RESUMEN
Minichromosome maintenance (MCM) proteins 2-7 are important in DNA replication licensing. Functional roles beyond licensing are speculated. In addition, significances in medulloblastoma (MB) remain unclear. In this study, we showed the frequent deregulation of MCM2 and MCM3 expression in 7 MB cell lines and 31 clinical samples. Moreover, DAOY and ONS76 and the clinical samples expressed elevated MCM7 transcripts with genomic gain of the gene. Immunopositivity restricted to tumor cells was found in 41, 37 and 53 out of 73 MB cases for MCM2, MCM3 and MCM7, respectively. High-MCM3 expression was associated with poor prognosis. Knockdowns of these MCMs significantly inhibited anchorage-dependent and -independent MB cell growth. The inhibition of MCM3 expression by small interfering RNA knockdown was related to G1 arrest with reduced cyclin A expression, whereas the MCM2- and MCM7-knocked-down cells arrested at G2/M with increased cyclin A expression. Interestingly, we demonstrated the links of these MCMs with cell migration and invasion using wound-healing and Transwell migration/invasion assays. Exogenous overexpression of MCM2, MCM3 and MCM7 increased anchorage-independent cell growth, and also cell migration and invasion capabilities in MB cells. The knockdown reduced the number of filopodial cells and the cells with intense stress fibers by blocking cdc42 and Rho activation. Taken together, deregulation of MCM2, MCM3 and MCM7 expression might be involved in MB tumorigenesis and we revealed undefined roles of these MCMs in control of MB cell migration and invasion.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Movimiento Celular/genética , Proteínas de Unión al ADN/metabolismo , Meduloblastoma/metabolismo , Proteínas Nucleares/metabolismo , Biomarcadores de Tumor/análisis , Proteínas de Ciclo Celular/genética , Separación Celular , Neoplasias Cerebelosas/genética , Neoplasias Cerebelosas/metabolismo , Neoplasias Cerebelosas/patología , Hibridación Genómica Comparativa , Proteínas de Unión al ADN/genética , Citometría de Flujo , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Inmunohistoquímica , Hibridación Fluorescente in Situ , Estimación de Kaplan-Meier , Meduloblastoma/genética , Meduloblastoma/patología , Componente 2 del Complejo de Mantenimiento de Minicromosoma , Componente 3 del Complejo de Mantenimiento de Minicromosoma , Componente 7 del Complejo de Mantenimiento de Minicromosoma , Invasividad Neoplásica/genética , Proteínas Nucleares/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Pronóstico , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Nasopharyngeal carcinoma is closely associated with Epstein-Barr virus (EBV) infection. The EBV-encoded LMP1 has cell transformation property. It suppresses cellular senescence and enhances cell survival in various cell types. Many of the downstream events of LMP1 expression are mediated through its ability to activate NF-kappaB. In this study, we report a novel function of LMP1 to induce Id1 expression in nasopharyngeal epithelial cells (NP69) and human embryonal kidney cells (HEK293). The Id1 is a basic helix-loop-helix (bHLH) protein and a negative transcriptional regulator of p16(INK4a). Expression of Id1 facilitates cellular immortalization and stimulates cell proliferation. With the combination of both specific chemical inhibitors and genetic inhibitors of cell signaling, we showed that induction of Id1 by LMP1 was dependent on its NF-kappaB activation domain at the carboxy-terminal region, CTAR1 and CTAR2. Induction of Id1 by LMP1 may facilitate clonal expansion of premalignant nasopharyngeal epithelial cells infected with EBV and may promote their malignant transformation.
Asunto(s)
Células Epiteliales/metabolismo , Regulación Viral de la Expresión Génica , Herpesvirus Humano 4/fisiología , Nasofaringe/citología , Proteínas Represoras , Factores de Transcripción/fisiología , Proteínas de la Matriz Viral/fisiología , Carcinoma/epidemiología , Carcinoma/etiología , Carcinoma/virología , Células Clonales/patología , Inhibidor p16 de la Quinasa Dependiente de Ciclina/biosíntesis , Células Epiteliales/virología , Infecciones por Virus de Epstein-Barr/genética , Genes p16 , Hong Kong/epidemiología , Humanos , Proteína 1 Inhibidora de la Diferenciación , FN-kappa B/fisiología , Neoplasias Nasofaríngeas/epidemiología , Neoplasias Nasofaríngeas/etiología , Neoplasias Nasofaríngeas/virología , Estructura Terciaria de Proteína , Eliminación de Secuencia , Transducción de Señal , Factores de Transcripción/biosíntesis , Factores de Transcripción/genética , Transcripción Genética , Proteínas de la Matriz Viral/químicaRESUMEN
Diffusely infiltrative astrocytic tumours are the most common neoplasms in the human brain. To localise putative tumour suppressor loci that are involved in low-grade astrocytomas, we performed high-resolution genome-wide allelotype analysis on 17 fibrillary astrocytomas. Non-random allelic losses were identified on chromosomal arms 10p (29%), 10q (29%), 14q (35%), 17p (53%), and 19q (29%), with their respective common regions of deletions delineated at 10p14-15.1, 10q25.1-qter, 14q212.2-qer, 17p11.2-pter and 19q12-13.4. These results suggest that alterations of these chromosomal regions play important roles in the development of astrocytoma. We also allelotyped 21 de novo glioblastoma multiforme with an aim to unveil genetic changes that are common to both types of astrocytic tumours. Non-random allelic losses were identified on 9p (67%), 10p (62%), 10q (76%), 13q (60%), 14q (50%), and 17p (65%). Allelic losses of 10p, 10q, 14q and 17p were common genetic alterations detectable in both fibrillary astrocytomas and glioblastoma multiforme. In addition, two common regions of deletions on chromosome 14 were mapped to 14q22.3-32.1 and 14q32.1-qter, suggesting the presence of two putative tumour suppressor genes. In conclusion, our comprehensive allelotype analysis has unveiled several critical tumour suppressor loci that are involved in the development of fibrillary astrocytomas and glioblastoma multiforme. Although these two types of brain tumours are believed to evolve from different genetic pathways, they do share some common genetic changes. Our results indicate that deletions of chromosome 14q is a recurrent genetic event in the development of astrocytoma and highlight the subchromosomal regions on this chromosome that are likely to contain putative tumour suppressor genes involved in the oncogenesis of astrocytic tumours.
Asunto(s)
Astrocitoma/genética , Neoplasias Encefálicas/genética , Cromosomas Humanos Par 14/genética , Adulto , Alelos , Desequilibrio Alélico , Astrocitoma/patología , Neoplasias Encefálicas/patología , Aberraciones Cromosómicas , Cromosomas Humanos/genética , Cromosomas Humanos/ultraestructura , Cromosomas Humanos Par 14/ultraestructura , ADN de Neoplasias/genética , Femenino , Eliminación de Gen , Genotipo , Glioblastoma/genética , Glioblastoma/patología , Humanos , Pérdida de Heterocigocidad , MasculinoRESUMEN
Epidermal growth factor receptor is overexpressed and/or amplified in up to 50% of glioblastomas, suggesting an important role of this gene in glial tumorigenesis and progression. In the present study we demonstrated that epidermal growth factor receptor is involved in regulation of telomerase activity in glioblastoma. Antisense-epidermal growth factor receptor approach was used to inhibit epidermal growth factor receptor expression of glioblastoma U87MG cells. Telomerase activity in antisense-epidermal growth factor receptor cells decreased by up to 54 folds compared with control cells. Moreover, the telomere lengths of antisense-epidermal growth factor receptor cells were shortened. In addition, the tumorigenicity of antisense-epidermal growth factor receptor cells was significantly inhibited. Taken together, there were strong correlations between tumorigenicity and epidermal growth factor receptor expression levels, and between tumorigenicity and telomerase activity. These results provide evidence that epidermal growth factor receptor plays an important role in the regulation of telomerase activity of glioma cells. Our findings provide new insights into both the biological functions of epidermal growth factor receptor and the regulation of telomerase activity. The inhibition of telomerase activity triggered by antisense-epidermal growth factor receptor treatment may reflect yet another mechanism of antisense-epidermal growth factor receptor approach in tumour suppression.
