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Antibiotic resistance ranks among the top threats to humanity. Due to the frequent use of antibiotics, society is facing a high prevalence of multidrug resistant pathogens, which have managed to evolve mechanisms that help them evade the last line of therapeutics. An alternative to antibiotics could involve the use of bacteriophages (phages), which are the natural predators of bacterial cells. In earlier times, phages were implemented as therapeutic agents for a century but were mainly replaced with antibiotics, and considering the menace of antimicrobial resistance, it might again become of interest due to the increasing threat of antibiotic resistance among pathogens. The current understanding of phage biology and clustered regularly interspaced short palindromic repeats (CRISPR) assisted phage genome engineering techniques have facilitated to generate phage variants with unique therapeutic values. In this review, we briefly explain strategies to engineer bacteriophages. Next, we highlight the literature supporting CRISPR-Cas9-assisted phage engineering for effective and more specific targeting of bacterial pathogens. Lastly, we discuss techniques that either help to increase the fitness, specificity, or lytic ability of bacteriophages to control an infection.
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BACKGROUND: The recent emergence of gene editing using Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated system (Cas) tools and advances in genomics and proteomics has revolutionized drug discovery and personalized medicine. PURPOSE AND SCOPE: The CRISPR-Cas system has enabled gene and cell-based therapies, screening for novel drug targets, a new generation of disease models, elucidation of drug resistance mechanisms, and drug efficacy testing. Here, we summarized recent investigations and strategies involved in cancer-related drug discovery using the CRISPR-Cas system. CONCLUSION: CRISPR-Cas-mediated gene editing has shown great potential in the development of next generation drugs for treatment of Mendelian disorders and various cancer types. In this review, we focused on the impact of the CRISPR-Cas system in drug discovery and its application to biomarker identification and validation, high-end target genes, and breakthrough anticancer cell therapies. We also highlighted the role of CRISPR-Cas in precision disease modeling and functional drug screening.
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Antineoplásicos , Neoplasias , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Sistemas CRISPR-Cas/genética , Descubrimiento de Drogas , Edición Génica , Genómica , Neoplasias/tratamiento farmacológico , Neoplasias/genéticaRESUMEN
Although many studies have been conducted on leukemia, only a few have analyzed the metabolomic profiles of various leukemic cells. In this study, the metabolomes of THP-1, U937, KG-1 (acute myelogenous leukemia, AML), K562 (chronic myelogenous leukemia, CML), and cord blood-derived CD34-positive hematopoietic stem cells (HSC) were analyzed using gas chromatography-mass spectrometry, and specific metabolic alterations were found using multivariate statistical analysis. Compared to HSCs, leukemia cell metabolomes were found to have significant alterations, among which three were related to amino acids, three to sugars, and five to fatty acids. Compared to CML, four metabolomes were observed specifically in AML. Given that overall more metabolites are present in leukemia cells than in HSCs, we observed that the activation of glycolysis and oxidative phosphorylation (OXPHOS) metabolism facilitated the incidence of leukemia and the proliferation of leukemic cells. Analysis of metabolome profiles specifically present in HSCs and leukemia cells greatly increases our basic understanding of cellular metabolic characteristics, which is valuable fundamental knowledge for developing novel anticancer drugs targeting leukemia metabolism.
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Parkinson's disease (PD) is a neurodegenerative disease characterized by progressive movement disturbances caused by the selective loss of dopamine (DA) neurons in the substantia nigra. Despite the identification of the causal mechanisms underlying the pathogenesis of PD, effective treatments remain elusive. In this study, we observed that a low level of fetal bovine serum (FBS) effectively induced DA neurons in rat neural precursor cells (NPCs) by enhancing nuclear receptor-related 1 protein (NURR1) expression. Among the various components of FBS, the thyroid hormones triiodothyronine (T3) and thyroxine (T4) were identified as key factors for the induction of DA neurons. Since an overdose of thyroid hormones can cause hyperthyroidism, we synthesized several thyroid hormone derivatives that can partially activate thyroid hormone receptors and induce the complete differentiation of NPCs into DA neurons. Two derivatives (#3 and #9) showed positive effects on the induction and maturation of DA neurons without showing significant affinity for the thyroid hormone receptor. They also effectively protected and restored DA neurons from neurotoxic insults. Taken together, these observations demonstrate that thyroid hormone derivatives can strongly induce DA neuron differentiation while avoiding excessive thyroid stimulation and might therefore be useful candidates for PD treatment.
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Neuronas Dopaminérgicas/citología , Células-Madre Neurales/citología , Hormonas Tiroideas/síntesis química , Tiroxina/farmacología , Triyodotironina/farmacología , Animales , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Medios de Cultivo/química , Neuronas Dopaminérgicas/efectos de los fármacos , Neuronas Dopaminérgicas/metabolismo , Femenino , Células-Madre Neurales/efectos de los fármacos , Células-Madre Neurales/metabolismo , Cultivo Primario de Células , Ratas , Ratas Sprague-Dawley , Receptores de Hormona Tiroidea/metabolismo , Hormonas Tiroideas/química , Hormonas Tiroideas/farmacologíaRESUMEN
Glioblastoma multiforme (GBM), a particularly aggressive type of malignant brain tumor, has a high mortality rate. Bcl-w, an oncogene, is reported to enhance cell survival, proliferation, epithelial-mesenchymal transition (EMT), migratory and invasive abilities, and stemness maintenance in a variety of cancer cell types, including GBM. In this study, we confirmed that Bcl-w-induced conditional medium (CM) enhances tumorigenic phenotypes of migration, invasiveness, and stemness maintenance. Notably, platelet-derived growth factor-A (PDGF-A) expression, among other factors of the tumor environment, was increased by CM of Bcl-w-overexpressing cells, prompting investigation of the potential correlation between Bcl-w and PDGF-A and their effects on GBM malignancy. Bcl-w and PDGF-A levels were positively regulated and increased tumorigenicity by Sox2 activation in GBM cells. miR-340-5p was further identified as a direct inhibitor of Bcl-w and Sox2. Overexpression of miR-340-5p reduced mesenchymal traits, cell migration, invasion, and stemness in GBM through attenuating Bcl-w and Sox2 expression. Our novel findings highlight the potential utility of miR-340-5p as a therapeutic agent for glioblastoma multiforme through inhibitory effects on Bcl-w-induced PDGF-A and Sox2 activation.
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Cell therapeutic agents for treating degenerative brain diseases using neural stem cells are actively being developed. However, few systems have been developed to monitor in real time whether the transplanted neural stem cells are actually differentiated into neurons. Therefore, it is necessary to develop a technology capable of specifically monitoring neuronal differentiation in vivo. In this study, we established a system that expresses cell membrane-targeting red fluorescent protein under control of the Synapsin promoter in order to specifically monitor differentiation from neural stem cells into neurons. In order to overcome the weak expression level of the tissue-specific promoter system, the partial 5' UTR sequence of Creb was added for efficient expression of the cell surface-specific antigen. This system was able to track functional neuronal differentiation of neural stem cells transplanted in vivo, which will help improve stem cell therapies.
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Otitis media (OM) is a group of inflammatory diseases of the middle ear (ME), regardless of cause or pathological mechanism. Among the molecular biological studies assessing the pathology of OM are investigations into the expression of C-type lectin receptors (CLR) in the ME and Eustachian tube (ET). To date, nine studies have evaluated CLR expression in the ME and ET. The expression of individual CLRs in mammalian ME and ET varies by species and model of OM. Assessments have shown that the patterns of CLR expression in the ME and ET vary; that CLR expression may vary by type of OM; and that the distribution and levels of expression of CLRs may depend on the presence or absence of inflammation, with variations even within the same species and same tissue. Infection of the ME and ET with various pathogens is a common cause of all types of OM, with host responses to pathogens mediated initially by the innate immune system. CLRs are important factors in the innate immune system because they act as both adhesion molecules and as pathogen recognition receptors. The expression of CLRs in OM tissues suggests that CLRs are associated with the pathogenesis of various types of OM.
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Oído Medio/metabolismo , Trompa Auditiva/metabolismo , Lectinas Tipo C/metabolismo , Otitis Media/metabolismo , Animales , HumanosRESUMEN
Generation of functional dopamine (DA) neurons is an essential step for the development of effective cell therapy for Parkinson's disease (PD). The generation of DA neurons can be accomplished by overexpression of DA-inducible genes using virus- or DNA-based gene delivery methods. However, these gene delivery methods often cause chromosomal anomalies. In contrast, mRNA-based gene delivery avoids this problem and therefore is considered safe to use in the development of cell-based therapy. Thus, we used mRNA-based gene delivery method to generate safe DA neurons. In this study, we generated transformation-free DA neurons by transfection of mRNA encoding DA-inducible genes Nurr1 and FoxA2. The delivery of mRNA encoding dopaminergic fate inducing genes proved sufficient to induce naive rat forebrain precursor cells to differentiate into neurons exhibiting the biochemical, electrophysiological, and functional properties of DA neurons in vitro. Additionally, the generation efficiency of DA neurons was improved by the addition of small molecules, db-cAMP, and the adjustment of transfection timing. The successful generation of DA neurons using an mRNA-based method offers the possibility of developing clinical-grade cell sources for neuronal cell replacement treatment for PD.
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Neuronas Dopaminérgicas/metabolismo , ARN Mensajero/síntesis química , ARN Mensajero/genética , Factores de Transcripción/genética , Animales , Línea Celular , Neuronas Dopaminérgicas/citología , Expresión Génica , Regulación de la Expresión Génica , Orden Génico , Genes Reporteros , Vectores Genéticos/genética , Factor Nuclear 3-beta del Hepatocito/genética , Factor Nuclear 3-beta del Hepatocito/metabolismo , Humanos , Miembro 2 del Grupo A de la Subfamilia 4 de Receptores Nucleares/genética , Miembro 2 del Grupo A de la Subfamilia 4 de Receptores Nucleares/metabolismo , Ratas , Transfección , Tirosina 3-Monooxigenasa/genéticaRESUMEN
The purpose of this research is to develop a method to screen a large number of potential driver mutations of acute myeloid leukemia (AML) using a retroviral cDNA library and murine bone marrow transduction-transplantation system. As a proof-of-concept, murine bone marrow (BM) cells were transduced with a retroviral cDNA library encoding well-characterized oncogenes and homeobox genes, and the virus-transduced cells were transplanted into lethally irradiated mice. The proto-oncogenes responsible for leukemia initiation were identified by PCR amplification of cDNA inserts from genomic DNA isolated from leukemic cells. In an initial screen of ten leukemic mice, the MYC proto-oncogene was detected in all the leukemic mice. Of ten leukemic mice, 3 (30%) had MYC as the only transgene, and seven mice (70%) had additional proto-oncogene inserts. We repeated the same experiment after removing MYC-related genes from the library to characterize additional leukemia-inducing gene combinations. Our second screen using the MYC-deleted proto-oncogene library confirmed MEIS1and the HOX family as cooperating oncogenes in leukemia pathogenesis. The model system we introduced in this study will be valuable in functionally screening novel combinations of genes for leukemogenic potential in vivo, and the system will help in the discovery of new targets for leukemia therapy.
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Genes Homeobox , Leucemia/genética , Proto-Oncogenes , Animales , Células de la Médula Ósea/metabolismo , Células de la Médula Ósea/patología , Trasplante de Médula Ósea , Línea Celular , Transformación Celular Neoplásica/genética , Modelos Animales de Enfermedad , Epistasis Genética , Femenino , Eliminación de Gen , Expresión Génica , Biblioteca de Genes , Vectores Genéticos/genética , Inmunofenotipificación , Leucemia/mortalidad , Leucemia/patología , Ratones , Familia de Multigenes , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-myc/genética , Retroviridae/genética , Transducción GenéticaRESUMEN
Mad2, a key component of the spindle checkpoint, is closely associated with chromosomal instability and poor prognosis in cancer. p31comet is a Mad2-interacting protein that serves as a spindle checkpoint silencer at mitosis. In this study, we showed that p31comet-induced apoptosis and senescence occur via counteraction of Mad2 activity. Upon retroviral transduction of p31comet, the majority of human cancer cell lines tested lost the ability to form colonies in a low-density seeding assay. Cancer cells with p31comet overexpression underwent distinct apoptosis and/or senescence, irrespective of p53 status, confirming the cytotoxicity of p31comet. Interestingly, both cytotoxic and Mad2 binding activities were eliminated upon deletion of the C-terminal 30 amino acids of p31comet. Point mutation or deletion of the region affecting Mad2 binding additionally abolished cytotoxic activity. Consistently, wild-type Mad2 interacting with p31comet, but not its non-binding mutant, inhibited cell death, indicating that the mechanism of p31comet-induced cell death involves Mad2 inactivation. Our results clearly suggest that the regions of p31comet affecting interactions with Mad2, including the C-terminus, are essential for induction of cell death. The finding that p31comet-induced cell death is mediated by interactions with Mad2 that lead to its inactivation is potentially applicable in anticancer therapy.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Apoptosis , Proteínas de Ciclo Celular/metabolismo , Proteínas Mad2/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Supervivencia Celular , Senescencia Celular , Células Clonales/citología , Humanos , Puntos de Control de la Fase M del Ciclo Celular , Ratones , Proteínas Nucleares/química , Proteínas Nucleares/genética , Unión Proteica , Eliminación de SecuenciaRESUMEN
Investigation of characteristics of cell- and nuclear-penetrating anti-double stranded (ds)DNA autoantibodies (autoAbs) is important to understand pathogenesis of lupus nephritis, but has not been clearly explored. The present study reports that three anti-dsDNA monoclonal autoAbs, which contain more than two arginine residues in their CDR3s of variable heavy domain (VH), penetrated into living murine mesangial cells and the cell nuclei. However, an anti-dsDNA monoclonal Ab (mAb) having only one arginine in the CDR3-VH did not penetrate cells. To assess the contribution of antigen-binding sites, especially the VH, in cell- and nuclear-penetration, we evaluated the characteristics of recombinant single chain Fv(scFv), VH, and variable light domain (VL) of a penetrating mAb. The scFv and VH domain, containing arginine in CDR3-VH maintained the ability to penetrate cells and the cell nuclei, whereas the VL domain, having no arginine in CDR3, did not penetrate cells. The penetratingm Abs, scFv, and VH activated ERK and increased cellular protein levels of Bcl-2, whereas the non-penetrating Ab and VL did not. The cell survival was decreased by the penetrating mAbs, scFv and VH, not by the non-penetrating mAb and VL. The data indicate that an antigen-binding site is required for cell-penetration and that positively-charged arginine residues in CDR3-VH contribute to the cell- and nuclear-penetrating ability of a subset of anti-dsDNA autoAbs. Furthermore,the nuclear-penetrating anti-dsDNA autoAbs could possibly function as a pathogenic factor for lupus nephritis by up-regulating ERK activation and Bcl-2 production in mesangial cells. The cell- and nuclear-penetrating VH domain may be exploited as a vehicle for the intra cellular delivery of various useful molecules.
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Arginina/metabolismo , Autoanticuerpos/química , Núcleo Celular/metabolismo , Regiones Determinantes de Complementariedad/química , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Región Variable de Inmunoglobulina/química , Células Mesangiales/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos Antinucleares/inmunología , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Autoanticuerpos/inmunología , Western Blotting , Línea Celular , Supervivencia Celular , Regiones Determinantes de Complementariedad/inmunología , Citometría de Flujo , Región Variable de Inmunoglobulina/inmunología , Ratones , Datos de Secuencia Molecular , Fosforilación , Unión Proteica , Homología de Secuencia de Aminoácido , Anticuerpos de Cadena Única/química , Anticuerpos de Cadena Única/inmunología , Relación Estructura-ActividadRESUMEN
CONCLUSION: Many patients with acoustic neuroma (AN) experience hearing loss and tinnitus. Time from first symptoms to diagnosis can be considerable. AN should be suspected, and MRI scans performed, in patients with hearing loss accompanied by asymmetry, tinnitus, low speech discrimination score (SDS), and abnormal auditory brainstem response (ABR). OBJECTIVES: To determine the otorhinolaryngological factors associated with AN by analyzing the clinical manifestations and diagnostic test results of patients with AN before MRI scanning. METHODS: This study enrolled 114 patients definitively diagnosed with AN after visiting the Ear-Nose-and-Throat and Neurosurgery Departments of Kyung Hee University Medical Center from 2001 to 2013. Factors retrospectively analyzed included patient age, gender, major symptoms, accompanying symptoms, symptom duration, pure-tone audiometry, SDS, asymmetry, tinnitogram, ABR, and MRI scan results. RESULTS: The main symptom of AN was hearing loss, and the most frequent accompanying symptom was tinnitus. More severe deafness correlated significantly with lower SDS (p < 0.05). Asymmetric hearing was observed in 75 of 116 patients (64.6%), and mean SDS was 73.1 ± 34.1%. Of patients with latencies of waves I, III, and V on ABR tests, 56.1%, 92.4%, and 92.4%, had interaural latency differences ≥0.2 ms. However, audiometry results did not correlate with lesion site or tumor size (p > 0.05).
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Neuroma Acústico/diagnóstico , Adulto , Anciano , Audiometría de Tonos Puros , Potenciales Evocados Auditivos del Tronco Encefálico , Femenino , Humanos , Masculino , Persona de Mediana Edad , Neuroma Acústico/fisiopatología , Estudios Retrospectivos , Pruebas de Discriminación del HablaRESUMEN
OBJECTIVES: To assess innate and humoral immune responses in middle ear effusion of obese pediatric patients with otitis media with effusion (OME). METHODS: We evaluated 219 children with OME, of whom 21 were obese and 198 were non-obese. We compared the expression in middle ear effusion of mRNAs encoding toll-like receptors (TLR) 2, 4, 5, and 9; nucleotide-binding oligomerization domains (NOD) 1 and 2; retinoic acid-inducible gene (RIG)-I; interleukins (IL)-6, -10, and -12; interferon (IFN)-γ; and tumor necrosis factor (TNF)-α mRNAs. We also compared the expression of immunoglobulins IgG, IgA, and IgM and the bacterial detection rate in the two groups. RESULTS: TLR2-mediated expression of IL-6 mRNA, TLR4-mediated expression of IL-6 and IL-10 mRNA, TLR5-mediated expression of IL-6, IL-10, and TNF-α mRNA, TLR9-mediated expression of IL-6 mRNA, and NOD2-mediated expression of IL-6, IL-12, and TNF-α mRNA were significantly lower in obese than in non-obese children (P<0.05). However, concentrations of IgG, IgA, and IgM in middle ear effusion were lower in obese than in non-obese children, but none of these differences was significant (P>0.05). CONCLUSION: Mean body mass index was higher and pattern-recognition receptor-mediated cytokine mRNA expression was lower in obese than in non-obese children with OME.
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The hematopoietic cell malignancy is one of the most prevalent type of cancer and the disease has multiple pathologic molecular signatures. Research on the origin of hematopoietic cancer stem cells and the mode of subsequent maintenance and differentiation needs robust animal models that can reproduce the transformation and differentiation event in vivo. Here, we show that co-transduction of MYC and PIM2 proto-oncogenes into mouse bone marrow cells readily establishes permanent cell lines that can induce lethal myeloid sarcoma in vivo. Unlike the previous doubly transgenic mouse model in which coexpression of MYC and PIM2 transgenes exclusively induced B cell lymphoma, we were able to show that the same combination of genes can also transform primary bone marrow myeloid cells in vitro resulting in permanent cell lines which induce myeloid sarcoma upon in vivo transplantation. By inducing cancerous transformation of fresh bone marrow cells in a controlled environment, the model we established will be useful for detailed study of the molecular events involved in initial transformation process of primary myeloid bone marrow cells and provides a model that can give insight to the molecular pathologic characteristics of human myeloid sarcoma, a rare presentation of solid tumors of undifferentiated myeloid blast cells associated with various types of myeloid leukemia.
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Células de la Médula Ósea/metabolismo , Proteínas Proto-Oncogénicas c-myc/biosíntesis , Sarcoma Mieloide/metabolismo , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/patología , Diferenciación Celular/fisiología , Procesos de Crecimiento Celular/fisiología , Línea Celular , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Modelos Animales de Enfermedad , Humanos , Ratones , Ratones Endogámicos C57BL , Proteínas Serina-Treonina Quinasas/biosíntesis , Proteínas Proto-Oncogénicas/biosíntesis , Sarcoma Mieloide/patologíaRESUMEN
PURPOSE: Whole-body radiation therapy can cause severe injury to the hematopoietic system, and therefore it is necessary to identify a novel strategy for overcoming this injury. METHODS AND MATERIALS: Mice were irradiated with 4.5 Gy after heat shock protein 25 (HSP25) gene transfer using an adenoviral vector. Then, peripheral blood cell counts, histopathological analysis, and Western blotting on bone marrow (BM) cells were performed. The interaction of HSP25 with Tie2 was investigated with mouse OP9 and human BM-derived mesenchymal stem cells to determine the mechanism of HSP25 in the hematopoietic system. RESULTS: HSP25 transfer increased BM regeneration and reduced apoptosis following whole-body exposure to ionizing radiation (IR). The decrease in Tie2 protein expression that followed irradiation of the BM was blocked by HSP25 transfer, and Tie2-positive cells were more abundant among the BM cells of HSP25-transferred mice, even after IR exposure. Following systemic RNA interference of Tie2 before IR, HSP25-mediated radioprotective effects were partially blocked in both mice and cell line systems. Stability of Tie2 was increased by HSP25, a response mediated by the interaction of HSP25 with Tie2. IR-induced tyrosine phosphorylation of Tie2 was augmented by HSP25 overexpression; downstream events in the Tie2 signaling pathway, including phosphorylation of AKT and EKR1/2, were also activated. CONCLUSIONS: HSP25 protects against radiation-induced BM damage by interacting with and stabilizing Tie2. This may be a novel strategy for HSP25-mediated radioprotection in BM.
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Médula Ósea/efectos de la radiación , Proteínas de Choque Térmico HSP27/fisiología , Células Madre Mesenquimatosas/metabolismo , Traumatismos Experimentales por Radiación/prevención & control , Proteínas Tirosina Quinasas Receptoras/fisiología , Adenoviridae/genética , Animales , Apoptosis/fisiología , Western Blotting , Médula Ósea/fisiología , Ensayo de Unidades Formadoras de Colonias/métodos , Técnicas de Silenciamiento del Gen , Técnicas de Transferencia de Gen , Vectores Genéticos/genética , Proteínas de Choque Térmico HSP27/genética , Proteínas de Choque Térmico , Humanos , Etiquetado Corte-Fin in Situ , Ratones , Chaperonas Moleculares , Fosforilación , Interferencia de ARN , Traumatismos Experimentales por Radiación/fisiopatología , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptor TIE-2 , Regeneración/fisiología , Transducción de Señal , Irradiación Corporal Total/efectos adversosRESUMEN
Metallothioneins (MTs) are intracellular low-molecular-weight, cysteine-rich proteins with potent metal-binding and redox functions, but with limited membrane permeativity. The aim of this study was to investigate whether we could enhance delivery of MT-1 to pancreatic islets or ß cells in vitro and in vivo. The second goal was to determine whether increased MT-1 could prevent cellular toxicity induced by high glucose and free fatty acids in vitro (glucolipotoxicity) and ameliorate the development of diabetes induced by streptozotocin in mice or delay the development of diabetes by improving insulin secretion and resistance in the OLETF rat model of type 2 diabetes. Expression of HIV-1 Tat-MT-1 enabled efficient delivery of MT into both INS-1 cells and rat islets. Intracellular MT activity increased in parallel with the amount of protein delivered to cells. The formation of reactive oxygen species, glucolipotoxicity, and DNA fragmentation due to streptozotocin decreased after treating pancreatic ß cells with Tat-MT in vitro. Importantly, in vivo, intraperitoneal injection resulted in delivery of the Tat-MT protein to the pancreas as well as liver, muscle, and white adipose tissues. Multiple injections increased radical-scavenging activity, decreased apoptosis, and reduced endoplasmic reticulum stress in the pancreas. Treatment with Tat-MT fusion protein delayed the development of diabetes in streptozotocin-induced mice and improved insulin secretion and resistance in OLETF rats. These results suggest that in vivo transduction of Tat-MT may offer a new strategy to protect pancreatic ß cells from glucolipotoxicity, may improve insulin resistance in type 2 diabetes, and may have a protective effect in preventing islet destruction in type 1 diabetes.
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Diabetes Mellitus Tipo 2/prevención & control , Productos del Gen tat/metabolismo , Técnicas de Transferencia de Gen , VIH-1/metabolismo , Metalotioneína/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Animales , Células Cultivadas , Diabetes Mellitus Tipo 2/inducido químicamente , Diabetes Mellitus Tipo 2/metabolismo , Modelos Animales de Enfermedad , Productos del Gen tat/genética , Productos del Gen tat/aislamiento & purificación , Insulina/metabolismo , Resistencia a la Insulina , Secreción de Insulina , Masculino , Metalotioneína/genética , Metalotioneína/aislamiento & purificación , Ratones , Ratones Endogámicos ICR , Ratas , Ratas Endogámicas OLETF , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , EstreptozocinaRESUMEN
A subpopulation of cancer cells with stem cell properties is responsible for tumor formation, maintenance, and malignant progression; however, the molecular mechanisms underlying the maintenance of cancer stem-like cell properties have remained unclear. Here, we show that the Rho family GTPase Rac1 is involved in the glioma stem-like cell (GSLC) maintenance and tumorigenicity in human glioma. The Rac1-Pak signaling was markedly activated in GSLCs. Knockdown of Rac1 caused reduction of expression of GSLC markers, self-renewal-related proteins and neurosphere formation. Moreover, down-regulation of Rac1 suppressed the migration, invasion, and malignant transformation in GSLCs. Furthermore, inhibition of Rac1 enhanced radiation sensitivity of GSLCs. These results indicate that the small GTPase Rac1 is involved in the maintenance of stemness and malignancies in GSLCs.
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Glioma/metabolismo , Glioma/patología , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Proteína de Unión al GTP rac1/metabolismo , Animales , Línea Celular Tumoral , Proliferación Celular , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Trasplante de Neoplasias , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal/fisiología , Quinasas p21 Activadas/metabolismo , Proteína de Unión al GTP rac1/genéticaRESUMEN
Mutations in the superoxide dismutase 1 (SOD1) gene are linked to glutamate excitotoxicity in familial amyotrophic lateral sclerosis (fALS), but the underlying mechanism remains unclear. We investigated whether nuclear factor-kappaB (NF-kappaB) activation is involved in glutamate excitotoxicity by using motor neuron-neuroblastoma hybrid cells that expressed a mutant (G93A) SOD1 (mtSOD1) or wild-type SOD1 (wtSOD1). MtSOD1 cells were more vulnerable to glutamate excitotoxicity than wtSOD1 cells and showed higher NF-kappaB activity, higher nuclear cRel expression, and lower nuclear RelA expression under basal conditions. Glutamate treatment increased NF-kappaB activation along with nuclear expressions of RelA and cRel in wtSOD1 cells but induced only weak nuclear RelA expression in mtSOD1 cells. Suppression of NF-kappaB activation using transfection of the superrepressive mutant form of IkappaBalpha (mIkappaBalpha) inhibited nuclear RelA expression in both types of SOD1 cells, which increased glutamate excitotoxicity in wtSOD1 cells but not in mtSOD1 cells. Furthermore, immunohistochemistry confirmed stronger RelA immunoreactivity in the nuclei of motor neurons of spinal cord in wild-type SOD1 transgenic mice than in those in SOD1 G93A transgenic mice. In addition, we found that glutamate treatment decreased XIAP expression and increased caspase-3 activity in mtSOD1 cells and mIkappaBalpha-overexpressing wtSOD1 cells. Our results suggest that glutamate excitotoxicity in motor neurons of SOD1-linked fALS is attributable, at least in part, to the impairment of IkappaBalpha-dependent RelA activation and subsequent apoptosis mediated by XIAP inhibition and caspase-3 activation.
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Ácido Glutámico/toxicidad , Neuronas Motoras/metabolismo , FN-kappa B/metabolismo , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Animales , Biotransformación/fisiología , Caspasa 3/metabolismo , Muerte Celular , Línea Celular , Núcleo Celular/enzimología , Núcleo Celular/metabolismo , Supervivencia Celular/efectos de los fármacos , Citoplasma/enzimología , Citoplasma/metabolismo , Colorantes Fluorescentes , Vectores Genéticos , Humanos , Células Híbridas , Inmunohistoquímica , Indoles , Luciferasas/metabolismo , Ratones , Ratones Transgénicos , Neuronas Motoras/efectos de los fármacos , Mutación/genética , Mutación/fisiología , Neuroblastoma/metabolismo , Receptores de Glutamato/metabolismo , Retroviridae/genéticaRESUMEN
We investigated whether expression of the IL-12 p35 subunit in membrane-bound form in tumor cells enhanced their immunogenicity. Since p35 is only secreted when associated with the IL-12 p40 subunit, we generated tumor cells expressing membrane-bound forms of p35 and p40 as chimeras with the transmembrane/cytoplasmic region of TNFα (mbIL-12p35 and mbIL-12p40). The relevant vectors were transfected into MethA fibrosarcoma cells, and mbIL-12p35 or mbIL-12p40-expressing tumor clones were isolated and their ability to induce antitumor immunity studied. Cells of the mbIL-12p35 tumor clone induced CD69 expression and IFNγ production in purified CD8(+) T cells in vitro, and their in vivo tumorigenicity was reduced. Cells of the mbIL-12p40 tumor clone failed to show either of these activities. Mice that had rejected cells of the mbIL-12p35 tumor clone possessed systemic antitumor immunity to wild type tumor cells. The growth rate of mbIL-12p35 tumor cells was greater in CD8(+) T cell-depleted mice than in CD4(+) T-cell- and NK cell-depleted mice or normal mice, suggesting that CD8(+) T cells were mainly responsible for the antitumor immunity. These results indicate that expression of mbIL-12p35 on tumor cells enhances their immunogenicity by increasing their ability to activate CD8(+) T cells, possibly by direct priming.
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Linfocitos T CD8-positivos/inmunología , Vacunas contra el Cáncer/inmunología , Fibrosarcoma/inmunología , Subunidad p35 de la Interleucina-12/inmunología , Activación de Linfocitos , Linfocitos T Citotóxicos/inmunología , Animales , Antígenos CD/genética , Antígenos CD/metabolismo , Antígenos de Diferenciación de Linfocitos T/genética , Antígenos de Diferenciación de Linfocitos T/metabolismo , Linfocitos T CD8-positivos/metabolismo , Vacunas contra el Cáncer/genética , Línea Celular Tumoral , Citometría de Flujo , Ingeniería Genética , Interferón gamma/genética , Interferón gamma/metabolismo , Subunidad p35 de la Interleucina-12/genética , Subunidad p40 de la Interleucina-12/genética , Subunidad p40 de la Interleucina-12/inmunología , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Ratones , Ratones Endogámicos BALB C , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes de Fusión/inmunología , Linfocitos T Citotóxicos/metabolismo , Transfección , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
In obesity, dysregulation of adipocytokines is involved in several pathological conditions including diabetes and certain cancers. As a member of the adipocytokines, adiponectin plays crucial roles in whole-body energy homeostasis. Recently, it has been reported that the level of plasma adiponectin is reduced in several types of cancer patients. However, it is largely unknown whether and how adiponectin affects colon cancer cell growth. Here, we show that adiponectin suppresses the proliferation of colon cancer cells including HCT116, HT29, and LoVo. In colon cancer cells, adiponectin attenuated cell cycle progression at the G(1)/S boundary and concurrently increased expression of cyclin-dependent kinase inhibitors such as p21 and p27. Adiponectin stimulated AMP-activated protein kinase (AMPK) phosphorylation whereas inhibition of AMPK activity blunted the effect of adiponectin on the proliferation of colon cancer cells. Furthermore, knockdown of adiponectin receptors such as AdipoR1 and AdipoR2 relieved the suppressive effect of adiponectin on the growth of colon cancer cells. In addition, adiponectin repressed the expression of sterol regulatory element binding protein-1c, which is a key lipogenic transcription factor associated with colon cancers. These results suggest that adiponectin could inhibit the growth of colon cancer cells through stimulating AMPK activity.