts of NPC and EBV-transformed B cells (lymphoblastoid cell lines [LCLs]). Using mutant proteins expressed in HeLa cells, LMP1 was shown to bind galectin 9 in a TRAF3-independent manner. Galectin 9 is abundant in NPC biopsies as well as in LCLs, whereas it is absent in Burkitt lymphoma cells. In subsequent experiments, NPC cells were treated with Simvastatin, a drug reported to dissociate LMP1 from membrane rafts in EBV-transformed B cells. We found no significant effects of Simvastatin on the distribution of LMP1 and galectin 9 in NPC cell rafts. However, Simvastatin was highly cytotoxic for NPC cells, regardless of the presence or absence of LMP1. This suggests that Simvastatin is a potentially useful agent for the treatment of NPCs although it has distinct mechanisms of action in NPC and LCL cells.


Asunto(s)
Carcinoma/metabolismo , Galectinas/metabolismo , Herpesvirus Humano 4/fisiología , Membranas Intracelulares/metabolismo , Neoplasias Nasofaríngeas/metabolismo , Simvastatina/farmacología , Proteínas de la Matriz Viral/metabolismo , Western Blotting , Carcinoma/virología , Línea Celular , Transformación Celular Viral , Citotoxinas , Resistencia a Medicamentos , Galectinas/genética , Humanos , Inmunoprecipitación , Membranas Intracelulares/efectos de los fármacos , Espectrometría de Masas , Microscopía Electrónica , Neoplasias Nasofaríngeas/virología , Unión Proteica/efectos de los fármacos , Células Tumorales Cultivadas
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