RESUMEN
BACKGROUND: Glioblastoma is the most aggressive and most lethal primary brain tumor in the adulthood. Current standard therapies are not curative and novel therapeutic options are urgently required. Present knowledge suggests that the continued glioblastoma growth and recurrence is determined by glioblastoma stem-like cells (GSCs), which display self-renewal, tumorigenic potential, and increased radio- and chemo-resistance. The G-quadruplex ligand RHPS4 displays in vitro radiosensitizing effect in GBM radioresistant cells through the targeting and dysfunctionalization of telomeres but RHPS4 and Ionizing Radiation (IR) combined treatment efficacy in vivo has not been explored so far. METHODS: RHPS4 and IR combined effects were tested in vivo in a heterotopic mice xenograft model and in vitro in stem-like cells derived from U251MG and from four GBM patients. Cell growth assays, cytogenetic analysis, immunoblotting, gene expression and cytofluorimetric analysis were performed in order to characterize the response of differentiated and stem-like cells to RHPS4 and IR in single and combined treatments. RESULTS: RHPS4 administration and IR exposure is very effective in blocking tumor growth in vivo up to 65 days. The tumor volume reduction and the long-term tumor control suggested the targeting of the stem cell compartment. Interestingly, RHPS4 treatment was able to strongly reduce cell proliferation in GSCs but, unexpectedly, did not synergize with IR. Lack of radiosensitization was supported by the GSCs telomeric-resistance observed as the total absence of telomere-involving chromosomal aberrations. Remarkably, RHPS4 treatment determined a strong reduction of CHK1 and RAD51 proteins and transcript levels suggesting that the inhibition of GSCs growth is determined by the impairment of the replication stress (RS) response and DNA repair. CONCLUSIONS: We propose that the potent antiproliferative effect of RHPS4 in GSCs is not determined by telomeric dysfunction but is achieved by the induction of RS and by the concomitant depletion of CHK1 and RAD51, leading to DNA damage and cell death. These data open to novel therapeutic options for the targeting of GSCs, indicating that the combined inhibition of cell-cycle checkpoints and DNA repair proteins provides the most effective means to overcome resistance of GSC to genotoxic insults.
Asunto(s)
Acridinas/administración & dosificación , Neoplasias Encefálicas/terapia , Glioblastoma/terapia , Células Madre Neoplásicas/efectos de los fármacos , Fármacos Sensibilizantes a Radiaciones/administración & dosificación , Acridinas/farmacología , Animales , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/efectos de la radiación , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/genética , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de la radiación , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , Ratones , Recombinasa Rad51/genética , Recombinasa Rad51/metabolismo , Fármacos Sensibilizantes a Radiaciones/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Glioblastoma (GBM) is one of the deadliest human cancers. Because of the extremely unfavorable prognosis of GBM, it is important to develop more effective diagnostic and therapeutic strategies based on biologically and clinically relevant subclassification systems. Analyzing a collection of seventeen patient-derived glioblastoma stem-like cells (GSCs) by gene expression profiling, NMR spectroscopy and signal transduction pathway activation, we identified two GSC clusters, one characterized by a pro-neural-like phenotype and the other showing a mesenchymal-like phenotype. Evaluating the levels of proteins differentially expressed by the two GSC clusters in the TCGA GBM sample collection, we found that SRC activation is associated with a GBM subgroup showing better prognosis whereas activation of RPS6, an effector of mTOR pathway, identifies a subgroup with a worse prognosis. The two clusters are also differentiated by NMR spectroscopy profiles suggesting a potential prognostic stratification based on metabolic evaluation. Our data show that the metabolic/proteomic profile of GSCs is informative of the genomic/proteomic GBM landscape, which differs among tumor subtypes and is associated with clinical outcome.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Glioblastoma/metabolismo , Glioblastoma/mortalidad , Proteínas de Neoplasias/biosíntesis , Células Madre Neoplásicas/metabolismo , Supervivencia sin Enfermedad , Femenino , Humanos , Masculino , Resonancia Magnética Nuclear Biomolecular , Proteómica , Tasa de SupervivenciaRESUMEN
Glioblastoma multiforme (GBM) is the most common and malignant primary brain tumour, with very poor prognosis. The high recurrence rate and failure of conventional treatments are expected to be related to the presence of radio-resistant cancer stem cells (CSCs) inside the tumour mass. CSCs can both self-renew and differentiate into the heterogeneous lineages of cancer cells. Recent evidence showed a higher effectiveness of C-ions and protons in inactivating CSCs, suggesting a potential advantage of Hadrontherapy compared with conventional radiotherapy for GBM treatment. To investigate the mechanisms involved in the molecular and cellular responses of CSCs to ionising radiations, two GBM stem cell (GSC) lines, named lines 1 and 83, which were derived from patients with different clinical outcomes and having different metabolic profiles (as shown by NMR spectroscopy), were irradiated with (137)Cs photons and with protons or C-ions of 62 MeV u(-1) in the dose range of 5-40 Gy. The biological effects investigated were: cell death, cell cycle progression, and DNA damage induction and repair. Preliminary results show a different response to ionising radiation between the two GSC lines for the different end points investigated. Further experiments are in progress to consolidate the data and to get more insights on the influence of radiation quality.
Asunto(s)
Neoplasias Encefálicas/radioterapia , Carbono/uso terapéutico , Radioisótopos de Cesio/uso terapéutico , Glioblastoma/radioterapia , Células Madre Neoplásicas/efectos de la radiación , Terapia de Protones , Radiación Ionizante , Apoptosis/efectos de la radiación , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/mortalidad , Neoplasias Encefálicas/patología , Ciclo Celular/efectos de la radiación , Daño del ADN/efectos de la radiación , Glioblastoma/metabolismo , Glioblastoma/mortalidad , Glioblastoma/patología , Histonas/metabolismo , Humanos , Espectroscopía de Resonancia Magnética , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Pronóstico , Radiobiología , Tasa de Supervivencia , Células Tumorales CultivadasRESUMEN
Glioblastoma (GBM) is the most common and deadly adult brain tumor. Despite aggressive surgery, radiation, and chemotherapy, the life expectancy of patients diagnosed with GBM is â¼14 months. The extremely aggressive nature of GBM results from glioblastoma stem-like cells (GSCs) that sustain GBM growth, survive intensive chemotherapy, and give rise to tumor recurrence. There is accumulating evidence revealing that GSC resilience is because of concomitant activation of multiple survival pathways. In order to decode the signal transduction networks responsible for the malignant properties of GSCs, we analyzed a collection of GSC lines using a dual, but complementary, experimental approach, that is, reverse-phase protein microarrays (RPPMs) and kinase inhibitor library screening. We treated GSCs in vitro with clinically relevant concentrations of temozolomide (TMZ) and performed RPPM to detect changes in phosphorylation patterns that could be associated with resistance. In addition, we screened GSCs in vitro with a library of protein and lipid kinase inhibitors to identify specific targets involved in GSC survival and proliferation. We show that GSCs are relatively insensitive to TMZ treatment in terms of pathway activation and, although displaying heterogeneous individual phospho-proteomic profiles, most GSCs are resistant to specific inhibition of the major signaling pathways involved in cell survival and proliferation. However, simultaneous multipathway inhibition by the staurosporin derivative UCN-01 results in remarkable inhibition of GSC growth in vitro. The activity of UCN-01 on GSCs was confirmed in two in vivo models of GBM growth. Finally, we used RPPM to study the molecular and functional effects of UCN-01 and demonstrated that the sensitivity to UCN-01 correlates with activation of survival signals mediated by PDK1 and the DNA damage response initiated by CHK1. Taken together, our results suggest that a combined inhibition of PDK1 and CHK1 represents a potentially effective therapeutic approach to reduce the growth of human GBM.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias Encefálicas/tratamiento farmacológico , Glioblastoma/tratamiento farmacológico , Células Madre Neoplásicas/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Animales , Neoplasias Encefálicas/enzimología , Neoplasias Encefálicas/patología , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Dacarbazina/análogos & derivados , Dacarbazina/farmacología , Relación Dosis-Respuesta a Droga , Resistencia a Antineoplásicos , Glioblastoma/enzimología , Glioblastoma/patología , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Terapia Molecular Dirigida , Células Madre Neoplásicas/enzimología , Células Madre Neoplásicas/patología , Análisis por Matrices de Proteínas , Proteínas Serina-Treonina Quinasas/metabolismo , Proteómica/métodos , Piruvato Deshidrogenasa Quinasa Acetil-Transferidora , Transducción de Señal/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas , Estaurosporina/análogos & derivados , Estaurosporina/farmacología , Temozolomida , Factores de Tiempo , Carga Tumoral/efectos de los fármacos , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
MicroRNAs (miRNAs) from the gene cluster miR-143-145 are diminished in cells of colorectal tumor origin when compared with normal colon epithelia. Until now, no report has addressed the coordinate action of these miRNAs in colorectal cancer (CRC). In this study, we performed a comprehensive molecular and functional analysis of the miRNA cluster regulatory network. First, we evaluated proliferation, migration, anchorage-independent growth and chemoresistance in the colon tumor cell lines after miR-143 and miR-145 restoration. Then, we assessed the contribution of single genes targeted by miR-143 and miR-145 by reinforcing their expression and checking functional recovery. Restoring miR-143 and miR-145 in colon cancer cells decreases proliferation, migration and chemoresistance. We identified cluster of differentiation 44 (CD44), Kruppel-like factor 5 (KLF5), Kirsten rat sarcoma 2 viral oncogene homolog (KRAS) and v-Raf murine sarcoma viral oncogene homolog B1 (BRAF) as proteins targeted by miR-143 and miR-145. Their re-expression can partially revert a decrease in transformation properties caused by the overexpression of miR-143 and miR-145. In addition, we determined a set of mRNAs that are diminished after reinforcing miR-143 and miR-145 expression. The whole transcriptome analysis ascertained that downregulated transcripts are enriched in predicted target genes in a statistically significant manner. A number of additional genes, whose expression decreases as a direct or indirect consequence of miR-143 and miR-145, reveals a complex regulatory network that affects cell signaling pathways involved in transformation. In conclusion, we identified a coordinated program of gene repression by miR-143 and miR-145, in CRC, where either of the two miRNAs share a target transcript, or where the target transcripts share a common signaling pathway. Major mediators of the oncosuppression by miR-143 and miR-145 are genes belonging to the growth factor receptor-mitogen-activated protein kinase network and to the p53 signaling pathway.
Asunto(s)
Neoplasias Colorrectales/genética , Regulación Neoplásica de la Expresión Génica , MicroARNs/fisiología , Oncogenes , Animales , División Celular/genética , Línea Celular Tumoral , Neoplasias Colorrectales/patología , Redes Reguladoras de Genes , Humanos , Ratones , Reacción en Cadena en Tiempo Real de la Polimerasa , Transcriptoma , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Drug treatment of malignant gliomas is limited by the intrinsic resistance of glioma stem cells (GSCs) to chemotherapy. GSCs isolated from human glioblastoma multiforme (GBM) expressed metabotropic glutamate receptors (mGlu3 receptors). The DNA-alkylating agent, temozolomide, killed GSCs only if mGlu3 receptors were knocked down or pharmacologically inhibited. In contrast, mGlu3 receptor blockade did not affect the action of paclitaxel, etoposide, cis-platinum, and irinotecan. mGlu3 receptor blockade enabled temozolomide toxicity by inhibiting a phosphatidylinositol-3-kinase/nuclear factor-κB pathway that supports the expression of O(6)-methylguanine-DNA methyltransferase (MGMT), an enzyme that confers resistance against DNA-alkylating agents. In mice implanted with GSCs into the brain, temozolomide combined with mGlu3 receptor blockade substantially reduced tumor growth. Finally, 87 patients with GBM undergoing surgery followed by adjuvant chemotherapy with temozolomide survived for longer time if tumor cells expressed low levels of mGlu3 receptors. In addition, the methylation state of the MGMT gene promoter in tumor extracts influenced survival only in those patients with low expression of mGlu3 receptors in the tumor. These data encourage the use of mGlu3 receptor antagonists as add-on drugs in the treatment of GBM, and suggest that the transcript of mGlu3 receptors should be measured in tumor specimens for a correct prediction of patients' survival in response to temozolomide treatment.
Asunto(s)
Glioblastoma/metabolismo , Células Madre Neoplásicas/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Aminoácidos/toxicidad , Animales , Antineoplásicos Alquilantes/farmacología , Antineoplásicos Alquilantes/uso terapéutico , Quimioterapia Adyuvante , Terapia Combinada , Metilación de ADN/efectos de los fármacos , Dacarbazina/análogos & derivados , Dacarbazina/farmacología , Dacarbazina/uso terapéutico , Resistencia a Antineoplásicos/efectos de los fármacos , Glioblastoma/tratamiento farmacológico , Glioblastoma/mortalidad , Humanos , Ratones , FN-kappa B/metabolismo , Células Madre Neoplásicas/citología , O(6)-Metilguanina-ADN Metiltransferasa/genética , O(6)-Metilguanina-ADN Metiltransferasa/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Regiones Promotoras Genéticas , ARN Mensajero/metabolismo , Receptores de Glutamato Metabotrópico/antagonistas & inhibidores , Receptores de Glutamato Metabotrópico/genética , Transducción de Señal , Tasa de Supervivencia , Temozolomida , Trasplante Heterólogo , Células Tumorales Cultivadas , Xantenos/toxicidadRESUMEN
Glioblastoma multiforme (GBM) is among the most aggressive tumor types and is essentially an incurable malignancy characterized by resistance to chemo-, radio-, and immunotherapy. GBM is maintained by a hierarchical cell organization that includes stem-like, precursor, and differentiated cells. Recurrence and maintenance of the tumor is attributed to a small population of undifferentiated tumor-initiating cells, defined as glioblastoma stem-like cells (GSLCs). This cellular hierarchy offers a potential treatment to induce differentiation of GSLCs away from tumor initiation to a more benign phenotype or to a cell type more amenable to standard therapies. Bone morphogenetic proteins (BMPs), members of the TGF-ß superfamily, have numerous biological activities including control of growth and differentiation. In vitro, a BMP7 variant (BMP7v) decreased primary human GSLC proliferation, endothelial cord formation, and stem cell marker expression while enhancing neuronal and astrocyte differentiation marker expression. In subcutaneous and orthotopic GSLC xenografts, which closely reproduce the human disease, BMP7v decreased tumor growth and stem cell marker expression, while enhancing astrocyte and neuronal differentiation compared with control mice. In addition, BMP7v reduced brain invasion, angiogenesis, and associated mortality in the orthotopic model. Inducing differentiation of GSLCs and inhibiting angiogenesis with BMP7v provides a potentially powerful and novel approach to the treatment of GBM.
Asunto(s)
Proteína Morfogenética Ósea 7/farmacología , Células Madre Neoplásicas/metabolismo , Animales , Proteína Morfogenética Ósea 7/genética , Proteína Morfogenética Ósea 7/uso terapéutico , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Citocinas/metabolismo , Glioblastoma/tratamiento farmacológico , Glioblastoma/metabolismo , Glioblastoma/patología , Células HCT116 , Humanos , Ratones , Células Madre Neoplásicas/efectos de los fármacos , Neovascularización Patológica , Trasplante HeterólogoRESUMEN
Neural stem cells (NSCs) isolated from the subventricular zone (SVZ) of postnatal mice, and cultured as neurospheres, expressed functional mGlu3 receptors. Following mitogen withdrawal and plating onto poly-ornitine-coated dishes, cells dissociated from the neurospheres differentiated into GFAP(+) astrocytes (about 85%), and a small percentage of beta-III tubulin(+)-neurons and O1(+)-oligodendrocytes. Activation of mGlu3 receptors with LY379268 (100 nM, applied every other day), during the differentiation period, impaired astrocyte differentiation, favoring the maintenance in culture of proliferating progenitors co-expressing GFAP with the immature markers, Sox1 and nestin. Co-treatment with the preferential mGlu2/3 receptor antagonist, LY341495 (100 nM), reversed this effect. We examined whether mGlu3 receptors could modulate the canonical signaling pathway activated by bone morphogenic proteins (BMPs), which are known to promote astrocyte differentiation of SVZ/NSCs. An acute challenge of cells isolated from the neurospheres with BMP4 (100 ng/mL) led to phosphorylation and nuclear translocation of the transcription factors, Smads. This effect was largely attenuated by the mGlu2/3 receptor agonist, LY379268. The interaction of mGlu3 and BMP4 receptors was mediated by the activation of the mitogen-activated protein kinase (MAPK) pathway. Accordingly, LY379268 failed to affect BMP receptor signaling when combined with the MAPK kinase inhibitor, UO-126 (30 muM). These data raise the intriguing possibility that glutamate regulates differentiation of SVZ/NSCs by activating mGlu3 receptors.
Asunto(s)
Astrocitos/metabolismo , Diferenciación Celular/fisiología , Linaje de la Célula/fisiología , Receptores de Glutamato Metabotrópico/metabolismo , Células Madre/metabolismo , Telencéfalo/metabolismo , Animales , Animales Recién Nacidos , Astrocitos/citología , Proteína Morfogenética Ósea 4/metabolismo , Proteína Morfogenética Ósea 4/farmacología , Receptores de Proteínas Morfogenéticas Óseas/agonistas , Receptores de Proteínas Morfogenéticas Óseas/metabolismo , Células Cultivadas , Agonistas de Aminoácidos Excitadores/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Proteína Ácida Fibrilar de la Glía/metabolismo , Ácido Glutámico/metabolismo , Proteínas de Filamentos Intermediarios/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/fisiología , Ratones , Proteínas del Tejido Nervioso/metabolismo , Nestina , Factores de Transcripción SOXB1/metabolismo , Proteínas Smad/metabolismo , Esferoides Celulares , Células Madre/citología , Telencéfalo/citologíaRESUMEN
Targeted-therapies enhancing differentiation of glioma-initiating cells (GICs) are potential innovative approaches to the treatment of malignant gliomas. These cells support tumour growth and recurrence and are resistant to radiotherapy and chemotherapy. We have found that GICs express mGlu3 metabotropic glutamate receptors. Activation of these receptors sustained the undifferentiated state of GICs in culture by negatively modulating the action of bone morphogenetic proteins, which physiologically signal through the phosphorylation of the transcription factors, Smads. The cross-talk between mGlu3 receptors and BMP receptors was mediated by the activation of the mitogen-activated protein kinase pathway. Remarkably, pharmacological blockade of mGlu3 receptors stimulated the differentiation of cultured GICs into astrocytes, an effect that appeared to be long lasting, independent of the growth conditions, and irreversible. In in vivo experiments, a 3-month treatment with the brain-permeant mGlu receptor antagonist, LY341495 limited the growth of infiltrating brain tumours originating from GICs implanted into the brain parenchyma of nude mice. While clusters of tumour cells were consistently found in the brain of control mice, they were virtually absent in a large proportion of mice treated with LY341495. These findings pave the way to a new non-cytotoxic treatment of malignant gliomas based on the use of mGlu3 receptor antagonists.
Asunto(s)
Receptores de Proteínas Morfogenéticas Óseas/metabolismo , Neoplasias Encefálicas/metabolismo , Glioma/metabolismo , Receptores de Glutamato Metabotrópico/fisiología , Transducción de Señal/fisiología , Aminoácidos/farmacología , Animales , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/fisiopatología , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/farmacología , Agonistas de Aminoácidos Excitadores/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Glioma/tratamiento farmacológico , Glioma/patología , Glioma/fisiopatología , Humanos , Imagen por Resonancia Magnética , Ratones , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Fosforilación/efectos de los fármacos , Receptores de Glutamato Metabotrópico/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Xantenos/farmacologíaRESUMEN
Glioblastoma multiforme is a severe form of cancer most likely arising from the transformation of stem or progenitor cells resident in the brain. Although the tumorigenic population in glioblastoma is defined as composed by cancer stem cells (CSCs), the cellular target of the transformation hit remains to be identified. Glioma stem cells (SCs) are thought to have a differentiation potential restricted to the neural lineage. However, using orthotopic versus heterotopic xenograft models and in vitro differentiation assays, we found that a subset of glioblastomas contained CSCs with both neural and mesenchymal potential. Subcutaneous injection of CSCs or single CSC clones from two of seven patients produced tumor xenografts containing osteo-chondrogenic areas in the context of glioblastoma-like tumor lesions. Moreover, CSC clones from four of seven cases generated both neural and chondrogenic cells in vitro. Interestingly, mesenchymal differentiation of the tumor xenografts was associated with reduction of both growth rate and mitotic index. These findings suggest that in a subclass of glioblastomas the tumorigenic hit occurs on a multipotent stem cell, which may reveal its plasticity under specific environmental stimuli. The discovery of such biological properties might provide considerable information to the development of new therapeutic strategies aimed at forcing glioblastoma stem cell differentiation.
Asunto(s)
Neoplasias Encefálicas/patología , Glioblastoma/patología , Mesodermo/citología , Células Madre Neoplásicas/citología , Adulto , Anciano , Animales , Diferenciación Celular , Células Clonales , Femenino , Humanos , Masculino , Ratones , Ratones SCID , Persona de Mediana Edad , Células Madre Neoplásicas/química , Células Madre Neoplásicas/patología , Neuronas/citología , Ensayos Antitumor por Modelo de XenoinjertoAsunto(s)
Neoplasias del Colon/patología , Células Madre Neoplásicas/patología , Adenoma/patología , Células Madre Adultas/patología , Animales , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Neoplasias del Colon/diagnóstico , Neoplasias del Colon/terapia , Modelos Animales de Enfermedad , Humanos , RatonesRESUMEN
Cancer is generally characterized by loss of CG dinucleotides methylation resulting in a global hypomethylation and the consequent genomic instability. The major contribution to the general decreased methylation levels seems to be due to demethylation of heterochromatin repetitive DNA sequences. In human immunodeficiency, centromeric instability and facial anomalies syndrome, demethylation of pericentromeric satellite 2 DNA sequences has been correlated to functional mutations of the de novo DNA methyltransferase 3b (DNMT3b), but the mechanism responsible for the hypomethylated status in tumors is poorly known. Here, we report that human glioblastoma is affected by strong hypomethylation of satellite 2 pericentromeric sequences that involves the stem cell compartment. Concomitantly with the integrity of the DNMTs coding sequences, we report aberrations in DNA methyltrasferases expression showing upregulation of the DNA methyltransferase 1 (DNMT1) and downregulation of the de novo DNA methyltransferase 3a (DNMT3a). Moreover, we show that DNMT3a is the major de novo methyltransferase expressed in normal neural progenitor cells (NPCs) and its forced re-expression is sufficient to partially recover the methylation levels of satellite 2 repeats in glioblastoma cell lines. Thus, we speculate that DNMT3a decreased expression may be involved in the early post-natal inheritance of an epigenetically altered NPC population that could be responsible for glioblastoma development later in adult life.
Asunto(s)
Neoplasias Encefálicas/genética , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Metilación de ADN , Epigénesis Genética , Glioblastoma/genética , Células Madre Neoplásicas/enzimología , Neoplasias Encefálicas/enzimología , Línea Celular Tumoral , ADN (Citosina-5-)-Metiltransferasas/genética , ADN Metiltransferasa 3A , ADN Satélite/metabolismo , Glioblastoma/enzimología , Humanos , Neuronas/enzimologíaAsunto(s)
Enfermedades Autoinmunes Desmielinizantes SNC/inmunología , Enfermedades Autoinmunes Desmielinizantes SNC/metabolismo , Inflamación/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Oligodendroglía/metabolismo , Células Madre/metabolismo , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/metabolismo , Animales , Células Cultivadas , Citotoxicidad Inmunológica , Enfermedades Autoinmunes Desmielinizantes SNC/patología , Humanos , Inflamación/inmunología , Inflamación/patología , Ratones , Oligodendroglía/patología , Células Madre/patología , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/antagonistas & inhibidoresAsunto(s)
Antineoplásicos/farmacología , Resistencia a Múltiples Medicamentos , Resistencia a Antineoplásicos , Células Madre Neoplásicas/efectos de los fármacos , Animales , Antineoplásicos/uso terapéutico , Apoptosis/efectos de los fármacos , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/patología , Cisplatino/farmacología , Cisplatino/uso terapéutico , Doxorrubicina/farmacología , Doxorrubicina/uso terapéutico , Etopósido/farmacología , Etopósido/uso terapéutico , Glioblastoma/tratamiento farmacológico , Glioblastoma/patología , Humanos , Células Jurkat , Ratones , Ratones Desnudos , Células Madre Neoplásicas/patología , Factores de Tiempo , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The use of neural progenitor cells (NPCs) is limited by the incomplete knowledge of the extracellular signals regulating their proliferation and survival. We report that cultured mouse NPCs express functional mGlu3 and mGlu5 metabotropic glutamate receptors. Pharmacological blockade of both receptors reduced NPC proliferation and survival, whereas activation of mGlu5 receptors substantially enhanced cell proliferation. Adult mice lacking mGlu5 receptors or treated with mGlu5 or mGlu3 receptor antagonists showed a dramatic reduction in the number of dividing neuroprogenitors present in the subventricular zone and in the dentate gyrus of the hippocampus. These data disclose a novel function of mGlu receptors and offer new potential strategies for the optimization of cell replacement therapy in neurodegenerative disorders.
Asunto(s)
Neuronas/citología , Receptores de Glutamato Metabotrópico/fisiología , Células Madre/citología , Animales , Western Blotting , Ciclo Celular/fisiología , Procesos de Crecimiento Celular/fisiología , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Inmunohistoquímica , Ratones , Ratones Noqueados , Neuronas/metabolismo , Prosencéfalo/citología , Prosencéfalo/metabolismo , Receptor del Glutamato Metabotropico 5 , Receptores de Glutamato Metabotrópico/antagonistas & inhibidores , Receptores de Glutamato Metabotrópico/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Madre/metabolismoRESUMEN
Exposure of immature rat cerebellar granule cell cultures to the type 4 metabotropic glutamate (mGlu4) receptor enhancer N-phenyl-7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxamide (PHCCC) reduced [3H]thymidine incorporation. Its action was sensitive to the growth conditions and was attenuated by two mGlu4 receptor antagonists. An antiproliferative action of PHCCC was also seen in cultures from wild-type, but not mGlu4, knock-out mice. At least in rat cultures, PHCCC was not neurotoxic and enhanced neuritogenesis. Although PHCCC reduced the increase in cAMP formation and phospho-AKT levels induced by forskolin, none of these transduction pathways significantly contributed to the reduction of [3H]thymidine incorporation. Interestingly, PHCCC reduced the expression of Gli-1, a transcription factor that mediates the mitogenic action of Sonic hedgehog. Finally, we treated newborn rats with PHCCC either intracerebrally (infusion of 5 nmol/2 microl in the cerebellar region once every other day) or systemically (5 mg/kg, i.p., once daily) from postnatal days 3-9. Local infusion of PHCCC induced substantial changes in the morphology of the developing cerebellum. In contrast, systemic injection of PHCCC induced only morphological abnormalities of the cerebellar lobule V, which became visible 11 d after the end of the treatment. These data suggest that mGlu4 receptors are involved in the regulation of cerebellar development.
Asunto(s)
Benzopiranos/farmacología , Cerebelo/citología , Neuronas/citología , Receptores de Glutamato Metabotrópico/agonistas , Células Madre/citología , Animales , Animales Recién Nacidos , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Cerebelo/efectos de los fármacos , Cerebelo/crecimiento & desarrollo , AMP Cíclico/biosíntesis , Depresión Química , Ratones , Ratones Noqueados , Neuritas/efectos de los fármacos , Neuritas/fisiología , Neuronas/efectos de los fármacos , Tamaño de los Órganos/efectos de los fármacos , Fosforilación , Proteínas Serina-Treonina Quinasas/biosíntesis , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas c-akt , Ratas , Ratas Sprague-Dawley , Receptores de Glutamato Metabotrópico/antagonistas & inhibidores , Receptores de Glutamato Metabotrópico/genética , Transducción de Señal , Células Madre/efectos de los fármacos , Timidina/metabolismoRESUMEN
Metabotropic glutamate (mGlu) receptors have been implicated in the regulation of developmental plasticity. Here, we examined the expression of mGlu1a-b, -2, -3, -4a-b, and -5a receptor subtypes from embryonic day 12 (E12) to the early and late postnatal life. While all transcripts (with the exception of mGlu4 mRNA) were detected prenatally, only the mGlu5 receptor protein was found in detectable amounts in the embryonic brain. Immunohistochemical analysis showed that the mGlu5 receptor was mainly expressed by cells surrounding the ventricles at E15, whereas it was more diffusely expressed at E18. In the postnatal life, besides its classical expression sites, the mGlu5 receptor was found in zones of active neurogenesis such as the external granular layer (EGL) of the cerebellar cortex and the subventricular zone. In these regions, the presence of actively proliferating progenitor cells was detected by BrdU staining. No other subtype (among those we have examined) was found to be expressed in regions enriched of BrdU(+) cells. These data suggest a role for mGlu5 receptors in the early brain development and in basic cellular processes such as proliferation and/or differentiation.
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Encéfalo/embriología , Encéfalo/crecimiento & desarrollo , Diferenciación Celular/genética , Neuronas/metabolismo , Receptores de Glutamato Metabotrópico/genética , Células Madre/metabolismo , Animales , Animales Recién Nacidos , Encéfalo/citología , Bromodesoxiuridina , División Celular/genética , Femenino , Feto , Regulación del Desarrollo de la Expresión Génica/genética , Ácido Glutámico/metabolismo , Masculino , Plasticidad Neuronal/genética , Neuronas/citología , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Receptor del Glutamato Metabotropico 5 , Receptores de Glutamato Metabotrópico/biosíntesis , Células Madre/citologíaRESUMEN
Hashimoto's thyroiditis is a common chronic autoimmune disease characterized by the loss of thyroid follicular cells (thyrocytes) that are gradually replaced by lymphocytic infiltration and diffuse fibrosis. These morphological findings suggested that autoreactive T-cell clones were responsible for thyrocyte destruction and hypothyroidism through effector-target cytotoxic recognition. Later, autonomous interaction between thyrocyte Fas and FasL has been proposed as a major mechanism of thyrocyte depletion in Hashimoto's thyroiditis. Here, we analyze the possible role of Fas and FasL in the pathogenesis of Hashimoto's thyroiditis. We suggest that the Fas-FasL system dictates the outcome of the autoimmune response by acting on both immune and target cells.
Asunto(s)
Glicoproteínas de Membrana/fisiología , Tiroiditis Autoinmune/etiología , Receptor fas/fisiología , Apoptosis , Proteína Ligando Fas , HumanosRESUMEN
OBJECTIVE: We investigated the distribution of MICA triplet repeat polymorphism in a random population and in patients with seronegative spondyloarthritis from Sardinia compared to continental Italy. METHODS: We analyzed the distribution of MICA triplet repeat polymorphism in HLA-B*2709 [not associated with ankylosing spondylitis (AS)] and B*2705 (associated with AS) haplotypes, to verify whether the strong association of MICA-A4 with HLA-B27 reported in other populations is maintained in Sardinia, and compared the distribution of MICA-A alleles in HLA-B27 negative versus HLA-B27+ patients with AS. RESULTS: We found that the frequency of MICA-A4 triplet repeat allele in a random Sardinia population is higher (53.2%) than in other Caucasian populations (around 20%); this allele is strongly associated with both HLA-B*2709 and B*2705. No significant difference between HLA-B27+ patients with AS and healthy controls was found: the MICA-A4 allele was present in more than 90% of subjects. MICA-A4 was found in 16 out of 20 HLA-B27 negative Sardinian patients with AS, with a frequency (80%) more similar to that of the HLA-B27+ group of patients than that of controls. CONCLUSION: The high frequency of MICA-A4 allele in HLA-B27 negative patients with AS from Sardinia, suggests the presence within the HLA region of a susceptibility factor other and certainly weaker than B27. This factor is likely to be more easily found by analyzing genetically homogeneous populations like the Sardinian, characterized by a small number of very frequent haplotypes.