RESUMEN
Diffuse intrinsic pontine glioma (DIPG) is an incurable paediatric malignancy. Identifying the molecular drivers of DIPG progression is of the utmost importance. Long non-coding RNAs (lncRNAs) represent a large family of disease- and tissue-specific transcripts, whose functions have not yet been elucidated in DIPG. Herein, we studied the oncogenic role of the development-associated H19 lncRNA in DIPG. Bioinformatic analyses of clinical datasets were used to measure the expression of H19 lncRNA in paediatric high-grade gliomas (pedHGGs). The expression and sub-cellular location of H19 lncRNA were validated in DIPG cell lines. Locked nucleic acid antisense oligonucleotides were designed to test the function of H19 in DIPG cells. We found that H19 expression was higher in DIPG vs. normal brain tissue and other pedHGGs. H19 knockdown resulted in decreased cell proliferation and survival in DIPG cells. Mechanistically, H19 buffers let-7 microRNAs, resulting in the up-regulation of oncogenic let-7 target (e.g., SULF2 and OSMR). H19 is the first functionally characterized lncRNA in DIPG and a promising therapeutic candidate for treating this incurable cancer.
Asunto(s)
Neoplasias del Tronco Encefálico/genética , Proliferación Celular , Glioma/genética , ARN Largo no Codificante/metabolismo , Neoplasias del Tronco Encefálico/metabolismo , Neoplasias del Tronco Encefálico/patología , Células Cultivadas , Regulación Neoplásica de la Expresión Génica , Glioma/metabolismo , Glioma/patología , Histonas/metabolismo , Humanos , MicroARNs/genética , MicroARNs/metabolismo , Mutación , ARN Largo no Codificante/genéticaRESUMEN
The blood-brain barrier (BBB) consists of endothelial cells, astrocytes, and pericytes embedded in basal lamina (BL). Most in vitro models use nonhuman, monolayer cultures for therapeutic-delivery studies, relying on transendothelial electrical resistance (TEER) measurements without other tight-junction (TJ) formation parameters. We aimed to develop reliable, reproducible, in vitro 3-dimensional (3D) models incorporating relevant human, in vivo cell types and BL proteins. The 3D BBB models were constructed with human brain endothelial cells, human astrocytes, and human brain pericytes in mono-, co-, and tricultures. TEER was measured in 3D models using a volt/ohmmeter and cellZscope. Influence of BL proteins-laminin, fibronectin, collagen type IV, agrin, and perlecan-on adhesion and TEER was assessed using an electric cell-substrate impedance-sensing system. TJ protein expression was assessed by Western blotting (WB) and immunocytochemistry (ICC). Perlecan (10 µg/ml) evoked unreportedly high, in vitro TEER values (1200 Ω) and the strongest adhesion. Coculturing endothelial cells with astrocytes yielded the greatest resistance over time. ICC and WB results correlated with resistance levels, with evidence of prominent occludin expression in cocultures. BL proteins exerted differential effects on TEER, whereas astrocytes in contact yielded higher TEER values and TJ expression.-Maherally, Z., Fillmore, H. L., Tan, S. L., Tan, S. F., Jassam, S. A., Quack, F. I., Hatherell, K. E., Pilkington, G. J. Real-time acquisition of transendothelial electrical resistance in an all-human, in vitro, 3-dimensional, blood-brain barrier model exemplifies tight-junction integrity.
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Barrera Hematoencefálica/metabolismo , Uniones Estrechas/metabolismo , Agrina/metabolismo , Astrocitos/citología , Astrocitos/metabolismo , Barrera Hematoencefálica/citología , Línea Celular , Técnicas de Cocultivo , Sistemas de Computación , Impedancia Eléctrica , Células Endoteliales/citología , Células Endoteliales/metabolismo , Proteoglicanos de Heparán Sulfato/metabolismo , Humanos , Imagenología Tridimensional , Modelos Biológicos , Modelos Neurológicos , Pericitos/metabolismoRESUMEN
PURPOSE: Metastatic non-small cell lung (NSCLC) cancer represents one of the most common types of brain metastasis. The mechanisms involved in how circulating cancer cells transmigrate into brain parenchyma are not fully understood. The aim of this work was to investigate the role of fucosylated carbohydrate epitopes CD15 and sialyated CD15s in cancer adhesion to brain-derived endothelial cells and determine their influence in blood-brain barrier (BBB) disruption METHODS: Three distinct, independent methods were used to measure brain endothelial integrity and include voltohmmeter (EVOM™), impedance spectroscopy (CellZscope®) and electric cell-substrate impedance sensing system (ECIS™). Two fucosyltransferases (FUT4 and 7) responsible for CD15 and CD15s synthesis were modulated in four human cancer cell lines (three lung cancer and one glioma). RESULTS: Overexpression of CD15 or CD15s epitopes led to increase in adhesion of cancer cells to cerebral endothelial cells compared with wild-type and cells with silenced CD15 or CD15s (p < 0.01). This overexpression led to the disruption of cerebral endothelial cell monolayers (p < 0.01). Knockdown of FUT4 and FUT7 in metastatic cancer cells prevented disruption of an in vitro BBB model. Surprisingly, although the cells characterised as 'non-metastatic', they became 'metastatic' -like when cells were forced to over-express either FUT4 or FUT7. CONCLUSIONS: Results from these studies suggest that overexpression of CD15 and CD15s could potentiate the transmigration of circulating NSCLC cells into the brain. The clinical significance of these studies includes the possible use of these epitopes as biomarkers for metastasis.
Asunto(s)
Barrera Hematoencefálica/patología , Neoplasias Encefálicas/secundario , Carcinoma de Pulmón de Células no Pequeñas/patología , Adhesión Celular , Células Endoteliales/patología , Fucosiltransferasas/metabolismo , Neoplasias Pulmonares/secundario , Barrera Hematoencefálica/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Movimiento Celular , Células Endoteliales/metabolismo , Fucosiltransferasas/genética , Humanos , Antígeno Lewis X/genética , Antígeno Lewis X/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Células Tumorales CultivadasRESUMEN
Expression of the cell adhesion molecule (CAM), Sialyl Lewis X (CD15s) correlates with cancer metastasis, while expression of E-selectin (CD62E) is stimulated by TNF-α. CD15s/CD62E interaction plays a key role in the homing process of circulating leukocytes. We investigated the heterophilic interaction of CD15s and CD62E in brain metastasis-related cancer cell adhesion. CD15s and CD62E were characterised in human brain endothelium (hCMEC/D3), primary non-small cell lung cancer (NSCLC) (COR-L105 and A549) and metastatic NSCLC (SEBTA-001 and NCI-H1299) using immunocytochemistry, Western blotting, flow cytometry and immunohistochemistry in human brain tissue sections. TNF-α (25 pg/mL) stimulated extracellular expression of CD62E while adhesion assays, under both static and physiological flow live-cell conditions, explored the effect of CD15s-mAb immunoblocking on adhesion of cancer cell-brain endothelium. CD15s was faintly expressed on hCMEC/D3, while high levels were observed on primary NSCLC cells with expression highest on metastatic NSCLC cells (p < 0.001). CD62E was highly expressed on hCMEC/D3 cells activated with TNF-α, with lower levels on primary and metastatic NSCLC cells. CD15s and CD62E were expressed on lung metastatic brain biopsies. CD15s/CD62E interaction was localised at adhesion sites of cancer cell-brain endothelium. CD15s immunoblocking significantly decreased cancer cell adhesion to brain endothelium under static and shear stress conditions (p < 0.001), highlighting the role of CD15s-CD62E interaction in brain metastasis.
Asunto(s)
Neoplasias Encefálicas/etiología , Neoplasias Encefálicas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Selectina E/metabolismo , Células Endoteliales/metabolismo , Antígeno Lewis X/metabolismo , Neoplasias Pulmonares/complicaciones , Neoplasias Pulmonares/metabolismo , Adhesión Celular/genética , Adhesión Celular/fisiología , Línea Celular , Supervivencia Celular/genética , Supervivencia Celular/fisiología , Selectina E/genética , Humanos , Inmunohistoquímica , Antígeno Lewis X/genética , Microscopía Confocal , Metástasis de la Neoplasia/genética , Metástasis de la Neoplasia/fisiopatología , Resistencia al Corte/fisiología , Antígeno Sialil Lewis XRESUMEN
Glioblastoma multiforme (GBM) is the most common type of primary brain tumor, and current treatment regimens are only marginally effective. One of the most vexing and malignant aspects of GBM is its pervasive infiltration into surrounding brain tissue. This review describes the role of the Wilms tumor 1 gene (WT1) and its relationship to GBM. WT1 has several alternative splicing products, one of which, the KTS(+) variant, has been demonstrated to be involved in the transcriptional activation of a variety of oncogenes as well as the inhibition of tumor suppressor genes. Further, this paper will examine the relationship of WT1 with CD97, a gene that codes for an epidermal growth factor receptor family member, an adhesion G-protein-coupled receptor, thought to promote tumor invasiveness and migration. The authors suggest that further research into WT1 and CD97 will allow clinicians to begin to deal more effectively with the infiltrative behavior displayed by GBM and design new therapies that target this deadly disease.
Asunto(s)
Antígenos CD/genética , Neoplasias Encefálicas/genética , Glioblastoma/genética , Tumor de Wilms/genética , Antígenos CD/metabolismo , Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Humanos , Receptores Acoplados a Proteínas G , Tumor de Wilms/metabolismoRESUMEN
Herein we continue the study of matrix metalloproteinase-1 (MMP-1) with respect to glioblastoma multiforme (GBM) cell tumorigenicity and angiogenesis. A model of tumorigenicity with cells stably altered to over-express or knock-down MMP-1 revealed that it significantly increases tumor incidence and size. Organized endothelial growth in human umbilical vein endothelial cell (HUVEC)-GBM co-cultures was significantly increased in the presence of MMP-1. CD31 analysis of model tumors elucidated a substantial recruitment of endothelium in MMP-1 enhanced samples. Antibody arrays indicated an inverse expression of certain anti-angiogenic factors with respect to MMP-1, the most notable of which was a significant increase in tissue inhibitor of metalloproteinases-4 (TIMP-4) in the absence of MMP-1, as validated by immunoblot.
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Neoplasias Encefálicas/genética , Regulación Neoplásica de la Expresión Génica/genética , Glioblastoma/genética , Metaloproteinasa 1 de la Matriz/genética , Inhibidores Tisulares de Metaloproteinasas/metabolismo , Animales , Línea Celular Tumoral , Transformación Celular Neoplásica/genética , Técnicas de Cocultivo , Modelos Animales de Enfermedad , Células Endoteliales/patología , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Metaloproteinasa 1 de la Matriz/metabolismo , Ratones , Ratones Desnudos , Mutación/genética , Neovascularización Patológica/genética , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Proteómica , Inhibidores Tisulares de Metaloproteinasas/genética , Venas Umbilicales/citología , Inhibidor Tisular de Metaloproteinasa-4RESUMEN
Brain metastases are the most common fatal complication of systemic cancer, especially of lung (40-50%) and breast (20-30%) cancers. In this era of personalized therapy, there is a critical need to uncover the signaling architecture of brain metastases; however, little is known about what signaling pathways are activated in the context of the brain microenvironment. Using a unique study set of 42 brain metastases from patients with breast or nonsmall cell lung cancer (NSCLC), the phosphorylation/activation states of 128 key signaling proteins involved in cancer signaling were measured in laser capture microdissected tumor epithelium using reverse phase protein microarray (RPMA) technology. Distinct pathway activation subgroups from both breast and lung metastases were underpinned by, among others, ERBB2, AKT, mTOR, EGFR, SMAD, and ERK-p38 signaling. Breast cancer metastases showed significantly (p < 0.05) higher activation of the c-ERBB2/IGFR-AKT pathway network compared to NSCLC metastases, whereas NSCLC metastases to the brain exhibited higher relative levels of many members of the EGFR-ERK signaling network. Protein pathway activation mapping using RPMA revealed both the heterogeneity of signaling networks in brain metastases that would require a prior stratification to targeted therapies as well as the requirement of direct analysis of the metastatic lesion.
Asunto(s)
Neoplasias Encefálicas/genética , Neoplasias de la Mama/genética , Carcinoma de Pulmón de Células no Pequeñas/genética , Neoplasias Pulmonares/genética , Análisis por Matrices de Proteínas/métodos , Mapeo de Interacción de Proteínas/métodos , Proteómica/métodos , Transducción de Señal , Biomarcadores de Tumor/análisis , Biomarcadores de Tumor/genética , Encéfalo/metabolismo , Encéfalo/patología , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/secundario , Mama/metabolismo , Mama/patología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Femenino , Humanos , Rayos Láser , Pulmón/metabolismo , Pulmón/patología , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Redes y Vías Metabólicas , Microdisección , Terapia Molecular Dirigida , Especificidad de Órganos , Fosforilación , Medicina de PrecisiónRESUMEN
Wilms' tumor 1 (WT1) is a transcription factor with a multitude of downstream targets that have wide-ranging effects in non-glioma cell lines. Though its expression in glioblastomas is now well-documented, the role of WT1 in these tumors remains poorly defined. We hypothesized that WT1 functions as an oncogene to enhance glioblastoma viability and chemoresistance. WT1's role was examined by studying the effect of WT1 silencing and overexpression on DNA damage, apoptosis and cell viability. Results indicated that WT1 silencing adversely affected glioblastoma viability, at times, in synergy with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and cisplatin. To investigate other mechanisms through which WT1 could affect viability, we measured cell cycle distribution, senescence, and autophagy. WT1 silencing had no effect on these processes. Lastly, we examined WT1 regulation of IGF-1R expression. Counterintuitively, upregulation of IGF-1R was evident after WT1 silencing. In conclusion, WT1 functions as a survival factor in glioblastomas, possibly through inhibition of IGF-1R expression.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Neoplasias Encefálicas/patología , Proliferación Celular/efectos de los fármacos , Resistencia a Antineoplásicos , Silenciador del Gen/efectos de los fármacos , Glioblastoma/patología , Receptor IGF Tipo 1/metabolismo , Proteínas WT1/genética , Apoptosis/efectos de los fármacos , Western Blotting , Neoplasias Encefálicas/genética , Carmustina/administración & dosificación , Ciclo Celular/efectos de los fármacos , Cisplatino/administración & dosificación , Regulación Neoplásica de la Expresión Génica , Glioblastoma/genética , Humanos , Técnicas In Vitro , Regiones Promotoras Genéticas/genética , ARN Interferente Pequeño/genética , Receptor IGF Tipo 1/genética , Células Tumorales CultivadasRESUMEN
In this communication, we describe the successful encapsulation of (177)Lu into the endohedral metallofullerene (177)Lu(x)Lu(3-x)N@C(80) (x = 1-3) starting with (177)LuCl(3) in a modified quartz Kraschmer-Huffman electric generator. We demonstrate that the (177)Lu (beta-emitter) in this fullerene cage is not significantly released for a period of up to at least one-half-life (6.7 days). We also demonstrate that this agent can be conjugated with an interleukin-13 peptide that is designed to target an overexpressed receptor in glioblastoma multiforme tumors. This nanoparticle delivery platform provides flexibility for a wide range of radiotherapeutic and radiodiagnostic multimodal applications.
Asunto(s)
Fulerenos/química , Interleucina-13/química , Lutecio/química , Radioisótopos/química , Marcaje IsotópicoRESUMEN
High grade gliomas invariably recur due in a large part to tumor cells permeating normal brain in an inaccessible, diffuse manner. Previous work demonstrates that the expression of matrix metalloproteinases (MMP) contributes to this characteristic. Not only can MMPs assist a cell in traversing its environment by clearing extracellular matrix molecules, but they can also impact non-traditional downstream signals that affect a cell's ability to interact and respond to its surroundings. Contributions to the induction of MMP expression and functional significance in glioma are still under investigation. Evidence in other cancer settings indicates that nitric oxide (NO) may play a role in tumor/cell progression that can influence MMP production. Matrix metalloproteinase-1 (MMP-1), also known as interstitial collagenase, and the constitutive nitric oxide synthases (NOS) have been shown to be over-expressed in high grade gliomas. In the current study we investigated the potential involvements of NO with regard to MMP-1 and functional glioma cell movement. With the treatment of the NO donor sodium nitroprusside (SNP), there was significant induction of MMP-1 mRNA, secreted MMP-1 protein and motility of glioma cell lines within 48 h. RNA inhibition of MMP-1 through transient transfection of three MMP-1 specific siRNAs revealed a marked abrogation of the NO-mediated induction of motility. In addition, application of the NOS inhibitor N(omega)-Nitro-L-arginine methyl ester (L-NAME) impaired movement of glioma cells. These data provide evidence for a regulatory axis of high grade glioma cell movement from NO through MMP-1, with NOS inhibitor results showing promise for future pharmacologic investigation.
Asunto(s)
Movimiento Celular/fisiología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glioma/metabolismo , Glioma/fisiopatología , Metaloproteinasa 1 de la Matriz/metabolismo , Óxido Nítrico/metabolismo , Análisis de Varianza , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Humanos , Metaloproteinasa 1 de la Matriz/genética , NG-Nitroarginina Metil Éster/farmacología , Donantes de Óxido Nítrico/farmacología , Nitroprusiato/farmacología , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Transfección/métodosRESUMEN
Glioblastoma multiforme (GBM) is one of the most highly vascularized of all human tumors. Our objective was to characterize a 3-dimensional (3-D) in vitro angiogenesis model by co-culturing HUVEC and GBM cells, and to study the role of VEGF in mediating capillary tubule formation in this model. HUVEC-coated dextran beads were suspended in fibrin gel with human glioma cells on top. The number of sprouts and the length of the processes were measured. HUVEC can be induced to form sprouts and longer processes with lumens, in co-culture with glioma cells that secrete VEGF. Addition of exogenous VEGF enhances this effect. In the absence of glioma cells, many single HUVEC migrate away from the beads, without significant tubule formation. Hypoxia further stimulated sprout formation by 50-100%. Anti-VEGF neutralizing antibody suppressed HUVEC sprouting by 75% in co-culture with glioma cells. This 3-D in vitro co-culture system provides a robust and useful model for analysis of the major steps of glioma-induced angiogenesis.
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Neoplasias Encefálicas/patología , Células Endoteliales/citología , Glioblastoma/patología , Neovascularización Patológica/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Western Blotting , Neoplasias Encefálicas/metabolismo , Hipoxia de la Célula/fisiología , Línea Celular Tumoral , Técnicas de Cocultivo/métodos , Células Endoteliales/metabolismo , Glioblastoma/metabolismo , Humanos , Venas Umbilicales/citologíaRESUMEN
Medulloblastoma (MB), the most common malignant paediatric brain tumour occurs in the cerebellum. Advances in molecular genomics have led to the identification of defined subgroups which are associated with distinct clinical prognoses. Despite this classification, standard therapies for all subgroups often leave children with life-long neurological deficits. New therapeutic approaches are therefore urgently needed to reduce current treatment toxicity and increase survival for patients. GD3 is a well-studied ganglioside which is known to have roles in the development of the cerebellum. Post-partum GD3 is not highly expressed in the brain. In some cancers however GD3 is highly expressed. In MB cells GD3 is largely acetylated to GD3A. GD3 is pro-apoptotic but GD3A can protect cells from apoptosis. Presence of these gangliosides has previously been shown to correlate with resistance to chemotherapy. Here we show that the GD3 acetylation pathway is dysregulated in MB and as a proof-of-principle we show that increased GD3 expression sensitises an MB cell line to etoposide.
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Acetilesterasa/metabolismo , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/enzimología , Etopósido/uso terapéutico , Meduloblastoma/tratamiento farmacológico , Meduloblastoma/enzimología , Acetilación/efectos de los fármacos , Acetiltransferasas/metabolismo , Línea Celular Tumoral , Niño , Células Clonales , Etopósido/farmacología , Gangliósidos/metabolismo , Humanos , Potencial de la Membrana Mitocondrial/efectos de los fármacosRESUMEN
Diffuse intrinsic pontine glioma (DIPG) is a devastating and incurable paediatric brain tumour with a median overall survival of 9 months. Until recently, DIPGs were treated similarly to adult gliomas, but due to the advancement in molecular and imaging technologies, our understanding of these tumours has increased dramatically. While extensive research is being undertaken to determine the function of the molecular aberrations in DIPG, there are significant gaps in understanding the biology and the influence of the tumour microenvironment on DIPG growth, specifically in regards to the developing pons. The precise orchestration and co-ordination of the development of the brain, the most complex organ in the body, is still not fully understood. Herein, we present a brief overview of brainstem development, discuss the developing microenvironment in terms of DIPG growth, and provide a basis for the need for studies focused on bridging pontine development and DIPG microenvironment. Conducting investigations in the context of a developing brain will lead to a better understanding of the role of the tumour microenvironment and will help lead to identification of drivers of tumour growth and therapeutic resistance.
RESUMEN
Medulloblastoma is the most common malignant paediatric brain tumour and current therapies often leave patients with severe neurological disabilities. Four major molecular groups of medulloblastoma have been identified (Wnt, Shh, Group 3 and Group 4), which include additional, recently defined subgroups with different prognosis and genetic characteristics. Lactate dehydrogenase A (LDHA) is a key enzyme in the aerobic glycolysis pathway, an abnormal metabolic pathway commonly observed in cancers, associated with tumour progression and metastasis. Studies indicate MBs have a glycolytic phenotype; however, LDHA has not yet been explored as a therapeutic target for medulloblastoma. LDHA expression was examined in medulloblastoma subgroups and cell lines. The effects of LDHA inhibition by oxamate or LDHA siRNA on medulloblastoma cell line metabolism, migration and proliferation were examined. LDHA was significantly overexpressed in Group 3 and Wnt MBs compared to non-neoplastic cerebellum. Furthermore, we found that oxamate significantly attenuated glycolysis, proliferation and motility in medulloblastoma cell lines, but LDHA siRNA did not. We established that aerobic glycolysis is a potential therapeutic target for medulloblastoma, but broader LDH inhibition (LDHA, B, and C) may be more appropriate than LDHA inhibition alone.
RESUMEN
Membrane-type 5 matrix metalloproteinase (MT5-MMP) expression is ubiquitous in brain development while restricted to regions of neuroplasticity in adult. In the multipotent NT2 model of neurogenesis and differentiation, MT5-MMP is differentially expressed with significantly higher mRNA levels in the differentiated neuronal hNT cells. MT5-MMP cDNA cloned from NT2 cells unexpectedly revealed a novel sequence (MT5-MMPvar) characterized by a 162bp deletion. Both transcripts were identified in NT2, hNT and adult human hippocampus. In vitro, MT5-MMPvar translated into a approximately 55 kDa protein. It was also detected in NT2, hNT and adult human hippocampus. These results suggest more than one human MT5-MMP transcript may exist in the central nervous system.
Asunto(s)
Empalme Alternativo/genética , Metaloproteinasas de la Matriz Asociadas a la Membrana/genética , Células Madre Multipotentes/metabolismo , Adulto , Secuencia de Aminoácidos , Bacterias , Secuencia de Bases , Encéfalo/metabolismo , Diferenciación Celular , Células Cultivadas , Clonación Molecular , ADN Complementario/genética , Regulación de la Expresión Génica , Humanos , Metaloproteinasas de la Matriz Asociadas a la Membrana/biosíntesis , Metaloproteinasas de la Matriz Asociadas a la Membrana/química , Datos de Secuencia Molecular , Peso Molecular , Células Madre Multipotentes/citología , Neuronas/citología , Neuronas/metabolismo , Biosíntesis de Proteínas , ARN Mensajero/genética , Homología de Secuencia de Ácido NucleicoRESUMEN
There is significant interest in the potential use of telomerase-immortalized cells in transplantation to replace neurons lost to neurodegenerative diseases and other central nervous system injuries. Neural progenitor cells (NPCs) transduced with human telomerase reverse transcriptase (hTERT), the catalytic component of telomerase, have the potential both to proliferate indefinitely in vitro and to respond to differentiation signals necessary for generating appropriate cells for transplantation. The purpose of this study was to evaluate the differentiation of neurons from NT2 cells, a model NPC cell line, following hTERT transduction. RT-PCR and telomerase activity data demonstrated that persistent exogenous hTERT expression significantly inhibited the differentiation of neurons from NT2 cells. Following retinoic acid induced differentiation, hTERT-NT2 cells produced only one fourth of the neurons generated by parental and vector-control cells. A differentiation-inhibiting effect of constitutive telomerase activity has not been reported previously in other hTERT-transduced progenitor cell lines, implying a unique role for telomerase in the proliferation and differentiation of NPCs that have tumorigenic potential. Elucidating the mechanism responsible for this effect may aid in understanding the potential role of telomerase activity in the tumorigenicity of NPCs, as well as in optimizing the production of safe, telomerase-engineered, transplantable neurons.
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Diferenciación Celular/fisiología , Expresión Génica/fisiología , Neuronas/metabolismo , Células Madre/metabolismo , Telomerasa/metabolismo , Antineoplásicos/farmacología , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Expresión Génica/efectos de los fármacos , Humanos , Proteínas Asociadas a Microtúbulos/metabolismo , Inhibición Neural/efectos de los fármacos , Inhibición Neural/fisiología , Neuroblastoma , ARN Mensajero/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Células Madre/efectos de los fármacos , Tretinoina/farmacología , Tubulina (Proteína)/metabolismoRESUMEN
OBJECT: The WT1 gene is overexpressed in many types of human cancer. It has been demonstrated that Wilms tumor 1 (WT1) promotes tumor cell proliferation and survival in some cell lines by inhibiting p53-mediated apoptosis; however, this relationship has not been investigated in gliomas. The goal in this study was to characterize the expression pattern of WT1 in human gliomas and to determine if a correlation exists between WT1 expression and p53 status. METHODS: The authors screened nine malignant glioma cell lines, 50 glioblastoma multiforme (GBM) samples, and 16 lower-grade glial tumors for WT1 expression. RESULTS: Five of nine cell lines, 44 of 50 GBM samples, and 13 of 16 lower-grade gliomas expressed WT1 mRNA on reverse transcriptase polymerase chain reaction (PCR) analysis. Expression of WT1 was not detected in normal astrocytes. Two WT1 isoforms, +/+ and -/+, were expressed in the majority of these samples. Real-time PCR analysis of the GBM cell lines revealed that the level of WT1 mRNA ranged from 6.33 to 214.70 ng per ng 18S ribosomal RNA. The authors screened the GBM samples for p53 mutation by using PCR and single-stranded conformational polymorphism analysis, and they demonstrated an association between WT1 expression and p53 status. Tumors that contained wild-type p53 were significantly more likely to express WT1 than tumors that contained mutant p53. CONCLUSIONS: The presence of WT1 in glioma cell lines and the majority of primary tumor samples and its absence in normal astrocytes support the suggestion that WT1 expression is important in glioma biology.
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Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Genes p53/genética , Glioma/genética , Glioma/metabolismo , Proteínas WT1/metabolismo , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Neoplasias Encefálicas/patología , Estudios de Casos y Controles , Línea Celular Tumoral , Femenino , Glioma/patología , Humanos , Masculino , Persona de Mediana Edad , Polimorfismo Conformacional Retorcido-Simple , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas WT1/genéticaRESUMEN
Microarray technologies have come into prominence for the assessment of molecular diagnostic profiles in cancer tissue biopsies. To better understand the effect of sampling bias, we paired image-guided stereotactic biopsy and microarray technology to study regional intratumoral differences in tumor periphery and core regions of untreated glioblastoma. RNA was extracted from serial frozen sections using an integral histopathologic scoring approach. Gene expression analysis was performed using high-density oligonucleotide microarrays (22,283 probe sets). A consensus list of 643 genes (784 probe sets) with greater than 2-fold difference between intratumoral periphery and core samples was obtained using Microarray Suite 5.0, model-based expression indexes, and robust multiarray analysis algorithms. Results were validated using quantitative polymerase chain reaction and Western blotting analyses. Reproducible profiles emerged, in which multiple therapeutic targets significant to glioblastoma [matrix metalloproteinases, AKT1 (v-akt murine thymoma viral oncogene homolog 1), epidermal growth factor receptor, vascular endothelial growth factor] showed significant differences in regional expression that may affect treatment response. This study suggests important intratumoral regional differences in the molecular phenotype of glioblastoma.
Asunto(s)
Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Glioblastoma/genética , Glioblastoma/patología , Imagen por Resonancia Magnética Intervencional , Análisis de Secuencia por Matrices de Oligonucleótidos , Algoritmos , Biopsia , Western Blotting , Análisis por Conglomerados , Humanos , Estudios Prospectivos , ARN/análisis , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Técnicas EstereotáxicasRESUMEN
There are over 120 types of brain tumor and approximately 45% of primary brain tumors are gliomas, of which glioblastoma multiforme (GBM) is the most common and aggressive with a median survival rate of 14 months. Despite progress in our knowledge, current therapies are unable to effectively combat primary brain tumors and patient survival remains poor. Tumor metabolism is important to consider in therapeutic approaches and is the focus of numerous research investigations. Lactate dehydrogenase A (LDHA) is a cytosolic enzyme, predominantly involved in anaerobic and aerobic glycolysis (the Warburg effect); however, it has multiple additional functions in non-neoplastic and neoplastic tissues, which are not commonly known or discussed. This review summarizes what is currently known about the function of LDHA and identifies areas that would benefit from further exploration. The current knowledge of the role of LDHA in the brain and its potential as a therapeutic target for brain tumors will also be highlighted. The Warburg effect appears to be universal in tumors, including primary brain tumors, and LDHA (because of its involvement with this process) has been identified as a potential therapeutic target. Currently, there are, however, no suitable LDHA inhibitors available for tumor therapies in the clinic.
Asunto(s)
Neoplasias Encefálicas/enzimología , Neoplasias Encefálicas/terapia , L-Lactato Deshidrogenasa/metabolismo , L-Lactato Deshidrogenasa/uso terapéutico , Animales , Neoplasias Encefálicas/genética , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Isoenzimas/uso terapéutico , L-Lactato Deshidrogenasa/genética , Lactato Deshidrogenasa 5RESUMEN
BACKGROUND: CD15, which is overexpressed on various cancers, has been reported as a cell adhesion molecule that plays a key role in non-CNS metastasis. However, the role of CD15 in brain metastasis is largely unexplored. This study provides a better understanding of CD15/CD62E interaction, enhanced by tumor necrosis factor-α (TNF-α), and its correlation with brain metastasis in non-small cell lung cancer (NSCLC). METHODS: CD15 and E-selectin (CD62E) expression was demonstrated in both human primary and metastatic NSCLC cells using flow cytometry, immunofluorescence, and Western blotting. The role of CD15 was investigated using an adhesion assay under static and physiological flow live-cell conditions. Human tissue sections were examined using immunohistochemistry. RESULTS: CD15, which was weakly expressed on hCMEC/D3 human brain endothelial cells, was expressed at high levels on metastatic NSCLC cells (NCI-H1299, SEBTA-001, and SEBTA-005) and at lower levels on primary NSCLC (COR-L105 and A549) cells (P < .001). The highest expression of CD62E was observed on hCMEC/D3 cells activated with TNF-α, with lower levels on metastatic NSCLC cells followed by primary NSCLC cells. Metastatic NSCLC cells adhered most strongly to hCMEC/D3 compared with primary NSCLC cells. CD15 immunoblocking decreased cancer cell adhesion to brain endothelium under static and shear stress conditions (P < .0001), confirming a correlation between CD15 and cerebral metastasis. Both CD15 and CD62E expression were detected in lung metastatic brain biopsies. CONCLUSION: This study enhances the understanding of cancer cell-brain endothelial adhesion and confirms that CD15 plays a crucial role in adhesion in concert with TNF-α activation of its binding partner, CD62E.