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1.
Elife ; 52016 07 26.
Artículo en Inglés | MEDLINE | ID: mdl-27458799

RESUMEN

When small phosphatidylcholine liposomes are added to perforated cells, they bind preferentially to the Golgi suggesting an exceptional avidity of this organelle for curved membranes without stereospecific interactions. We show that the cis golgin GMAP-210 accounts for this property. First, the liposome tethering properties of the Golgi resembles that of the amphipathic lipid-packing sensor (ALPS) motif of GMAP-210: both preferred small (radius < 40 nm) liposomes made of monounsaturated but not saturated lipids. Second, reducing GMAP-210 levels or redirecting its ALPS motif to mitochondria decreased liposome capture by the Golgi. Extensive mutagenesis analysis suggests that GMAP-210 tethers authentic transport vesicles via the same mechanism whereby the ALPS motif senses lipid-packing defects at the vesicle surface through its regularly spaced hydrophobic residues. We conclude that the Golgi uses GMAP-210 as a filter to select transport vesicles according to their size and bulk lipid composition.


Asunto(s)
Aparato de Golgi/metabolismo , Liposomas/química , Liposomas/metabolismo , Proteínas Nucleares/metabolismo , Proteínas del Citoesqueleto , Análisis Mutacional de ADN , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteínas Nucleares/genética , Vesículas Transportadoras/metabolismo
2.
Dev Cell ; 26(1): 86-100, 2013 Jul 15.
Artículo en Inglés | MEDLINE | ID: mdl-23810513

RESUMEN

It was recently demonstrated that embryonic glucagon-producing cells in the pancreas can regenerate and convert into insulin-producing ß-like cells through the constitutive/ectopic expression of the Pax4 gene. However, whether α cells in adult mice display the same plasticity is unknown. Similarly, the mechanisms underlying such reprogramming remain unclear. We now demonstrate that the misexpression of Pax4 in glucagon(+) cells age-independently induces their conversion into ß-like cells and their glucagon shortage-mediated replacement, resulting in islet hypertrophy and in an unexpected islet neogenesis. Combining several lineage-tracing approaches, we show that, upon Pax4-mediated α-to-ß-like cell conversion, pancreatic duct-lining precursor cells are continuously mobilized, re-express the developmental gene Ngn3, and successively adopt a glucagon(+) and a ß-like cell identity through a mechanism involving the reawakening of the epithelial-to-mesenchymal transition. Importantly, these processes can repeatedly regenerate the whole ß cell mass and thereby reverse several rounds of toxin-induced diabetes, providing perspectives to design therapeutic regenerative strategies.


Asunto(s)
Reprogramación Celular , Diabetes Mellitus Experimental/metabolismo , Células Secretoras de Insulina/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Glucemia/análisis , Diferenciación Celular , Linaje de la Célula , Movimiento Celular , Diabetes Mellitus Experimental/genética , Transición Epitelial-Mesenquimal , Regulación de la Expresión Génica , Células Secretoras de Glucagón/metabolismo , Células Secretoras de Glucagón/patología , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Hipertrofia/metabolismo , Hipertrofia/patología , Células Secretoras de Insulina/patología , Ratones , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Factores de Transcripción Paired Box/genética , Factores de Transcripción Paired Box/metabolismo , Conductos Pancreáticos/efectos de los fármacos , Conductos Pancreáticos/metabolismo , Conductos Pancreáticos/patología , Estreptozocina
3.
Front Oncol ; 2: 18, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-22649778

RESUMEN

The hypoxia-inducible factor 1 (HIF-1), in addition to genetic and epigenetic changes, is largely responsible for alterations in cell metabolism in hypoxic tumor cells. This transcription factor not only favors cell proliferation through the metabolic shift from oxidative phosphorylation to glycolysis and lactic acid production but also stimulates nutrient supply by mediating adaptive survival mechanisms. In this study we showed that glycogen synthesis is enhanced in non-cancer and cancer cells when exposed to hypoxia, resulting in a large increase in glycogen stores. Furthermore, we demonstrated that the mRNA and protein levels of the first enzyme of glycogenesis, phosphoglucomutase1 (PGM1), were increased in hypoxia. We showed that induction of glycogen storage as well as PGM1 expression were dependent on HIF-1 and HIF-2. We established that hypoxia-induced glycogen stores are rapidly mobilized in cells that are starved of glucose. Glycogenolysis allows these "hypoxia-preconditioned" cells to confront and survive glucose deprivation. In contrast normoxic control cells exhibit a high rate of cell death following glucose removal. These findings point to the important role of hypoxia and HIF in inducing mechanisms of rapid adaptation and survival in response to a decrease in oxygen tension. We propose that a decrease in pO(2) acts as an "alarm" that prepares the cells to face subsequent nutrient depletion and to survive.

4.
Cancer Res ; 72(8): 2140-50, 2012 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-22389449

RESUMEN

Resistance to chemotherapy-induced apoptosis of tumor cells represents a major hurdle to efficient cancer therapy. Although resistance is a characteristic of tumor cells that evolve in a low oxygen environment (hypoxia), the mechanisms involved remain elusive. We observed that mitochondria of certain hypoxic cells take on an enlarged appearance with reorganized cristae. In these cells, we found that a major mitochondrial protein regulating metabolism and apoptosis, the voltage-dependent anion channel 1 (VDAC1), was linked to chemoresistance when in a truncated (VDAC1-ΔC) but active form. The formation of truncated VDAC1, which had a similar channel activity and voltage dependency as full-length, was hypoxia-inducible factor-1 (HIF-1)-dependent and could be inhibited in the presence of the tetracycline antibiotics doxycycline and minocycline, known inhibitors of metalloproteases. Its formation was also reversible upon cell reoxygenation and associated with cell survival through binding to the antiapoptotic protein hexokinase. Hypoxic cells containing VDAC1-ΔC were less sensitive to staurosporine- and etoposide-induced cell death, and silencing of VDAC1-ΔC or treatment with the tetracycline antibiotics restored sensitivity. Clinically, VDAC1-ΔC was detected in tumor tissues of patients with lung adenocarcinomas and was found more frequently in large and late-stage tumors. Together, our findings show that via induction of VDAC1-ΔC, HIF-1 confers selective protection from apoptosis that allows maintenance of ATP and cell survival in hypoxia. VDAC1-ΔC may also hold promise as a biomarker for tumor progression in chemotherapy-resistant patients.


Asunto(s)
Resistencia a Antineoplásicos/fisiología , Neoplasias Pulmonares/metabolismo , Canal Aniónico 1 Dependiente del Voltaje/biosíntesis , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Apoptosis/fisiología , Hipoxia de la Célula , Línea Celular Tumoral , Supervivencia Celular , Progresión de la Enfermedad , Femenino , Citometría de Flujo , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Immunoblotting , Neoplasias Pulmonares/patología , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Isoformas de Proteínas/metabolismo , Transfección
5.
Blood ; 119(19): 4527-31, 2012 May 10.
Artículo en Inglés | MEDLINE | ID: mdl-22452982

RESUMEN

Autophagy is the process by which superfluous or damaged macromolecules or organelles are degraded by the lysosome. Pharmacologic and genetic evidence indicates that autophagy plays pleiotropic functions in cellular homeostasis, development, survival, and differentiation. The differentiation of human blood monocytes into macrophages is a caspase-dependent process when triggered ex vivo by colony stimulating factor-1. We show here, using pharmacologic inhibitors, siRNA approaches, and Atg7-/- mice, that autophagy initiated by ULK1 is required for proper colony stimulating factor-1-driven differentiation of human and murine monocytes. We also unravel a role for autophagy in macrophage acquisition of phagocytic functions. Collectively, these findings highlight an unexpected and essential role of autophagy during monocyte differentiation and acquisition of macrophage functions.


Asunto(s)
Autofagia/fisiología , Diferenciación Celular/efectos de los fármacos , Factor Estimulante de Colonias de Macrófagos/farmacología , Macrófagos/efectos de los fármacos , Fagocitosis/efectos de los fármacos , Animales , Autofagia/efectos de los fármacos , Autofagia/genética , Proteína 7 Relacionada con la Autofagia , Homólogo de la Proteína 1 Relacionada con la Autofagia , Catepsina B/farmacología , Células Cultivadas , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Macrófagos/fisiología , Ratones , Ratones Noqueados , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/fisiología , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Monocitos/fisiología , Fagocitosis/fisiología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/fisiología , ARN Interferente Pequeño/farmacología
6.
Infect Immun ; 79(6): 2396-403, 2011 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-21402759

RESUMEN

The SecA2 auxiliary secretion system of Gram-positive bacteria promotes the export of virulence proteins essential for colonization of the host in the case of both Mycobacterium tuberculosis and Listeria monocytogenes, two intracellular bacteria causing diseases in humans. We and others have demonstrated that this secretion system is also linked to the onset of long-term CD8(+) T cell-mediated protective immunity in mice. In the case of L. monocytogenes, expression of SecA2 inside the cytosol of infected cells correlates with the generation of CCL3-secreting memory CD8(+) T cells that are required for protection against secondary challenge with wild-type (wt) L. monocytogenes. Since the SecA2 ATPase is well conserved among Gram-positive pathogenic bacteria, we hypothesized that SecA2 itself bears evolutionarily conserved motifs recognized by cytosolic pattern recognition receptors, leading to signaling events promoting the differentiation of CCL3(+) memory CD8(+) T cells. To test this possibility, we generated a stable L. monocytogenes chromosomal mutant that expressed a SecA2 ATPase bearing a mutated nucleotide binding site (NBS). Similarly to a SecA2 deletion mutant, the NBS mutant exhibited rough colonies, a bacterial chaining phenotype, an impaired protein secretion profile, and in vivo virulence in comparison to wt L. monocytogenes. Importantly, mice immunized with the SecA2 NBS mutant were not protected against secondary infection with wt L. monocytogenes and did not develop CCL3(+) memory CD8(+) T cells. NBS mutant and wt SecA2 proteins were expressed to comparable extents by bacteria, suggesting that SecA2 itself is unlikely to promote the induction of these cells. Rather, one or several of the SecA2 substrate proteins released inside the cytosol of infected cells may be involved.


Asunto(s)
Adenosina Trifosfatasas/fisiología , Proteínas Bacterianas/fisiología , Linfocitos T CD8-positivos/inmunología , Memoria Inmunológica/inmunología , Listeria monocytogenes/inmunología , Listeriosis/inmunología , Proteínas de Transporte de Membrana/fisiología , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/inmunología , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Western Blotting , Clonación Molecular , Femenino , Citometría de Flujo , Listeria monocytogenes/genética , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/inmunología , Ratones , Ratones Endogámicos BALB C , Mutagénesis Sitio-Dirigida , Canales de Translocación SEC , Proteína SecA
7.
PLoS One ; 6(2): e14682, 2011 Feb 14.
Artículo en Inglés | MEDLINE | ID: mdl-21339824

RESUMEN

Bacillus sphaericus strains that produce the binary toxin (Bin) are highly toxic to Culex and Anopheles mosquitoes, and have been used since the late 1980s as a biopesticide for the control of these vectors of infectious disease agents. The Bin toxin produced by these strains targets mosquito larval midgut epithelial cells where it binds to Cpm1 (Culex pipiens maltase 1) a digestive enzyme, and causes severe intracellular damage, including a dramatic cytoplasmic vacuolation. The intoxication of mammalian epithelial MDCK cells engineered to express Cpm1 mimics the cytopathologies observed in mosquito enterocytes following Bin ingestion: pore formation and vacuolation. In this study we demonstrate that Bin-induced vacuolisation is a transient phenomenon that affects autolysosomes. In addition, we show that this vacuolisation is associated with induction of autophagy in intoxicated cells. Furthermore, we report that after internalization, Bin reaches the recycling endosomes but is not localized either within the vacuolating autolysosomes or within any other degradative compartment. Our observations reveal that Bin elicits autophagy as the cell's response to intoxication while protecting itself from degradation through trafficking towards the recycling pathways.


Asunto(s)
Autofagia/efectos de los fármacos , Infecciones por Bacillaceae/patología , Toxinas Bacterianas/toxicidad , Animales , Anopheles/enzimología , Anopheles/genética , Infecciones por Bacillaceae/metabolismo , Bacillus/metabolismo , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/farmacología , Células Cultivadas , Culex/enzimología , Culex/genética , Perros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Interacciones Huésped-Patógeno/efectos de los fármacos , Interacciones Huésped-Patógeno/fisiología , Fagosomas/efectos de los fármacos , Fagosomas/metabolismo , Fagosomas/patología , Proteínas Recombinantes de Fusión/metabolismo , Transfección , Vacuolas/efectos de los fármacos , Vacuolas/metabolismo , Vacuolas/patología , alfa-Glucosidasas/genética , alfa-Glucosidasas/metabolismo
8.
PLoS Pathog ; 7(12): e1002457, 2011 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-22241983

RESUMEN

Immunological memory is a hallmark of B and T lymphocytes that have undergone a previous encounter with a given antigen. It is assumed that memory cells mediate better protection of the host upon re-infection because of improved effector functions such as antibody production, cytotoxic activity and cytokine secretion. In contrast to cells of the adaptive immune system, innate immune cells are believed to exhibit a comparable functional effector response each time the same pathogen is encountered. Here, using mice infected by the intracellular bacterium Listeria monocytogenes, we show that during a recall bacterial infection, the chemokine CCL3 secreted by memory CD8+ T cells drives drastic modifications of the functional properties of several populations of phagocytes. We found that inflammatory ly6C+ monocytes and neutrophils largely mediated memory CD8+ T cell bacteriocidal activity by producing increased levels of reactive oxygen species (ROS), augmenting the pH of their phagosomes and inducing antimicrobial autophagy. These events allowed an extremely rapid control of bacterial growth in vivo and accounted for protective immunity. Therefore, our results provide evidence that cytotoxic memory CD8+ T cells can license distinct antimicrobial effector mechanisms of innate cells to efficiently clear pathogens.


Asunto(s)
Linfocitos T CD8-positivos/inmunología , Inmunidad Celular , Memoria Inmunológica , Listeria monocytogenes/inmunología , Listeriosis/inmunología , Monocitos/inmunología , Neutrófilos/inmunología , Animales , Quimiocina CCL3/genética , Quimiocina CCL3/inmunología , Listeriosis/genética , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados
9.
PLoS Pathog ; 6(10): e1001154, 2010 Oct 14.
Artículo en Inglés | MEDLINE | ID: mdl-20976202

RESUMEN

Protozoa and bacteria infect various types of phagocytic cells including macrophages, monocytes, dendritic cells and eosinophils. However, it is not clear which of these cells process and present microbial antigens in vivo and in which cellular compartments parasite peptides are loaded onto Major Histocompatibility Complex molecules. To address these issues, we have infected susceptible BALB/c (H-2d) mice with a recombinant Leishmania major parasite expressing a fluorescent tracer. To directly visualize the antigen presenting cells that present parasite-derived peptides to CD4+ T cells, we have generated a monoclonal antibody that reacts to an antigenic peptide derived from the parasite LACK antigen bound to I-Ad Major Histocompatibility Complex class II molecule. Immunogold electron microscopic analysis of in vivo infected cells showed that intracellular I-Ad/LACK complexes were present in the membrane of amastigote-containing phagosomes in dendritic cells, eosinophils and macrophages/monocytes. In both dendritic cells and macrophages, these complexes were also present in smaller vesicles that did not contain amastigote. The presence of I-Ad/LACK complexes at the surface of dendritic cells, but neither on the plasma membrane of macrophages nor eosinophils was independently confirmed by flow cytometry and by incubating sorted phagocytes with highly sensitive LACK-specific hybridomas. Altogether, our results suggest that peptides derived from Leishmania proteins are loaded onto Major Histocompatibility Complex class II molecules in the phagosomes of infected phagocytes. Although these complexes are transported to the cell surface in dendritic cells, therefore allowing the stimulation of parasite-specific CD4+ T cells, this does not occur in other phagocytic cells. To our knowledge, this is the first study in which Major Histocompatibility Complex class II molecules bound to peptides derived from a parasite protein have been visualized within and at the surface of cells that were infected in vivo.


Asunto(s)
Membrana Celular/metabolismo , Antígenos de Histocompatibilidad Clase II/metabolismo , Leishmania major/inmunología , Leishmaniasis Cutánea/inmunología , Leishmaniasis Cutánea/metabolismo , Fragmentos de Péptidos/metabolismo , Secuencia de Aminoácidos , Animales , Compartimento Celular/inmunología , Membrana Celular/inmunología , Células Cultivadas , Técnica del Anticuerpo Fluorescente/métodos , Antígenos de Histocompatibilidad Clase II/inmunología , Membranas Intracelulares/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Fragmentos de Péptidos/química , Fragmentos de Péptidos/inmunología , Fagosomas/inmunología , Fagosomas/metabolismo
10.
Cancer Res ; 70(19): 7514-22, 2010 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-20841472

RESUMEN

Cisplatin is an antineoplastic drug, mostly documented to cause cell death through the formation of DNA adducts. In patients, it exhibits a range of short-term side effects that are unlikely to be related to its genomic action. As cisplatin has been shown to modify membrane properties in different cell systems, we investigated its effects on mechanosensitive ion transporters and channels. We show here that cisplatin is a noncompetitive inhibitor of the mechanosensitive Na(+)/H(+) exchanger NHE-1, with a half-inhibition concentration of 30 µg/mL associated with a decrease in V(max) and Hill coefficient. We also showed that it blocks the Cl(-) and K(+) mechanosensitive channels VSORC and TREK-1 at similar concentrations. In contrast, the nonmechanosensitive Cl(-) and K(+) channels CFTR and TASK-1 and the Na(+)-coupled glucose transport, which share functional features with VSORC, TREK-1, and NHE-1, respectively, were insensitive to cisplatin. We next investigated whether cisplatin action was due to a direct effect on membrane or to cortical actin remodeling that would affect mechanosensors. Using scanning electron microscopy, in vivo actin labeling, and atomic force microscopy, we did not observe any modification of the Young's modulus and actin cytoskeleton for up to 60 and 120 µg/mL cisplatin, whereas these concentrations modified membrane morphology. Our results reveal a novel mechanism for cisplatin, which affects mechanosensitive channels and transporters involved in cell fate programs and/or expressed in mechanosensitive organs in which cisplatin elicits strong secondary effects, such as the inner ear or the peripheral nervous system. These results might constitute a common denominator to previously unrelated effects of this drug.


Asunto(s)
Actinas/metabolismo , Cisplatino/farmacología , Canales Iónicos/antagonistas & inhibidores , Intercambiadores de Sodio-Hidrógeno/antagonistas & inhibidores , Animales , Antineoplásicos/farmacología , Fenómenos Biomecánicos , Células COS , Forma de la Célula/efectos de los fármacos , Chlorocebus aethiops , Regulador de Conductancia de Transmembrana de Fibrosis Quística/antagonistas & inhibidores , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Microscopía de Fuerza Atómica , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Canales de Potasio de Dominio Poro en Tándem/antagonistas & inhibidores
11.
Cancer Res ; 70(6): 2465-75, 2010 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-20215500

RESUMEN

Targeting cancer cell metabolism is a new promising strategy to fight cancer. Metformin, a widely used antidiabetic agent, exerts antitumoral and antiproliferative action. In this study, the addition of metformin to 2-deoxyglucose (2DG) inhibited mitochondrial respiration and glycolysis in prostate cancer cells leading to a severe depletion in ATP. The combination of the two drugs was much more harmful for cancer cells than the treatment with metformin or 2DG alone, leading to 96% inhibition of cell viability in LNCaP prostate cancer cells. In contrast, a moderate effect on cell viability was observed in normal prostate epithelial cells. At the cellular level, the combination of metformin and 2DG induced p53-dependent apoptosis via the energy sensor pathway AMP kinase, and the reexpression of a functional p53 in p53-deficient prostate cancer cells restored caspase-3 activity. In addition to apoptosis, the combination of metformin and 2DG arrested prostate cancer cells in G(2)-M. This G(2)-M arrest was independent of p53 and correlated with a stronger decrease in cell viability than obtained with either drug. Finally, metformin inhibited 2DG-induced autophagy, decreased beclin 1 expression, and triggered a switch from a survival process to cell death. Our study reinforces the growing interest of metabolic perturbators in cancer therapy and highlights the potential use of the combination of metformin and 2DG as an anticancerous treatment.


Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Apoptosis/efectos de los fármacos , Desoxiglucosa/farmacología , Metformina/farmacología , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Adenosina Trifosfato/deficiencia , Adenosina Trifosfato/metabolismo , Adenilato Quinasa/metabolismo , Autofagia/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Desoxiglucosa/administración & dosificación , Desoxiglucosa/antagonistas & inhibidores , Sinergismo Farmacológico , Humanos , Masculino , Metformina/administración & dosificación , Neoplasias de la Próstata/patología
12.
Int Urogynecol J ; 21(3): 261-70, 2010 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-20052576

RESUMEN

INTRODUCTION AND HYPOTHESIS: Currently, most implants used for reinforcement in surgical treatment of pelvic floor disorders are knitted monofilament polypropylene (PP). While previously recognized as inert, PP is associated with high complication rates. Some recent literature suggests polyester prosthetics based on poly(ethylene terephthalate) (PET), which may be more inert in vivo. METHODS: A sample of 100 implants explanted from patients due to complications was examined to evaluate the relative degradation characteristics of PP and PET prosthetics. Histological, microscopic (scanning electron microscopy, SEM) and chemical analysis (Fourier transform infrared (FTIR) spectroscopy and differential scanning calorimetry (DSC)) were conducted on these explants. RESULTS: Poly(ethylene terephtahlate) explants appeared to sustain less degradation in vivo than the PP explants observed in this cohort. CONCLUSIONS: This is the first study to evaluate synthetic implants used in a vaginal approach for pelvic floor reinforcement. The study provides evidence contrary to published literature characterizing PP as inert in such applications. Additionally, the study suggests the need for clinical trials comparatively investigating the performance of new types of monofilament prosthetics, such as those comprising PET.


Asunto(s)
Reacción a Cuerpo Extraño/etiología , Polipropilenos/efectos adversos , Complicaciones Posoperatorias/etiología , Mallas Quirúrgicas/efectos adversos , Remoción de Dispositivos , Femenino , Humanos , Microscopía Electrónica de Rastreo , Prolapso de Órgano Pélvico/cirugía , Estudios Prospectivos , Espectroscopía Infrarroja por Transformada de Fourier , Incontinencia Urinaria de Esfuerzo/cirugía
13.
J Cell Physiol ; 222(3): 648-57, 2010 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-19957303

RESUMEN

It is well established that cells exposed to the limiting oxygen microenvironment (hypoxia) of tumors acquire resistance to chemotherapy, through mechanisms not fully understood. We noted that a large number of cell lines showed protection from apoptotic stimuli, staurosporine, or etoposide, when exposed to long-term hypoxia (72 h). In addition, these cells had unusual enlarged mitochondria that were induced in a HIF-1-dependent manner. Enlarged mitochondria were functional as they conserved their transmembrane potential and ATP production. Here we reveal that mitochondria of hypoxia-induced chemotherapy-resistant cells undergo a HIF-1-dependent and mitofusin-1-mediated change in morphology from a tubular network to an enlarged phenotype. An imbalance in mitochondrial fusion/fission occurs since silencing of not only the mitochondrial fusion protein mitofusin 1 but also BNIP3 and BNIP3L, two mitochondrial HIF-targeted genes, reestablished a tubular morphology. Hypoxic cells were insensitive to staurosporine- and etoposide-induced cell death, but the silencing of mitofusin, BNIP3, and BNIP3L restored sensitivity. Our results demonstrate that some cancer cells have developed yet another way to evade apoptosis in hypoxia, by inducing mitochondrial fusion and targeting BNIP3 and BNIP3L to mitochondrial membranes, thereby giving these cells a selective growth advantage.


Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Apoptosis/efectos de los fármacos , Resistencia a Antineoplásicos , Etopósido/farmacología , Mitocondrias/patología , Dilatación Mitocondrial , Neoplasias/patología , Estaurosporina/farmacología , Adenosina Trifosfato/metabolismo , Hipoxia de la Célula , Proliferación Celular/efectos de los fármacos , GTP Fosfohidrolasas/genética , GTP Fosfohidrolasas/metabolismo , Células HeLa , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Mitocondrias/metabolismo , Proteínas de Transporte de Membrana Mitocondrial , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Fenotipo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Interferencia de ARN , Factores de Tiempo , Transfección , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo
14.
PLoS One ; 4(11): e7889, 2009 Nov 18.
Artículo en Inglés | MEDLINE | ID: mdl-19924252

RESUMEN

CML is an hematopoietic stem cell disease characterized by the t(9;22) (q34;q11) translocation encoding the oncoprotein p210BCR-ABL. The effect of acadesine (AICAR, 5-Aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside) a compound with known antileukemic effect on B cell chronic lymphoblastic leukemia (B-CLL) was investigated in different CML cell lines. Acadesine triggered loss of cell metabolism in K562, LAMA-84 and JURL-MK1 and was also effective in killing imatinib-resistant K562 cells and Ba/F3 cells carrying the T315I-BCR-ABL mutation. The anti-leukemic effect of acadesine did not involve apoptosis but required rather induction of autophagic cell death. AMPK knock-down by Sh-RNA failed to prevent the effect of acadesine, indicating an AMPK-independent mechanism. The effect of acadesine was abrogated by GF109203X and Ro-32-0432, both inhibitor of classical and new PKCs and accordingly, acadesine triggered relocation and activation of several PKC isoforms in K562 cells. In addition, this compound exhibited a potent anti-leukemic effect in clonogenic assays of CML cells in methyl cellulose and in a xenograft model of K562 cells in nude mice. In conclusion, our work identifies an original and unexpected mechanism by which acadesine triggers autophagic cell death through PKC activation. Therefore, in addition to its promising effects in B-CLL, acadesine might also be beneficial for Imatinib-resistant CML patients.


Asunto(s)
Aminoimidazol Carboxamida/análogos & derivados , Autofagia , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Ribonucleósidos/farmacología , Proteínas Quinasas Activadas por AMP/metabolismo , Aminoimidazol Carboxamida/farmacología , Animales , Apoptosis , Línea Celular Tumoral , Proliferación Celular , Humanos , Células K562 , Ratones , Ratones Desnudos , Proteína Quinasa C/metabolismo , ARN Interferente Pequeño/metabolismo , Translocación Genética
15.
Development ; 136(21): 3647-55, 2009 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-19820183

RESUMEN

The size of the mammalian body is determined by genetic and environmental factors differentially modulating pre- and postnatal growth. We now report a control of growth acting in the mouse from the first cleavages to the postnatal stages. It was evidenced by a hereditary epigenetic modification (paramutation) created by injection of a miR-124 microRNA into fertilized eggs. From the blastocyst to the adult, mouse pups born after microinjection of this miRNA showed a 30% increase in size. At the blastocyst stage, frequent duplication of the inner cell mass resulted in twin pregnancies. A role of sperm RNA as a transgenerational signal was confirmed by the giant phenotype of the progeny of transgenic males expressing miR-124 during spermiogenesis. In E2.5 to E8.5 embryos, increased levels of several transcripts with sequence homology to the microRNA were noted, including those of Sox9, a gene known for its crucial role in the progenitors of several adult tissues. A role in embryonic growth was confirmed by the large size of embryos expressing a Sox9 DNA transgene. Increased expression in the paramutants was not related to a change in miR-124 expression, but to the establishment of a distinct, heritable chromatin structure in the promoter region of Sox9. While the heritability of body size is not readily accounted for by Mendelian genetics, our results suggest the alternate model of RNA-mediated heritable epigenetic modifications.


Asunto(s)
Tamaño Corporal/genética , Epigénesis Genética , Ratones/embriología , MicroARNs/metabolismo , Factor de Transcripción SOX9/metabolismo , Animales , Masculino , Ratones/genética
16.
Autophagy ; 5(8): 1092-8, 2009 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-19786835

RESUMEN

Autophagy is a highly conserved catabolic process for the elimination and recycling of organelles and macromolecules, characterized by the formation of double-membrane vesicles called autophagosomes. To date, the function of autophagy in cell differentiation is poorly documented. Here, we investigated the possibility that megakaryocytic differentiation of the Chronic Myelogenous Leukemia (CML) cell line K562, a process known to be accompanied by accumulation of vacuoles inside the cells, might involve autophagy. Using various complementary approaches, we show that the combination of the phorbol ester PMA and the p38(MAPK) inhibitor SB202190 (SB), which engaged a majority of K562 cells towards the megakaryocytic lineage, also triggered vacuolization and autophagy. The combination of PMA + SB appears to induce both increase in autophagic fluxes and an autophagic degradation blockage. Induction of autophagy was accompanied also by increased expression of Beclin 1 and p62/SQSTM1 and was found to precede the onset of megakaryocytic differentiation. Moreover, knockdown of LC3 and Beclin 1 by specific siRNAs impaired PMA + SB-mediated vacuolization, LC3-II accumulation and megakaryocytic differentiation, as well. To the best of our knowledge, this is the first description that induction of autophagy is involved in megakaryocytic differentiation of K562 CML cells.


Asunto(s)
Autofagia , Diferenciación Celular , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Megacariocitos/citología , Diferenciación Celular/efectos de los fármacos , Humanos , Imidazoles/farmacología , Células K562 , Megacariocitos/efectos de los fármacos , Megacariocitos/ultraestructura , Piridinas/farmacología , Acetato de Tetradecanoilforbol/farmacología , Vacuolas/efectos de los fármacos , Vacuolas/metabolismo
17.
J Biol Chem ; 284(28): 18699-706, 2009 Jul 10.
Artículo en Inglés | MEDLINE | ID: mdl-19389708

RESUMEN

Melanins are synthesized in melanocytes within specialized organelles called melanosomes. Numerous studies have shown that the pH of melanosome plays a key role in the regulation of melanin synthesis. However, until now, acute regulation of melanosome pH by a physiological stimulus has never been demonstrated. In the present study, we show that the activation of the cAMP pathway by alphaMSH or forskolin leads to an alkalinization of melanosomes and a concomitant regulation of vacuolar ATPases and ion transporters of the solute carrier family. The solute carrier family members include SLC45A2, which is mutated in oculocutaneous albinism type IV, SLC24A4 and SLC24A5, proteins implicated in the control of eye, hair, and skin pigmentation, and the P protein, encoded by the oculocutaneous albinism type II locus. Interestingly, H89, a pharmacological inhibitor of protein kinase A (PKA), prevents the cAMP-induced pigmentation and induces acidification of melanosomes. The drastic depigmenting effect of H89 is not due to an inhibition of tyrosinase expression. Indeed, H89 blocks the induction of melanogenesis induced by LY294002, a potent inhibitor of the PI 3-kinase pathway, without any effect on tyrosinase expression. Furthermore, PKA is not involved in the inhibition of pigmentation promoted by H89 because LY294002 induces pigmentation independently of PKA. Also, other PKA inhibitors do not affect pigmentation. Taken together, our results strengthen the support for a key role of melanosome pH in the regulation of melanin synthesis and, for the first time, demonstrate that melanosome pH is regulated by cAMP and alphaMSH. Notably, these are both mediators of the response to solar UV radiation, the main physiological stimulus of skin pigmentation.


Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/química , AMP Cíclico/metabolismo , Melanosomas/metabolismo , alfa-MSH/metabolismo , Animales , Cromonas/farmacología , Inhibidores Enzimáticos/farmacología , Regulación Enzimológica de la Expresión Génica , Humanos , Concentración de Iones de Hidrógeno , Isoquinolinas/farmacología , Melaninas/química , Melanoma Experimental , Ratones , Modelos Biológicos , Monofenol Monooxigenasa/antagonistas & inhibidores , Monofenol Monooxigenasa/metabolismo , Morfolinas/farmacología , Pigmentación de la Piel , Sulfonamidas/farmacología
18.
Cardiovasc Res ; 83(1): 61-71, 2009 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-19351742

RESUMEN

AIMS: Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors. PPARbeta agonists were suggested as potential drugs for the treatment of metabolic syndrome, but effects of PPARbeta activation on cardiac growth and vascularization are unknown. Thus, we investigated the consequences of pharmacological PPARbeta activation on the heart and the underlying molecular mechanisms. METHODS AND RESULTS: Male C57/Bl6 mice were injected with the specific PPARbeta agonists GW0742 or GW501516, or vehicle. Cardiomyocyte size and vascularisation were determined at different time points. Expression differences were investigated by quantitative reverse transcriptase-polymerase chain reaction and western blotting. In addition, the effects of PPARbeta stimulation were compared with hearts of mice undergoing long-term voluntary exercise or pharmacological PPARalpha activation. Five hours after GW0742 injection, we detected an enhanced angiogenesis compared with vehicle-injected controls. After 24 h, the heart-to-body weight ratios were higher in mice injected with either GW0742 or GW501516 vs. controls. The increased heart size was due to cardiomyocyte enlargement. No signs of pathological cardiac hypertrophy (i.e. apoptosis, fibrosis, or deteriorated cardiac function) could be detected. The effects are mediated via calcineurin A (CnA) activation as: (i) CnA was upregulated, (ii) GW0742 administration or co-transfection of PPARbeta significantly stimulated the activity of the CnA promoter, (iii) PPARbeta protein bound directly to the CnA promoter, (iv) the CnA target genes NFATc3, Hif-1alpha, and Cdk 9 were upregulated in response to PPARbeta stimulation, and (v) the inhibition of CnA activity by cyclosporine A abolished the hypertrophic and angiogenic responses to PPARbeta stimulation. CONCLUSION: Our data suggest PPARbeta pharmacological activation as a novel approach to increase cardiac vascularization and cardiac muscle mass.


Asunto(s)
Calcineurina/metabolismo , Corazón/crecimiento & desarrollo , Neovascularización Fisiológica/efectos de los fármacos , PPAR-beta/agonistas , Tiazoles/farmacología , Animales , Inhibidores de la Calcineurina , Células Cultivadas , Vasos Coronarios/citología , Vasos Coronarios/efectos de los fármacos , Vasos Coronarios/metabolismo , Quinasa 9 Dependiente de la Ciclina/metabolismo , Ciclosporina/farmacología , Inhibidores Enzimáticos/farmacología , Corazón/efectos de los fármacos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Animales , Miocardio/citología , Miocardio/metabolismo , Miocitos Cardíacos/citología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Factores de Transcripción NFATC/metabolismo , PPAR-beta/farmacología
19.
Mol Cell Biol ; 29(10): 2570-81, 2009 May.
Artículo en Inglés | MEDLINE | ID: mdl-19273585

RESUMEN

While hypoxia-inducible factor (HIF) is a major actor in the cell survival response to hypoxia, HIF also is associated with cell death. Several studies implicate the HIF-induced putative BH3-only proapoptotic genes bnip3 and bnip3l in hypoxia-mediated cell death. We, like others, do not support this assertion. Here, we clearly demonstrate that the hypoxic microenvironment contributes to survival rather than cell death by inducing autophagy. The ablation of Beclin1, a major actor of autophagy, enhances cell death under hypoxic conditions. In addition, the ablation of BNIP3 and/or BNIP3L triggers cell death, and BNIP3 and BNIP3L are crucial for hypoxia-induced autophagy. First, while the small interfering RNA-mediated ablation of either BNIP3 or BNIP3L has little effect on autophagy, the combined silencing of these two HIF targets suppresses hypoxia-mediated autophagy. Second, the ectopic expression of both BNIP3 and BNIP3L in normoxia activates autophagy. Third, 20-mer BH3 peptides of BNIP3 or BNIP3L are sufficient in initiating autophagy in normoxia. Herein, we propose a model in which the atypical BH3 domains of hypoxia-induced BNIP3/BNIP3L have been designed to induce autophagy by disrupting the Bcl-2-Beclin1 complex without inducing cell death. Hypoxia-induced autophagy via BNIP3 and BNIP3L is clearly a survival mechanism that promotes tumor progression.


Asunto(s)
Autofagia/fisiología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Hipoxia de la Célula , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Proteínas de la Membrana/metabolismo , Fragmentos de Péptidos/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Beclina-1 , Línea Celular Tumoral , Fibroblastos/citología , Fibroblastos/fisiología , Regulación de la Expresión Génica , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Fragmentos de Péptidos/genética , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/genética , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Alineación de Secuencia , Proteínas Supresoras de Tumor/química , Proteínas Supresoras de Tumor/genética
20.
Blood ; 113(2): 347-57, 2009 Jan 08.
Artículo en Inglés | MEDLINE | ID: mdl-18849489

RESUMEN

By presenting antigenic peptides on the cell surface, human leukocyte antigen (HLA) class I molecules are critical for immune defense. Their surface density determines, to a large extent, the level of CD8(+) T cell-dependent immune reactions; their loss is a major mechanism of immune escape. Therefore, powerful processes should regulate their surface expression. Here we document the mechanisms used by CD99 to mediate HLA class I modulation. Up-regulation of HLA class I by IFN-gamma requires CD99. In the trans Golgi network (TGN), and up to the cell surface, CD99 and HLA class I are physically associated via their transmembrane domain. CD99 also binds p230/golgin-245, a coiled-coil protein that recycles between the cytosol and buds/vesicles of the TGN and which plays a fundamental role in trafficking transport vesicles. p230/golgin-245 is anchored within TGN membranes via its Golgin-97, RanBP1, IMh1p, P230 (GRIP) domain and the overexpression of which leads to surface and intracellular down-modulation of HLA class I molecules.


Asunto(s)
Antígenos CD/inmunología , Autoantígenos/inmunología , Linfocitos T CD8-positivos/inmunología , Moléculas de Adhesión Celular/inmunología , Aparato de Golgi/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Proteínas de la Membrana/inmunología , Regulación hacia Arriba/inmunología , Antígeno 12E7 , Antígenos CD/metabolismo , Antivirales/inmunología , Antivirales/farmacología , Autoantígenos/metabolismo , Linfocitos T CD8-positivos/metabolismo , Moléculas de Adhesión Celular/metabolismo , Citosol/inmunología , Citosol/metabolismo , Aparato de Golgi/metabolismo , Antígenos HLA/biosíntesis , Antígenos HLA/inmunología , Antígenos de Histocompatibilidad Clase I/biosíntesis , Humanos , Inmunidad Celular/fisiología , Interferón gamma/inmunología , Interferón gamma/farmacología , Células Jurkat , Proteínas de la Membrana/metabolismo , Estructura Terciaria de Proteína/fisiología , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/inmunología , Vesículas Transportadoras/inmunología , Vesículas Transportadoras/metabolismo , Regulación hacia Arriba/efectos de los fármacos
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