RESUMEN
Immune mediators affect multiple biological functions of intestinal epithelial cells (IECs) and, like Paneth and Paneth-like cells, play an important role in intestinal epithelial homeostasis. IFN-γ a prototypical proinflammatory cytokine disrupts intestinal epithelial homeostasis. However, the mechanism underlying the process remains unknown. In this study, using in vivo and in vitro models we demonstrate that IFN-γ is spontaneously secreted in the small intestine. Furthermore, we observed that this cytokine stimulates mitochondrial activity, ROS production, and Paneth and Paneth-like cell secretion. Paneth and Paneth-like secretion downstream of IFN-γ, as identified here, is mTORC1 and necroptosis-dependent. Thus, our findings revealed that the pleiotropic function of IFN-γ also includes the regulation of Paneth cell function in the homeostatic gut.
RESUMEN
Epithelial barrier impairment is a hallmark of several pathologic processes in the gut, including inflammatory bowel diseases. Several intracellular signals prevent apoptosis in intestinal epithelial cells. Herein, we show that in colonocytes, rictor/mammalian target of rapamycin complex 2 (mTORC2) signaling is a prosurvival stimulus. Mechanistically, mTORC2 activates Akt, which, in turn, inhibits apoptosis by phosphorylating B-cell lymphoma 2 (BCL2) associated agonist of cell death (Bad) and preventing caspase-3 activation. Nevertheless, during inflammation, rictor/mTORC2 signaling declines and Akt activity is reduced. Consequently, active caspase-3 increases in surface colonocytes undergoing apoptosis/anoikis and causes epithelial barrier breakdown. Likewise, Rictor ablation in intestinal epithelial cells interrupts mTORC2/Akt signaling and increases apoptosis/anoikis of surface colonocytes without affecting the crypt architecture. The increase in epithelial permeability induced by Rictor ablation produces a mild inflammatory response in the colonic mucosa, but minimally affects the development/establishment of colitis. The data identify a previously unknown mechanism by which rictor/mTORC2 signaling regulates apoptosis/anoikis in intestinal epithelial cells during colitis and clarify its role in the maintenance of the intestinal epithelial barrier.
Asunto(s)
Apoptosis/fisiología , Colitis/patología , Células Epiteliales/metabolismo , Mucosa Intestinal/patología , Proteína Asociada al mTOR Insensible a la Rapamicina/metabolismo , Animales , Colitis/metabolismo , Células Epiteliales/patología , Mucosa Intestinal/metabolismo , Ratones , Transducción de Señal/fisiologíaRESUMEN
A single layer of polarized epithelial cells lining the colonic mucosa create a semipermeable barrier indispensable for gut homeostasis. The role of intestinal epithelial cell (IEC) polarization in the maintenance of the epithelial homeostasis and in the development of inflammatory bowel diseases is not fully understood. In this review, now we report that IEC polarization plays an essential role in the regulation of IL-6/STAT3 signaling in the colonic mucosa. Our results demonstrate that autocrine STAT3 activation in IECs is mediated by the apical secretion of IL-6 in response to the basolateral stimulation with IFN-γ. This process relies on the presence of functional, IFN-γ-producing CD4+ T cells. In the absence of basolateral IFN-γ, the compartmentalization of the IL-6/STAT3 signaling is disrupted, and STAT3 is activated mainly in macrophages. Thus, in this study, we show that during inflammation, IFN-γ regulates IL-6/STAT3 signaling in IEC in the colonic mucosa.
Asunto(s)
Colitis/metabolismo , Colon/metabolismo , Interleucina-6/metabolismo , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Animales , Células CACO-2 , Células Cultivadas , Células Epiteliales/metabolismo , Humanos , Inflamación/metabolismo , Interferón gamma/metabolismo , Mucosa Intestinal/metabolismo , Ratones , Ratones Endogámicos C57BLRESUMEN
The Na+, K+-ATPase transports Na+ and K+ across the membrane of all animal cells. In addition to its ion transporting function, the Na+, K+-ATPase acts as a homotypic epithelial cell adhesion molecule via its ß1 subunit. The extracellular region of the Na+, K+-ATPase ß1 subunit includes a single globular immunoglobulin-like domain. We performed Molecular Dynamics simulations of the ectodomain of the ß1 subunit and a refined protein-protein docking prediction. Our results show that the ß1 subunit Ig-like domain maintains an independent structure and dimerizes in an antiparallel fashion. Analysis of the putative interface identified segment Lys221-Tyr229. We generated triple mutations on YFP-ß1 subunit fusion proteins to assess the contribution of these residues. CHO fibroblasts transfected with mutant ß1 subunits showed a significantly decreased cell-cell adhesion. Association of ß1 subunits in vitro was also reduced, as determined by pull-down assays. Altogether, we conclude that two Na+, K+-ATPase molecules recognize each other by a large interface spanning residues 221-229 and 198-207 on their ß1 subunits.
Asunto(s)
Mutagénesis Sitio-Dirigida , ATPasa Intercambiadora de Sodio-Potasio/química , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Secuencias de Aminoácidos , Animales , Células CHO , Cricetulus , Modelos Moleculares , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Unión Proteica , Conformación Proteica , ATPasa Intercambiadora de Sodio-Potasio/genéticaRESUMEN
The mechanisms controlling degradation of cytosolic ß-catenin are important for regulating ß-catenin co-transcriptional activity. Loss of von Hippel-Lindau protein (pVHL) has been shown to stabilize ß-catenin, increasing ß-catenin transactivation and ß-catenin-mediated cell proliferation. However, the role of phosphoinositide 3-kinase (PI3K)/Akt in the regulation of ß-catenin signaling downstream from pVHL has never been addressed. Here, we report that hyperactivation of PI3K/Akt in cells lacking pVHL contributes to the stabilization and nuclear accumulation of active ß-catenin. PI3K/Akt hyperactivation is facilitated by the up-regulation of 14-3-3ζ and the down-regulation of 14-3-3ε, 14-3-3η and 14-3-3θ. Up-regulation of 14-3-3ζ in response to pVHL is important for the recruitment of PI3K to the cell membrane and for stabilization of soluble ß-catenin. In contrast, 14-3-3ε and 14-3-3η enhanced PI3K/Akt signaling by inhibiting PI3K and PDK1, respectively. Thus, our results demonstrated that 14-3-3 family members enhance PI3K/Akt/ß-catenin signaling in order to increase proliferation. Inhibition of Akt activation and/or 14-3-3 function strongly reduces ß-catenin signaling and decreases cell proliferation. Thus, inhibition of Akt and 14-3-3 function efficiently reduces cell proliferation in 786-0 cells characterized by hyperactivation of ß-catenin signaling due to pVHL loss.
Asunto(s)
Proteínas 14-3-3/biosíntesis , Proliferación Celular/fisiología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/fisiología , Regulación hacia Arriba/fisiología , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismo , beta Catenina/metabolismo , Proteínas 14-3-3/genética , Animales , Perros , Humanos , Células de Riñón Canino Madin Darby , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genética , beta Catenina/genéticaRESUMEN
Radiotherapy is an important treatment option for non-small cell lung carcinoma patients. Despite the appropriate use of radiotherapy, radioresistance is a biological behavior of cancer cells that limits the efficacy of this treatment. Deregulation of microRNAs contributes to the molecular mechanism underlying resistance to radiotherapy in cancer cells. Although the functional roles of microRNAs have been well described in lung cancer, their functional roles in radioresistance are largely unclear. In this study, we established a non-small cell lung carcinoma Calu-1 radioresistant cell line by continuous exposure to therapeutic doses of ionizing radiation as a model to investigate radioresistance-associated microRNAs. Our data show that 50 microRNAs were differentially expressed in Calu-1 radioresistant cells (16 upregulated and 34 downregulated); furthermore, well-known and novel microRNAs associated with resistance to radiotherapy were identified. Gene ontology and enrichment analysis indicated that modulated microRNAs might regulate signal transduction, cell survival, and apoptosis. Accordingly, Calu-1 radioresistant cells were refractory to radiation by increasing cell survival and reducing the apoptotic response. Among deregulated microRNAs, miR-29c was significantly suppressed. Reestablishment of miR-29c expression in Calu-1 radioresistant cells overcomes the radioresistance through the activation of apoptosis and downregulation of Bcl-2 and Mcl-1 target genes. Analysis of The Cancer Genome Atlas revealed that miR-29c is also suppressed in tumor samples of non-small cell lung carcinoma patients. Notably, we found that low miR-29c levels correlated with shorter relapse-free survival of non-small cell lung carcinoma patients treated with radiotherapy. Together, these results indicate a new role of miR-29c in radioresistance, highlighting their potential as a novel biomarker for outcomes of radiotherapy in lung cancer.
Asunto(s)
Apoptosis/genética , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/radioterapia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/radioterapia , MicroARNs/genética , Tolerancia a Radiación/genética , Biomarcadores de Tumor/genética , Carcinoma de Pulmón de Células no Pequeñas/mortalidad , Línea Celular Tumoral , Supervivencia Celular/efectos de la radiación , Regulación hacia Abajo/genética , Regulación Neoplásica de la Expresión Génica , Genes Supresores de Tumor , Humanos , Neoplasias Pulmonares/mortalidad , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/biosíntesis , Recurrencia Local de Neoplasia/genética , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Resultado del TratamientoRESUMEN
The gastrointestinal tract is the largest hormone-producing organ in the body due to a specialized cell population called enteroendocrine cells (EECs). The number of EECs increases in the mucosa of inflammatory bowel disease patients; however, the mechanisms responsible for these changes remain unknown. Here, we show that the pro-inflammatory cytokines interferon γ (IFNγ) and tumor necrosis factor α (TNFα) or dextran sulfate sodium (DSS)-induced colitis increase the number of EECs producing chromogranin A (CgA) in the colonic mucosa of C57BL/6J mice. CgA-positive cells were non-proliferating cells enriched with inactive phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and autophagy markers. Moreover, inhibition of Akt and autophagy prevented the increase in CgA-positive cells after IFNγ/TNFα treatment. Similarly, we observed that CgA-positive cells in the colonic mucosa of patients with colitis expressed Akt and autophagy markers. These findings suggest that Akt signaling and autophagy control differentiation of the intestinal EEC lineage during inflammation.
Asunto(s)
Cromogranina A/metabolismo , Colon/citología , Citocinas/farmacología , Epitelio/efectos de los fármacos , Epitelio/metabolismo , Células Neuroendocrinas/efectos de los fármacos , Células Neuroendocrinas/metabolismo , Animales , Autofagia/efectos de los fármacos , Western Blotting , Células CACO-2 , Colitis/metabolismo , Técnica del Anticuerpo Fluorescente , Humanos , Interferón gamma/farmacología , Interleucina-1beta/farmacología , Mucosa Intestinal/citología , Mucosa Intestinal/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas Proto-Oncogénicas c-akt/metabolismo , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
BACKGROUND: One of the main phenomena occurring in cellular membranes during virus infection is a change in membrane permeability. It has been observed that numerous viral proteins can oligomerize and form structures known as viroporins that alter the permeability of membranes. Previous findings have identified such proteins in cells infected with Japanese encephalitis virus (JEV), a member of the same family that Dengue virus (DENV) belongs to (Flaviviridae). In the present work, we investigated whether the small hydrophobic DENV protein NS2B serves a viroporin function. METHODS: We cloned the DENV NS2B sequence and expressed it in a bacterial expression system. Subsequently, we evaluated the effect of DENV NS2B on membranes when NS2B was overexpressed, measured bacterial growth restriction, and evaluated changes of permeability to hygromycin. The NS2B protein was purified by affinity chromatography, and crosslinking assays were performed to determine the presence of oligomers. Hemolysis assays and transmission electron microscopy were performed to identify structures involved in permeability changes. RESULTS: The DENV-2 NS2B protein showed similitude with the JEV viroporin. The DENV-2 NS2B protein possessed the ability to change the membrane permeability in bacteria, to restrict bacterial cell growth, and to enable membrane permeability to hygromycin B. The NS2B protein formed trimers that could participate in cell lysis and generate organized structures on eukaryotes membranes. CONCLUSIONS: Our data suggest that the DENV-2 NS2B viral protein is capable of oligomerizing and organizing to form pore-like structures in different lipid environments, thereby modifying the permeability of cell membranes.
Asunto(s)
Permeabilidad de la Membrana Celular/efectos de los fármacos , Virus del Dengue/metabolismo , Proteínas Recombinantes de Fusión/farmacología , Proteínas no Estructurales Virales/farmacología , Secuencia de Aminoácidos , Virus del Dengue/genética , Membrana Eritrocítica/efectos de los fármacos , Membrana Eritrocítica/ultraestructura , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Humanos , Datos de Secuencia Molecular , Conformación Proteica , Multimerización de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Alineación de Secuencia , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/aislamiento & purificaciónRESUMEN
INTRODUCTION: Patients with cervical cancer (CC) may experience local recurrence very often after treatment; when only clinical parameters are used, most cases are diagnosed in late stages, which decreases the chance of recovery. Molecular markers can improve the prediction of clinical outcome. Glycolysis is altered in 70% of CCs, so molecular markers of this pathway associated with the aggressiveness of CC can be identified. METHODS: The expression of 14 glycolytic genes was analyzed in 97 CC and 29 healthy cervical tissue (HCT) with microarray; only LDHA and PFKP were validated at the mRNA and protein levels in 36 of those CC samples and in 109 new CC samples, and 31 HCT samples by qRT-PCR, Western blotting, or immunohistochemistry. A replica analysis was performed on 295 CC from The Cancer Genome Atlas (TCGA) database. RESULTS: The protein expression of LDHA and PFKP was associated with poor overall survival [OS: LDHA HR = 4.0 (95% CI = 1.4-11.1); p = 8.0 × 10-3 ; PFKP HR = 3.3 (95% CI = 1.1-10.5); p = 4.0 × 10-2 ] and disease-free survival [DFS: LDHA HR = 4.5 (95% CI = 1.9-10.8); p = 1.0 × 10-3 ; PFKP HR = 3.2 (95% CI = 1.2-8.2); p = 1.8 × 10-2 ] independent of FIGO clinical stage, and the results for mRNA expression were similar. The risk of death was greater in patients with overexpression of both biomarkers than in patients with advanced FIGO stage [HR = 8.1 (95% CI = 2.6-26.1; p = 4.3 × 10-4 ) versus HR = 7 (95% CI 1.6-31.1, p = 1.0 × 10-2 )] and increased exponentially as the expression of LDHA and PFKP increased. CONCLUSIONS: LDHA and PFKP overexpression at the mRNA and protein levels was associated with poor OS and DFS and increased risk of death in CC patients regardless of FIGO stage. The measurement of these two markers could be very useful for evaluating clinical evolution and the risk of death from CC and could facilitate better treatment decision making.
Asunto(s)
Fosfofructoquinasas , Neoplasias del Cuello Uterino , Femenino , Humanos , Biomarcadores/metabolismo , Glucólisis/genética , L-Lactato Deshidrogenasa/genética , L-Lactato Deshidrogenasa/metabolismo , Lactato Deshidrogenasa 5/metabolismo , Fosfofructoquinasas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Neoplasias del Cuello Uterino/genéticaRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) has the worst prognosis among all human cancers as it is highly resistant to chemotherapy. K-Ras mutations usually trigger the development and progression of PDAC. We hypothesized that compounds stabilizing the KRas4B/PDE6δ complex could serve as PDAC treatments. Using in silico approaches, we identified the small molecules C14 and P8 that reduced K-Ras activation in primary PDAC cells. Importantly, C14 and P8 significantly prevented tumor growth in patient-derived xenotransplants. Combined treatment with C14 and P8 strongly increased cytotoxicity in PDAC cell lines and primary cultures and showed strong synergistic antineoplastic effects in preclinical murine PDAC models that were superior to conventional therapeutics without causing side effects. Mechanistically, C14 and P8 reduced tumor growth by inhibiting AKT and ERK signaling downstream of K-RAS leading to apoptosis, specifically in PDAC cells. Thus, combined treatment with C14 and P8 may be a superior pharmaceutical strategy to improve the outcome of PDAC.
Asunto(s)
Antineoplásicos , Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Ratones , Animales , Línea Celular Tumoral , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Carcinoma Ductal Pancreático/tratamiento farmacológico , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patología , Antineoplásicos/farmacología , Neoplasias PancreáticasRESUMEN
BACKGROUND: The ATP7A gene encodes the ATP7A protein, which is a trans-Golgi network copper transporter expressed in the brain and other organs. Mutations in this gene cause disorders of copper metabolism, such as Menkes disease. Here we describe the novel and unusual mutation (p.T1048I) in the ATP7A gene of a child with Menkes disease. The mutation affects a conserved DKTGT1048 phosphorylation motif that is involved in the catalytic activity of ATP7A. We also describe the clinical course and the response to copper treatment in this patient. CASE PRESENTATION: An 11-month-old male Caucasian infant was studied because of hypotonia, ataxia and global developmental delay. The patient presented low levels of serum copper and ceruloplasmin, and was shown to be hemizygous for the p.T1048I mutation in ATP7A. The diagnosis was confirmed when the patient was 18 months old, and treatment with copper-histidinate (Cu-His) was started immediately. The patient showed some neurological improvement and he is currently 8 years old. Because the p.T1048I mutation affects its catalytic site, we expected a complete loss of functional ATP7A and a classical Menkes disease presentation. However, the clinical course of the patient was mild, and he responded to Cu-His treatment, which suggests that this mutation leads to partial conservation of the activity of ATP7A. CONCLUSION: This case emphasizes the important correlation between genotype and phenotype in patients with Menkes disease. The prognosis in Menkes disease is associated with early detection, early initiation of treatment and with the preservation of some ATP7A activity, which is necessary for Cu-His treatment response. The description of this new mutation and the response of the patient to Cu-His treatment will contribute to the growing body of knowledge about treatment response in Menkes disease.
Asunto(s)
Adenosina Trifosfatasas/genética , Proteínas de Transporte de Catión/genética , Síndrome del Pelo Ensortijado/genética , Mutación , ATPasas Transportadoras de Cobre , Histidina/análogos & derivados , Histidina/uso terapéutico , Humanos , Lactante , Masculino , Síndrome del Pelo Ensortijado/tratamiento farmacológico , Compuestos Organometálicos/uso terapéutico , LinajeRESUMEN
The HPV-16 E6/E7 early transcripts are first produced as bicistronic or polycistronic mRNAs, and about 90% of the original pre-mRNA is spliced to produce three new alternative mRNAs. HPV-16 spliced transcripts are expressed heterogeneously in tumors and cell lines. Our results suggest that suboptimal splicing acceptor sites in E6/E7 intron 1 and the differential expression of splicing factors are involved in the production of the heterogeneous splicing profile in cell lines. The unspliced pre-mRNA and the alternative spliced transcripts contribute differentially to the production of E7 in stably transfected C33-A cells. The highest level of E7 was produced from the least prevalent transcript, the unspliced E6/E7(pre-mRNA). The order of relative expression of E7 was unspliced E6/E7(pre-mRNA) > E6*I/E7 > E6*II/E7. Our findings suggest that E6/E7 alternative splicing may be a mechanism for differential expression of the E6 and E7 oncoproteins, which also affects the expression of their targets, the proteins p53 and pRb.
Asunto(s)
Regulación Viral de la Expresión Génica , Papillomavirus Humano 16/fisiología , Proteínas E7 de Papillomavirus/biosíntesis , Línea Celular Tumoral , Femenino , Humanos , Biosíntesis de Proteínas , Empalme del ARN , ARN Mensajero/metabolismo , ARN Viral/metabolismo , Transcripción GenéticaRESUMEN
Dengue is the most prevalent and rapidly transmitted mosquito-borne viral disease of humans. One of the fundamental innate immune responses to viral infections includes the processing and release of pro-inflammatory cytokines such as interleukin (IL-1ß and IL-18) through the activation of inflammasome. Dengue virus stimulates the Nod-like receptor (NLRP3-specific inflammasome), however, the specific mechanism(s) by which dengue virus activates the NLRP3 inflammasome is unknown. In this study, we investigated the activation of the NLRP3 inflammasome in endothelial cells (HMEC-1) following dengue virus infection. Our results showed that dengue infection as well as the NS2A and NS2B protein expression increase the NLRP3 inflammasome activation, and further apoptosis-associated speck-like protein containing caspase recruitment domain (ASC) oligomerization, and IL-1ß secretion through caspase-1 activation. Specifically, we have demonstrated that NS2A and NS2B, two proteins of dengue virus that behave as putative viroporins, were sufficient to stimulate the NLRP3 inflammasome complex in lipopolysaccharide (LPS)-primed endothelial cells. In summary, our observations provide insight into the dengue-induced inflammatory response mechanism and highlight the importance of DENV-2 NS2A and NS2B proteins in activation of the NLRP3 inflammasome during dengue virus infection.
Asunto(s)
Virus del Dengue/inmunología , Dengue/inmunología , Células Endoteliales/fisiología , Inflamasomas/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Proteínas no Estructurales Virales/metabolismo , Proteínas Viroporinas/metabolismo , Proteínas Adaptadoras de Señalización CARD/metabolismo , Caspasa 1/metabolismo , Línea Celular Transformada , Dengue/virología , Virus del Dengue/patogenicidad , Humanos , Inmunidad Innata , Interleucina-1beta/metabolismo , Proteínas no Estructurales Virales/genética , Proteínas Viroporinas/genética , VirulenciaRESUMEN
Cumulative studies have demonstrated that dengue virus infection results in the induction of apoptosis of certain cells in vitro. Moreover, apoptosis of microvascular endothelial cells in the brain and in the intestinal serosa has been demonstrated postmortem in dengue virus (DENV)-infected patients. In this work, human microvascular endothelial cells (HMEC-1) infected with a DENV-2 clinical isolate, or HMEC-1 cells transfected with its protease sequence (NS3pro) or its complex (NS2BNS3pro) were able to trigger apoptosis after 24 h of infection or transfection. The infected or transfected HMEC-1 cells displayed the distinctive apoptotic hallmarks, which include cytoplasmic shrinkage and plasma membrane blebbing. In addition, the transfected HMEC-1 cells showed biochemical changes such as exposure of phosphatidylserine on the outer leaflet of the plasma membrane, TUNEL positivity, caspase 3 activation and cleaved PARP, a central regulator of apoptosis. These findings suggest the role of such proteins from the clinical isolate in the induction of apoptosis.
Asunto(s)
Apoptosis , Virus del Dengue/patogenicidad , Proteínas Virales/toxicidad , Caspasa 3/metabolismo , Línea Celular , Membrana Celular/química , Membrana Celular/ultraestructura , Colágeno Tipo XI/metabolismo , Células Endoteliales/virología , Humanos , Etiquetado Corte-Fin in Situ , Fosfatidilserinas/análisis , Transducción Genética , TransfecciónRESUMEN
Calicivirus infection causes intrinsic apoptosis, leading to viral propagation in the host. During murine norovirus infection, a reduction in the anti-apoptotic protein survivin has been documented. Here we report that in feline calicivirus infection, a downregulation of the anti-apoptotic proteins survivin and XIAP occur, which correlates with the translocation of the pro-apoptotic protein Smac/DIABLO from the mitochondria to the cytoplasm and the activation of caspase-3. Inhibition of survivin degradation by lactacystin treatment caused a delay in apoptosis progression, reducing virus release, without affecting virus production. However, the overexpression of survivin caused a negative effect in viral progeny production. Overexpression of the leader of the capsid protein (LC), but not of the protease-polymerase NS6/7, results in the downregulation of survivin and XIAP, caspase activation and mitochondrial damage. These results indicate that LC is responsible for the induction of apoptosis in transfected cells and most probably in FCV infection.
Asunto(s)
Apoptosis , Infecciones por Caliciviridae/metabolismo , Calicivirus Felino/fisiología , Proteínas de la Cápside/metabolismo , Regulación hacia Abajo , Survivin/genética , Proteína Inhibidora de la Apoptosis Ligada a X/genética , Animales , Infecciones por Caliciviridae/virología , Proteínas de la Cápside/química , Gatos , Línea Celular , Expresión Génica , Interacciones Huésped-Patógeno , Proteínas Mitocondriales/metabolismo , Transporte de Proteínas , Survivin/metabolismo , Proteínas Virales/biosíntesis , Replicación ViralRESUMEN
Radioresistance of tumor cells gives rise to local recurrence and disease progression in many patients. MicroRNAs (miRNAs) are master regulators of gene expression that control oncogenic pathways to modulate the radiotherapy response of cells. In the present study, differential expression profiling assays identified 16 deregulated miRNAs in acquired radioresistant breast cancer cells, of which miR-122 was observed to be up-regulated. Functional analysis revealed that miR-122 has a role as a tumor suppressor in parental cells by decreasing survival and promoting radiosensitivity. However, in radioresistant cells, miR-122 functions as an oncomiR by promoting survival. The transcriptomic landscape resulting from knockdown of miR-122 in radioresistant cells showed modulation of the ZNF611, ZNF304, RIPK1, HRAS, DUSP8 and TNFRSF21 genes. Moreover, miR-122 and the set of affected genes were prognostic factors in breast cancer patients treated with radiotherapy. Our data indicate that up-regulation of miR-122 promotes cell survival in acquired radioresistant breast cancer and also suggest that miR-122 differentially controls the response to radiotherapy by a dual function as a tumor suppressor an and oncomiR dependent on cell phenotype.
Asunto(s)
Neoplasias de la Mama , Regulación Neoplásica de la Expresión Génica/efectos de la radiación , Genes Supresores de Tumor , MicroARNs/biosíntesis , ARN Neoplásico/biosíntesis , Tolerancia a Radiación , Regulación hacia Arriba/efectos de la radiación , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Neoplasias de la Mama/radioterapia , Femenino , Humanos , Células MCF-7 , MicroARNs/genética , Proteínas de Neoplasias , ARN Neoplásico/genéticaRESUMEN
OBJECTIVE: Loss of expression of apoptotic regulatory proteins in many neoplasias might result in defective or delayed apoptosis, thus facilitating tumor growth or survival. We analyzed here, the basal expression of precursors of apoptotic Caspases in normal cervical epithelium, HPV+ cervical tumor samples and HPV+ tumor-derived cell lines. METHODS: Expression of initiator and effector Caspases was analyzed by immunochemistry in normal cervical epithelium and three types of cervical tumors (squamous cell carcinoma, adenocarcinoma and adenosquamous cell carcinoma) whereas expression of Caspases in HeLa, SiHa and CaSki cells was by immunofluorescence, Western blot and RT-PCR. Besides, the effect of the HPV-16 E6/E7 oncogenes on Caspases expression in cervical cells was evaluated by transfecting C33-A (HPV-) cells. RESULTS: Expression of Caspases 3 and 9 was undetectable in adenocarcinoma and adenosquamous cell carcinoma, respectively. Whereas in squamous cell carcinoma, the expression of Caspases was similar those observed in normal samples. Expression of Caspases 3 and 6 was low in HeLa and CaSki cells, while Caspase 8 was low in SiHa and it was not detected in C33-A cells. All Caspases were detected in the cytoplasm and nucleus of the cells. We did not observe an effect of the E6/E7 oncogenes on the expression of Caspases in C33-A cell. CONCLUSION: Our results showed a differential expression of several Caspases in carcinoma samples and cell lines, suggesting multiple alterations of the Caspase pathways in cervical cancer.
Asunto(s)
Caspasas/biosíntesis , Infecciones por Papillomavirus/enzimología , Neoplasias del Cuello Uterino/enzimología , Neoplasias del Cuello Uterino/virología , Adenocarcinoma/enzimología , Adenocarcinoma/patología , Adenocarcinoma/virología , Western Blotting , Carcinoma Adenoescamoso/enzimología , Carcinoma Adenoescamoso/patología , Carcinoma Adenoescamoso/virología , Carcinoma de Células Escamosas/enzimología , Carcinoma de Células Escamosas/patología , Carcinoma de Células Escamosas/virología , Línea Celular Tumoral , Femenino , Técnica del Anticuerpo Fluorescente , Células HeLa , Papillomavirus Humano 16 , Papillomavirus Humano 18 , Humanos , Inmunohistoquímica , Isoenzimas/biosíntesis , Infecciones por Papillomavirus/patología , Adhesión en Parafina , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Neoplasias del Cuello Uterino/patologíaRESUMEN
Noncoding circular RNAs are widespread in the tree of life. Particularly, intron-containing circular RNAs which apparently upregulate their parental gene expression. Entamoeba histolytica, the causative agent of dysentery and liver abscesses in humans, codes for several noncoding RNAs, including circular ribosomal RNAs, but no intron containing circular RNAs have been described to date. Divergent RT-PCR and diverse molecular approaches, allowed us to detect bona fide full-length intronic circular RNA (flicRNA) molecules. Self-splicing reactions, RNA polymerase II inhibition with Actinomycin D, and second step of splicing-inhibition with boric acid showed that the production of flicRX13 (one of the flicRNAs found in this work, and our test model) depends on mRNA synthesis and pre-mRNA processing instead of self-splicing. To explore the cues and factors involved in flicRX13 biogenesis in vivo, splicing assays were carried out in amoeba transformants where splicing factors and Dbr1 (intron lariat debranching enzyme 1) were silenced or overexpressed, or where Rabx13 wild-type and mutant 5'ss (splice site) and branch site minigene constructs were overexpressed. Whereas SF1 (splicing factor 1) is not involved, the U2 auxiliary splicing factor, Dbr1, and the GU-rich 5'ss are involved in postsplicing flicRX13 biogenesis, probably by Dbr1 stalling, in a similar fashion to the formation of ciRNAs (circular intronic RNAs), but with distinctive 5'-3'ss ligation points. Different from the reported functions of ciRNAs, the 5'ss GU-rich element of flicRX13 possibly interacts with transcription machinery to silence its own gene in cis. Furthermore, introns of E. histolytica virulence-related genes are also processed as flicRNAs.
Asunto(s)
Entamoeba histolytica/genética , Entamoeba histolytica/metabolismo , Intrones , Empalme del ARN , ARN/genética , ARN/metabolismo , Silenciador del Gen , Factores de Empalme de ARN/genética , Factores de Empalme de ARN/metabolismo , ARN CircularRESUMEN
Intestinal macrophages are highly mobile cells with extraordinary plasticity and actively contribute to cytokine-mediated epithelial cell damage. The mechanisms triggering macrophage polarization into a proinflammatory phenotype are unknown. Here, we report that during inflammation macrophages enhance its intercellular adhesion properties in order to acquire a M1-phenotype. Using in vitro and in vivo models we demonstrate that intercellular adhesion is mediated by integrin-αVß3 and relies in the presence of the unconventional class I myosin 1F (Myo1F). Intercellular adhesion mediated by αVß3 stimulates M1-like phenotype in macrophages through hyperactivation of STAT1 and STAT3 downstream of ILK/Akt/mTOR signaling. Inhibition of integrin-αVß3, Akt/mTOR, or lack of Myo1F attenuated the commitment of macrophages into a pro-inflammatory phenotype. In a model of colitis, Myo1F deficiency strongly reduces the secretion of proinflammatory cytokines, decreases epithelial damage, ameliorates disease activity, and enhances tissue repair. Together our findings uncover an unknown role for Myo1F as part of the machinery that regulates intercellular adhesion and polarization in macrophages.
Asunto(s)
Colitis Ulcerosa/inmunología , Integrina alfaVbeta3/metabolismo , Activación de Macrófagos , Macrófagos/inmunología , Miosina Tipo I/metabolismo , Animales , Línea Celular Tumoral , Colitis Ulcerosa/inducido químicamente , Citoesqueleto/inmunología , Citoesqueleto/metabolismo , Sulfato de Dextran/administración & dosificación , Sulfato de Dextran/toxicidad , Modelos Animales de Enfermedad , Humanos , Integrina alfaVbeta3/inmunología , Interleucina-1beta/inmunología , Interleucina-1beta/metabolismo , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Miosina Tipo I/genética , Miosina Tipo I/inmunología , Cultivo Primario de Células , Células RAW 264.7RESUMEN
Curcumin is a phytochemical with potent anti-neoplastic properties. The antitumoral effects of curcumin in cells derived from chronic or acute myeloid leukemia have been already described. However, a comparative study of the cytostatic and cytotoxic effects of curcumin on chronic and acute myeloid leukemia cells has not yet been performed. In the present study, the cellular effects of curcumin on cell lines derived from chronic or acute myeloid leukemia were examined. Dose and time-response assays were performed with curcumin on HL-60 and K562 cells. Cell viability was evaluated with trypan blue exclusion test and cell death by flow cytometry using a fluorescent molecular probe. A cell cycle profile was analyzed, and protein markers of cell cycle progression and cell death were investigated. In the present study, the K562 cells showed a higher sensitivity to the cytostatic and cytotoxic effects of curcumin compared with HL-60. In addition, curcumin induced G1 phase arrest in HL-60 cells and G2/M phase arrest in K562 cells. Furthermore, curcumin-related cell death in HL-60 was associated with the processed forms of caspases-9 and -3 proteins, whereas in K562 cells, both the processed and the unprocessed forms were present. Accordingly, activity of these caspases was significantly higher in HL-60 cells compared with that in K562. In conclusion, curcumin elicits different cellular mechanisms in chronic or acute myeloid leukemia cells and the powerful antitumoral effect was more potent in K562 compared with HL-60 cells.