RESUMEN
The gain-of-function mutation in the pleckstrin homology domain of AKT1 (AKT1E17K) occurs in lung and breast cancer. Through the use of human cellular models and of a AKT1E17K transgenic Cre-inducible murine strain (R26-AKT1E17K mice), we have demonstrated that AKT1E17K is a bona fide oncogene for lung epithelial cells. However, the role of AKT1E17K in breast cancer remains to be determined. Here, we report the generation and the characterization of a MMTV-CRE; R26-AKT1E17K mouse strain that expresses the mutant AKT1E17K allele in the mammary epithelium. We observed that AKT1E17K stimulates the development of mammary tumors classified as ductal adenocarcinoma of medium-high grade and presented a variety of proliferative alterations classified as adenosis with low-to-high grade dysplasia in the mammary epithelium. A subsequent immunohistochemical characterization suggested they were PR-/HER2-/ER+, basal-like and CK8-/CK10-/CK5+/CK14+. We also observed that, in parallel with an increased proliferation rate, tumors expressing mutant AKT1E17K presented an activation of the GSK3/cyclin D1 pathway in the mammary epithelium and cluster significantly with the human basal-like tumors. In conclusion, we demonstrate AKT1E17K is a bona fide oncogene that can initiate tumors at high efficiency in murine mammary epithelium in vivo.
RESUMEN
The molecular complexity of human breast cancer (BC) renders the clinical management of the disease challenging. Long non-coding RNAs (lncRNAs) are promising biomarkers for BC patient stratification, early detection, and disease monitoring. Here, we identified the involvement of the long intergenic non-coding RNA 01087 (LINC01087) in breast oncogenesis. LINC01087 appeared significantly downregulated in triple-negative BCs (TNBCs) and upregulated in the luminal BC subtypes in comparison to mammary samples from cancer-free women and matched normal cancer pairs. Interestingly, deregulation of LINC01087 allowed to accurately distinguish between luminal and TNBC specimens, independently of the clinicopathological parameters, and of the histological and TP53 or BRCA1/2 mutational status. Moreover, increased expression of LINC01087 predicted a better prognosis in luminal BCs, while TNBC tumors that harbored lower levels of LINC01087 were associated with reduced relapse-free survival. Furthermore, bioinformatics analyses were performed on TNBC and luminal BC samples and suggested that the putative tumor suppressor activity of LINC01087 may rely on interferences with pathways involved in cell survival, proliferation, adhesion, invasion, inflammation and drug sensitivity. Altogether, these data suggest that the assessment of LINC01087 deregulation could represent a novel, specific and promising biomarker not only for the diagnosis and prognosis of luminal BC subtypes and TNBCs, but also as a predictive biomarker of pharmacological interventions.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/metabolismo , ARN Largo no Codificante/metabolismo , Neoplasias de la Mama Triple Negativas/metabolismo , Biomarcadores de Tumor/genética , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Redes Reguladoras de Genes , Humanos , Células MCF-7 , Metástasis de la Neoplasia , Recurrencia Local de Neoplasia , Supervivencia sin Progresión , Mapas de Interacción de Proteínas , ARN Largo no Codificante/genética , Transducción de Señal , Factores de Tiempo , Transcriptoma , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/patologíaRESUMEN
Familial hypercholesterolemia (FH) is a genetic hyperlipidemia characterized by elevated concentrations of plasma LDL cholesterol. Statins are not always effective for the treatment of FH patients; unresponsive patients have poor prognosis and rely on LDL apheresis. In the past, we developed safe and effective gene therapy strategies for the expression of anti-atherogenic proteins using PEGylated helper-dependent adenoviral (HD-Ad) vectors. We recently developed a HD-Ad vector for the expression of the soluble form of the extracellular portion of the human LDL receptor (LDLR) fused with a rabbit transferrin dimer (LDLR-TF). We evaluated the efficacy of the LDLR-TF chimeric protein in CHOLDLA7, a cell line lacking LDLR expression, restoring the ability to uptake LDL. Subsequently, we administered intravenously 1 × 10E13 vp/kg of this vector in LDLR-deficient mice and observed amelioration of lipid profile and reduction of aortic atherosclerosis. Finally, we studied LDL distribution after HD-Ad vector-mediated expression of LDLR-TF in LDLR-deficient mice and found LDL accumulation in liver, and in heart and intestine. These results support the possibility of lowering LDL-C levels and reducing aortic atherosclerosis using a secreted therapeutic transgene; the present strategy potentially can be modified and adapted to non-systemic gene transfer with expression of the secreted chimeric protein in muscle or other tissues. Intramuscular or local administration strategies could improve the safety profile of this strategy and facilitate applicability.
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Terapia Genética/métodos , Receptores de LDL/genética , Transferrina/genética , Adenoviridae/genética , Infecciones por Adenoviridae/genética , Animales , Aorta/patología , Aterosclerosis/genética , Línea Celular , LDL-Colesterol/sangre , Enfermedad de la Arteria Coronaria/genética , Enfermedad de la Arteria Coronaria/fisiopatología , Técnicas de Transferencia de Gen , Vectores Genéticos/genética , Humanos , Lípidos/sangre , Ratones , Receptores de LDL/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/uso terapéutico , Transferrina/metabolismo , TransgenesRESUMEN
The CDK inhibitor, p27kip1, encoded by the Cdkn1b gene can negatively modulate cell proliferation. The control of p27 activity during the cell cycle is regulated at multiple levels, including transcription, translation, and protein stability. The last residue of p27 (threonine 198 in human, threonine 197 in mouse) is involved in the control of protein stability. We have generated a murine knock-in model (Cdkn1b T197A) in which threonine 197 is replaced by alanine, which renders p27 protein highly unstable due to a high rate of proteasomal degradation. Expectedly, Cdkn1b T197A/T197A mice present with increased body size and weight, organomegaly, and multiple organ hyperplasia, similar to what is observed in Cdkn1b KO/KO mice. We investigated the effects exerted by the restoration of normal levels of p27 protein in the tissue of Cdkn1b T197A/T197A mice. We found that proteasome inhibition with bortezomib rescues the hyperplasia induced by the lack of p27 expression in Cdkn1b T197A/T197A but not in Cdkn1b KO/KO mice. However, BAY 11-7082, a proteasome inhibitor that stabilizes IκB but not p27, fails to rescue hyperplasia in Cdkn1b T197A/T197A mice. Bortezomib increases p27 half-life and reduces the proliferation in MEFs derived from Cdkn1b T197A/T197A but not from Cdkn1b WT/WT mice, whereas BAY 11-7082 had no effect on the protein levels of p27 and on the proliferation rate of Cdkn1b T197A/T197A MEFs.The results presented here demonstrate that Cdkn1b T197A/T197A mice represent an attractive in vivo model to investigate whether the targeting of p27 degradation machinery might prove beneficial in the treatment of a variety of human proliferative disorders caused by increased turnover of p27 protein.
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Sustitución de Aminoácidos , Bortezomib/farmacología , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/química , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética , Modelos Animales , Animales , Técnicas de Sustitución del Gen , Hiperplasia , Ratones , Nitrilos/farmacología , Complejo de la Endopetidasa Proteasomal/metabolismo , Estabilidad Proteica , Proteolisis , Sulfonas/farmacologíaRESUMEN
Genetic modifications during development of paediatric groups 3 and 4 medulloblastoma are responsible for their highly metastatic properties and poor patient survival rates. PRUNE1 is highly expressed in metastatic medulloblastoma group 3, which is characterized by TGF-ß signalling activation, c-MYC amplification, and OTX2 expression. We describe the process of activation of the PRUNE1 signalling pathway that includes its binding to NME1, TGF-ß activation, OTX2 upregulation, SNAIL (SNAI1) upregulation, and PTEN inhibition. The newly identified small molecule pyrimido-pyrimidine derivative AA7.1 enhances PRUNE1 degradation, inhibits this activation network, and augments PTEN expression. Both AA7.1 and a competitive permeable peptide that impairs PRUNE1/NME1 complex formation, impair tumour growth and metastatic dissemination in orthotopic xenograft models with a metastatic medulloblastoma group 3 cell line (D425-Med cells). Using whole exome sequencing technology in metastatic medulloblastoma primary tumour cells, we also define 23 common 'non-synonymous homozygous' deleterious gene variants as part of the protein molecular network of relevance for metastatic processes. This PRUNE1/TGF-ß/OTX2/PTEN axis, together with the medulloblastoma-driver mutations, is of relevance for future rational and targeted therapies for metastatic medulloblastoma group 3.10.1093/brain/awy039_video1awy039media15742053534001.
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Proteínas Portadoras/metabolismo , Neoplasias Cerebelosas/metabolismo , Regulación Neoplásica de la Expresión Génica/fisiología , Meduloblastoma/metabolismo , Metástasis de la Neoplasia/fisiopatología , Fosfohidrolasa PTEN/metabolismo , Adolescente , Animales , Proteínas Portadoras/genética , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Neoplasias Cerebelosas/patología , Niño , Preescolar , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Redes Reguladoras de Genes , Humanos , Lactante , Masculino , Meduloblastoma/patología , Ratones , Ratones Endogámicos BALB C , Modelos Moleculares , Metástasis de la Neoplasia/genética , Fosfohidrolasa PTEN/genética , Monoéster Fosfórico Hidrolasas , Pirimidinonas/química , Pirimidinonas/farmacología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Factores de Transcripción de la Familia Snail/metabolismo , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
AIM: HtrA1, a member of the High Temperature Requirement Factor A family of oxidative stress-response proteases seems to play a role as a tumor suppressor, being down-regulated in a series of human cancers during their progression. Particularly, low HtrA1 mRNA levels have been observed in breast cancer patients with more aggressive clinical features. These have been shown to relate to a longer disease free and overall survival, with more pronounced effects in axillary nodes positive patients. SUBJECTS AND METHODS: We have analyzed for immunohistochemical HtrA1 expression a series of 66 sentinel node positive breast cancers through Tissue Micro Array technology. RESULTS: HtrA1 was absent to low in 29 cases, medium in 19 cases and high in 18 cases. Our data revealed a positive significant relation between HtrA1 expression level and estrogen (p=0,002) and progestinic receptor expression (p=0.003) and a negative correlation with histological grading (p=0.028), proliferation index (p=0.05), common BC histotypes (p=0.040), luminal A and B subtypes (p=0.001), metastasis development (p<0.0001) and local relapse (p<0.0001). Finally, no correlation was recorded between HtrA1 expression level and breast cancer histology type and metastasis to non sentinel nodes. Interestingly HtrA1 loss in SLN metastasis was able to predict positive non sentinel nodes (p=0.001). CONCLUSIONS: Low HtrA1 expression is significantly related to breast cancer poor prognosis parameters, and HtrA1 loss in sentinel nodes is related to metastasis of non sentinel nodes, offering a further marker useful for BC prognostic stratification.
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Neoplasias de la Mama/metabolismo , Carcinoma Ductal de Mama/metabolismo , Ganglios Linfáticos/metabolismo , Invasividad Neoplásica/patología , Serina Endopeptidasas/metabolismo , Adulto , Anciano , Neoplasias de la Mama/patología , Carcinoma Ductal de Mama/patología , Femenino , Serina Peptidasa A1 que Requiere Temperaturas Altas , Humanos , Ganglios Linfáticos/patología , Metástasis Linfática/patología , Persona de Mediana Edad , Pronóstico , Receptores de Estrógenos/metabolismo , Receptores de Progesterona/metabolismo , Biopsia del Ganglio Linfático CentinelaRESUMEN
BACKGROUND: Significant and sustained excess weight loss (EWL) appears to reduce the risk of obesity-related comorbidities (insulin resistance, hyperlipidemia, and inflammation), but this has been primarily shown in adult diabetic obese patients. We evaluated whether the EWL obtained 3 years after laparoscopic adjustable gastric banding (LAGB) improves the metabolic phenotype in nondiabetic morbidly obese (NDMO) individuals from south Italy. METHODS: Serum and subcutaneous adipose tissue (SAT) samples from 20 obese individuals (median BMI=41.5 kg/m(2)) before (T0) and after LAGB (T1) and from 10 controls (median BMI=22.8 kg/m(2)) were taken. Serum leptin, adiponectin, C reactive protein (CRP), and main analyte levels were evaluated by routine methods or immunoassay. In SAT, adipocyte size was measured by hematoxylin/eosin staining, cluster of differentiation 68 (CD68) macrophage infiltration marker by immunohistochemistry, and adiponectin, adiponectin receptors 1 and 2, and interleukin 6 (IL6) messenger RNAs by qRT-PCR. RESULTS: The average EWL was 66.7 %, and CRP, triglycerides, hepatic markers, leptin levels, homeostasis model assessment, and the leptin/adiponectin ratio were lower (p<0.05) at T1 than at T0. The expression of small adipocytes and adiponectin was increased (p<0.05), and inflammation markers (CD68 and IL6) decreased (p<0.05) at T1 vs. T0. At linear regression multivariate analysis, over 90 % (R (2)=0.905) of EWL (dependent variable) was explained by CD68, adiponectinemia, triglyceridemia, CRP, and total protein levels. CONCLUSIONS: The EWL obtained 3 years after LAGB resulted in an improvement of lipid metabolism and a reduction of inflammation in NDMO patients, thereby decreasing the risk of obesity-associated diseases.
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Diabetes Mellitus Tipo 2 , Inflamación/fisiopatología , Obesidad Mórbida/cirugía , Grasa Subcutánea/fisiopatología , Adulto , Estudios de Casos y Controles , Colesterol/sangre , Femenino , Gastroplastia/métodos , Humanos , Inflamación/prevención & control , Italia , Masculino , Obesidad Mórbida/sangre , Pérdida de PesoRESUMEN
Autosomal dominant dehydrated hereditary stomatocytosis (DHSt) usually presents as a compensated hemolytic anemia with macrocytosis and abnormally shaped red blood cells (RBCs). DHSt is part of a pleiotropic syndrome that may also exhibit pseudohyperkalemia and perinatal edema. We identified PIEZO1 as the disease gene for pleiotropic DHSt in a large kindred by exome sequencing analysis within the previously mapped 16q23-q24 interval. In 26 affected individuals among 7 multigenerational DHSt families with the pleiotropic syndrome, 11 heterozygous PIEZO1 missense mutations cosegregated with disease. PIEZO1 is expressed in the plasma membranes of RBCs and its messenger RNA, and protein levels increase during in vitro erythroid differentiation of CD34(+) cells. PIEZO1 is also expressed in liver and bone marrow during human and mouse development. We suggest for the first time a correlation between a PIEZO1 mutation and perinatal edema. DHSt patient red cells with the R2456H mutation exhibit increased ion-channel activity. Functional studies of PIEZO1 mutant R2488Q expressed in Xenopus oocytes demonstrated changes in ion-channel activity consistent with the altered cation content of DHSt patient red cells. Our findings provide direct evidence that R2456H and R2488Q mutations in PIEZO1 alter mechanosensitive channel regulation, leading to increased cation transport in erythroid cells.
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Anemia Hemolítica Congénita/genética , Hidropesía Fetal/genética , Canales Iónicos/genética , Mutación , Adulto , Secuencia de Aminoácidos , Anemia Hemolítica Congénita/clasificación , Anemia Hemolítica Congénita/diagnóstico , Animales , Embrión de Mamíferos , Femenino , Regulación del Desarrollo de la Expresión Génica , Humanos , Hidropesía Fetal/clasificación , Hidropesía Fetal/diagnóstico , Ratones , Ratones Transgénicos , Modelos Biológicos , Datos de Secuencia Molecular , Mutación/fisiología , Linaje , Homología de Secuencia de Aminoácido , Transfección , Xenopus laevisRESUMEN
Bone morphogenetic Proteins (BMPs) are growth factors also involved in ossification and chondrogenesis that have generated interest for their efficiency in inducing bone neo-synthesis. BMPs expression in engineered cells has been successful in stimulating osteoblastic differentiation and ectopic and orthotopic bone formation in vivo. We have previously shown that an adenoviral vector expressing bone morphogenetic protein type-4 (BMP-4) is able to efficiently drive bone formation in a rabbit model of discontinuous bone lesions. However, unregulated secretion of BMPs has also been implicated in bone overproduction and exostosis. We have constructed a replication-defective first generation adenoviral (FG-Ad) vector containing a cassette for the expression of BMP-4 associated with the Herpes Simplex virus thymidine kinase (TK) gene (FG-B4TK) in order to shut down BMP-4 expression and, therefore, regulate bone production. TK expression does not interfere with BMP-4 ability to induce ectopic bone formation in athymic nude mice. Administration of ganciclovir blocks ectopic bone production in quadriceps muscle transduced with the FG-B4TK with no effect on the contralateral muscle transduced with a vector expressing only BMP-4. Histological findings confirmed the pro-apoptotic activity of TK and the reduction of mineralized areas in the quadriceps transduced with FG-B4TK in mice treated with ganciclovir. We have generated a system to block BMP-4 secretion by inducing apoptosis in transduced cells therefore blocking unwanted bone formation. This system is an additional tool to generate regulated amount of bone in discontinuous bone lesions and can be easily coupled with biomaterials capable of recruiting cells and generating a local bioreactor.
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Desarrollo Óseo/genética , Proteína Morfogenética Ósea 4/genética , Vectores Genéticos/farmacología , Timidina Quinasa/genética , Animales , Apoptosis/genética , Desarrollo Óseo/efectos de los fármacos , Proteína Morfogenética Ósea 4/metabolismo , Huesos/metabolismo , Ganciclovir/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Técnicas de Transferencia de Gen , Vectores Genéticos/genética , Humanos , Masculino , Ratones , Ratones Desnudos , Osteogénesis/genética , Músculo Cuádriceps/fisiología , Simplexvirus/enzimología , TransgenesRESUMEN
Carbonic anhydrase IX (CA IX) is a transmembrane protein affecting pH regulation, cell migration/invasion, and survival in hypoxic tumors. Although the pathways related to CA IX have begun to emerge, molecular partners mediating its functions remain largely unknown. Here we characterize the CA IX interactome in hypoxic HEK-293 cells. Most of the identified CA IX-binding partners contain the HEAT/ARM repeat domain and belong to the nuclear transport machinery. We show that the interaction with two of these proteins, namely XPO1 exportin and TNPO1 importin, occurs via the C-terminal region of CA IX and increases with protein phosphorylation. We also demonstrate that nuclear CA IX is enriched in hypoxic cells and is present in renal cell carcinoma tissues. These data place CA IX among the cell-surface signal transducers undergoing nuclear translocation. Accordingly, CA IX interactome involves also CAND1, which participates in both gene transcription and assembly of SCF ubiquitin ligase complexes. It is noteworthy that down-regulation of CAND1 leads to decreased CA IX protein levels apparently via affecting its stability. Our findings provide the first evidence that CA IX interacts with proteins involved in nuclear/cytoplasmic transport, gene transcription, and protein stability, and suggest the existence of nuclear CA IX protein subpopulations with a potential intracellular function, distinct from the crucial CA IX role at the cell surface.
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Antígenos de Neoplasias , Anhidrasas Carbónicas , Carcinoma de Células Renales , Proteínas , Factores de Transcripción , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Anhidrasa Carbónica IX , Anhidrasas Carbónicas/genética , Anhidrasas Carbónicas/metabolismo , Carcinoma de Células Renales/genética , Carcinoma de Células Renales/metabolismo , Carcinoma de Células Renales/patología , Hipoxia de la Célula , Células HEK293 , Humanos , Fosforilación , Mapas de Interacción de Proteínas , Estabilidad Proteica , Proteínas/química , Proteínas/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The PATZ1 gene encoding a POZ/AT-hook/Kruppel zinc finger (PATZ) transcription factor, is considered a cancer-related gene because of its loss or misexpression in human neoplasias. As for other POZ/domain and Kruppel zinc finger (POK) family members, the transcriptional activity of PATZ is due to the POZ-mediated oligomer formation, suggesting that it might be not a typical transactivator but an architectural transcription factor, thus functioning either as activator or as repressor depending on the presence of proteins able to interact with it. Therefore, to better elucidate PATZ function, we searched for its molecular partners. By yeast two-hybrid screenings, we found a specific interaction between PATZ and BCL6, a human oncogene that plays a key role in germinal center (GC) derived neoplasias. We demonstrate that PATZ and BCL6 interact in germinal center-derived B lymphoma cells, through the POZ domain of PATZ. Moreover, we show that PATZ is able to bind the BCL6 regulatory region, where BCL6 itself acts as a negative regulator, and to contribute to negatively modulate its activity. Consistently, disruption of one or both Patz1 alleles in mice causes focal expansion of thymus B cells, in which BCL6 is up-regulated. This phenotype was almost completely rescued by crossing Patz1(+/-) with Bcl6(+/-) mice, indicating a key role for Bcl6 expression in its development. Finally, a significant number of Patz1 knock-out mice (both heterozygous and homozygous) also develop BCL6-expressing lymphomas. Therefore, the disruption of one or both Patz1 alleles may favor lymphomagenesis by activating the BCL6 pathway.
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Proteínas de Unión al ADN/metabolismo , Factores de Transcripción de Tipo Kruppel/metabolismo , Proteínas Represoras/metabolismo , Animales , Secuencia de Bases , Línea Celular , Inmunoprecipitación de Cromatina , Cartilla de ADN , Humanos , Linfoma de Células B/genética , Linfoma de Células B/patología , Ratones , Ratones Noqueados , Unión Proteica , Proteínas Proto-Oncogénicas c-bcl-6 , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
MiRNAs play a relevant role in regulating gene expression in a variety of physiological and pathological conditions including autoimmune disorders. MiRNAs are also important in the differentiation and function of the mouse intestinal epithelium. Our study was aimed to look for miRNA-based modulation of gene expression in celiac small intestine, and particularly for genes involved in cell intestinal differentiation/proliferation mechanisms. A cohort of 40 children (20 with active CD, 9 on a gluten-free diet (GFD), and 11 controls), were recruited at the Paediatrics Department (University of Naples Federico II). The expression of 365 human miRNAs was quantified by TaqMan low-density arrays. We used bioinformatics to predict putative target genes of miRNAs and to select biological pathways. The presence of NOTCH1, HES1, KLF4, MUC-2, Ki67 and beta-catenin proteins in the small intestine of CD and control children was tested by immunohistochemistry. The expression of about 20% of the miRNAs tested differed between CD and control children. We found that high miR-449a levels targeted and reduced both NOTCH1 and KLF4 in HEK-293 cells. NOTCH1, KLF4 signals and the number of goblet cells were lower in small intestine of children with active CD and in those on a GFD than in controls, whereas more nuclear beta-catenin staining, as a sign of the WNT pathway activation, and more Ki67 staining, as sign of proliferation, were present in crypts from CD patients than in controls. In conclusion we first demonstrate a miRNA mediated gene regulation in small intestine of CD patients. We also highlighted a reduced NOTCH1 pathway in our patients, irrespective of whether the disease was active or not. We suggest that NOTCH pathway could be constitutively altered in the celiac small intestine and could drive the increased proliferation and the decreased differentiation of intestinal cells towards the secretory goblet cell lineage.
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Enfermedad Celíaca/genética , Células Caliciformes/patología , Intestino Delgado/metabolismo , Intestino Delgado/patología , MicroARNs/genética , Receptor Notch1/metabolismo , Transducción de Señal , Regiones no Traducidas 3'/genética , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Estudios de Casos y Controles , Enfermedad Celíaca/patología , Recuento de Células , Niño , Preescolar , Biología Computacional , Dieta Sin Gluten , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Células Caliciformes/metabolismo , Células HEK293 , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/metabolismo , Masculino , Ratones , MicroARNs/metabolismo , Mucina 2/metabolismo , Unión Proteica/genética , Receptor Notch1/genética , Transducción de Señal/genética , Factor de Transcripción HES-1 , beta Catenina/metabolismoRESUMEN
BACKGROUND: Mortality is high in patients with esophageal carcinoma as tumors are rarely detected before the disease has progressed to an advanced stage. Here, we sought to isolate cell-free DNA released into the plasma of patients with esophageal carcinoma, to analyze copy number variations of marker genes in the search for early detection of tumor progression. METHODS: Plasma of 41 patients with esophageal carcinoma was prospectively collected before tumor resection and chemotherapy. Our dataset resulted heterogeneous for clinical data, resembling the characteristics of the tumor. DNA from the plasma was extracted to analyze copy number variations of the erbB2 gene using real-time PCR assays. RESULTS: The real-time PCR assays for erbB2 gene showed significant (P = 0.001) copy number variations in the plasma of patients with esophageal carcinoma, as compared to healthy controls with high sensitivity (80%) and specificity (95%). These variations in erbB2 were negatively correlated to the progression free survival of these patients (P = 0.03), and revealed a further risk category stratification of patients with low VEGF expression levels. CONCLUSION: The copy number variation of erbB2 gene from plasma can be used as prognostic marker for early detection of patients at risk of worse clinical outcome in esophageal cancer.
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Biomarcadores de Tumor/sangre , Carcinoma/diagnóstico , ADN/sangre , Neoplasias Esofágicas/diagnóstico , Genes erbB-2/genética , Anciano , Carcinoma/genética , Carcinoma/patología , Carcinoma/fisiopatología , Supervivencia sin Enfermedad , Detección Precoz del Cáncer , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patología , Neoplasias Esofágicas/fisiopatología , Femenino , Dosificación de Gen/genética , Humanos , Masculino , Persona de Mediana Edad , Pronóstico , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sensibilidad y Especificidad , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Experimental animal models of bacterial meningitis are useful to study the host-pathogen interactions occurring at the cerebral level and to analyze the pathogenetic mechanisms behind this life-threatening disease. In this study, we have developed a mouse model of meningococcal meningitis based on the intracisternal inoculation of bacteria. Experiments were performed with mouse-passaged serogroup C Neisseria meningitidis. Survival and clinical parameters of infected mice and microbiological and histological analysis of the brain demonstrated the establishment of meningitis with features comparable to those of the disease in humans. When using low bacterial inocula, meningococcal replication in the brain was very efficient, with a 1,000-fold increase of viable counts in 18 h. Meningococci were also found in the blood, spleens, and livers of infected mice, and bacterial loads in different organs were dependent on the infectious dose. As glutamate uptake from the host has been implicated in meningococcal virulence, mice were infected intracisternally with an isogenic strain deficient in the ABC-type L-glutamate transporter GltT. Noticeably, the mutant was attenuated in virulence in mixed infections, indicating that wild-type bacteria outcompeted the GltT-deficient meningococci. The data show that the GltT transporter plays a role in meningitis and concomitant systemic infection, suggesting that meningococci may use L-glutamate as a nutrient source and as a precursor to synthesize the antioxidant glutathione.
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Transportadoras de Casetes de Unión a ATP/fisiología , Sistema de Transporte de Aminoácidos X-AG/fisiología , Proteínas Bacterianas/fisiología , Meningitis Meningocócica/etiología , Neisseria meningitidis/patogenicidad , Animales , Femenino , Ácido Glutámico/metabolismo , Meningitis Meningocócica/patología , Ratones , Neisseria meningitidis/crecimiento & desarrollo , VirulenciaRESUMEN
The use of adult stem cells in tissue engineering approaches will benefit from the establishment of culture conditions that allow the expansion and maintenance of cells with stem cell-like activity and high differentiation potential. In the field of adult stem cells, bone marrow stromal cells (BMSCs) are promising candidates. In the present study, we define, for the first time, conditions for optimizing the yields of cultures enriched for specific progenitors of bone marrow. Using four distinct culture conditions, supernatants from culture of bone fragments, marrow stroma cell line MS-5, embryonic fibroblast cell line NIH3T3, and a cocktail of epidermal growth factor (EGF) and platelet-derived growth factor (PDGF), we isolated four different sub-populations of murine BMSCs (mBMSCs). These cells express a well-known marker of undifferentiated embryonic stem cells (Nanog) and show interesting features in immunophenotype, self-renewal ability, and differentiation potency. In particular, using NIH3T3 conditioned medium, we obtained cells that showed impairment in osteogenic and chondrogenic differentiation while retaining high adipogenic potential during passages. Our results indicate that the choice of the medium used for isolation and expansion of mBMSCs is important for enriching the culture of desired specific progenitors.
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Envejecimiento/fisiología , Células de la Médula Ósea/citología , Células Madre Mesenquimatosas/citología , Adipogénesis/efectos de los fármacos , Envejecimiento/efectos de los fármacos , Animales , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/metabolismo , Proliferación Celular/efectos de los fármacos , Forma de la Célula/efectos de los fármacos , Células Cultivadas , Condrogénesis/efectos de los fármacos , Medios de Cultivo Condicionados/farmacología , Proteínas de Homeodominio/metabolismo , Factor Inhibidor de Leucemia/metabolismo , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/metabolismo , Ratones , Ratones Endogámicos C57BL , Células 3T3 NIH , Proteína Homeótica Nanog , Osteogénesis/efectos de los fármacosRESUMEN
The molecular characterization of balanced chromosomal rearrangements has often been a powerful tool for the positional identification of genes associated with specific diseases. In some instances, these rearrangements may be associated with a variety of different phenotypes, and thus establishing a genotype-phenotype correlation may be a complex process. However, molecular characterization of the rearrangement remains a useful tool for diagnoses or prognoses, or for identifying new genes and establishing a gene-to-function relationship. In this work we describe the characterization of a de novo balanced translocation t(2;6)(q24.3;q22.31) found in a patient with a complex phenotype. The major clinical finding was a severe neurological involvement. Thanks to the molecular characterization of this translocation we found that the rearrangement led to the truncation of the TCBA1 gene on chromosome 6q. We found that the gene is transcribed in different splice variants and is highly specific for the central nervous system. TCBA1 does not show any similarity with other known genes, and no information is available about its function. However, the gene appears to be well conserved among species, and we were able to infer the sequence of a putative mouse homolog of TCBA1. This allowed us to perform a more detailed expression study in mice, thus confirming its specificity for the nervous system. This finding is of particular interest because it suggests that TCBA1 may be correlated with the neurological phenotype of our patient, and possibly mutated in genetic diseases with a neurological phenotype.
Asunto(s)
Proteínas de la Membrana/genética , Enfermedades del Sistema Nervioso/genética , Eliminación de Secuencia , Translocación Genética , Empalme Alternativo , Animales , Secuencia de Bases , Encéfalo/metabolismo , Encéfalo/patología , Cromosomas Humanos Par 2 , Cromosomas Humanos Par 6 , Análisis Mutacional de ADN , Embrión de Mamíferos/metabolismo , Embrión de Mamíferos/patología , Humanos , Hibridación Fluorescente in Situ , Lactante , Cariotipificación , Masculino , Ratones , Datos de Secuencia Molecular , Enfermedades del Sistema Nervioso/diagnóstico , Enfermedades del Sistema Nervioso/patología , Fenotipo , ARN Mensajero/metabolismoRESUMEN
Nonmuscle myosin heavy chain II-A is responsible for MYH9-related disease, which is characterized by macrothrombocytopenia, granulocyte inclusions, deafness, cataracts, and renal failure. Since another two highly conserved nonmuscle myosins, II-B and II-C, are known, an analysis of their tissue distribution is fundamental for the understanding of their biological roles. In mouse, we found that all forms are ubiquitously expressed. However, megakaryocytic and granulocytic lineages express only II-A, suggesting that congenital features, macrothrombocytopenia, and leukocyte inclusions correlate with its exclusive presence. In kidney, eye, and ear, where clinical manifestations have a late onset, as well as in other tissues apparently not affected in patients, II-A and at least one of the other two isoforms are expressed, suggesting that II-B and II-C can partially compensate for each other. We hypothesize that cells expressing only II-A manifest the congenital defects, while tissues expressing additional myosin II isoforms show either late onset of abnormalities or no pathological sign.
Asunto(s)
Proteínas Motoras Moleculares/genética , Cadenas Pesadas de Miosina/genética , Cadenas Pesadas de Miosina/metabolismo , Miosina Tipo II/metabolismo , Trombocitopenia/genética , Animales , Médula Ósea/metabolismo , Médula Ósea/ultraestructura , Catarata/genética , Cóclea/metabolismo , Cóclea/ultraestructura , Sordera/genética , Embrión de Mamíferos/metabolismo , Embrión de Mamíferos/ultraestructura , Ojo/metabolismo , Ojo/ultraestructura , Expresión Génica , Granulocitos/ultraestructura , Humanos , Hibridación in Situ , Cuerpos de Inclusión/genética , Riñón/metabolismo , Riñón/ultraestructura , Hígado/metabolismo , Hígado/ultraestructura , Ratones , Miosina Tipo II/genética , Nefritis/genética , Fenotipo , ARN Mensajero/análisis , Síndrome , Trombocitopenia/diagnóstico , Distribución TisularRESUMEN
Connexin genes are involved in several human diseases such as hearing and dermatological and peripheral nerve disorders. Connexins are protein units of gap junctions and form homotypic, heterotypic, or heteromeric complexes known as connexons. Data on the expression patterns of members of this family are partial and fragmentary. We therefore cloned all the identifiable murine homologs of human CONNEXIN genes and analyzed their expression patterns in embryonic and neonatal mouse tissues. We found that connexins are preferentially expressed in tissues derived from ectoderm and/or endoderm. Our data provide a comprehensive and detailed atlas of expression of connexin genes and in some cases suggest possible interactions of proteins that are coexpressed in the same tissue. Knowledge of temporal and spatial distribution of connexins also allows the identification of candidate genes for human diseases and provides important insight into mechanisms that lead to human disorders due to mutations in CONNEXIN genes.