RESUMEN
Polycomb group proteins are essential regulators of stem cell function during embryonic development and in adult tissue homeostasis. Bmi1, a key component of the Polycomb Repressive Complex 1, is highly expressed in undifferentiated neural stem cells (NSC) as well as in several human cancers including high-grade gliomas--highly aggressive brain tumors. Using a conditional gene activation approach in mice, we show that overexpression of Bmi1 induces repressive epigenetic regulation of the promoter of Survivin, a well-characterized antiapoptotic protein. This phenomenon is cell type-specific and it leads to apoptotic death of progenitor cells exclusively upon commitment toward a neuronal fate. Moreover, we show that this is triggered by increased oxidative stress-induced DNA damage. In contrast, undifferentiated NSC as well as glioma-initiating cells display an open chromatin configuration at the Survivin promoter and do not undergo apoptotic death. These findings raise the possibility that normal and neoplastic stem cells depend on the same mechanism for surviving the hyperproliferative state induced by increased Bmi1 expression.
Asunto(s)
Glioma/metabolismo , Proteínas Inhibidoras de la Apoptosis/metabolismo , Células-Madre Neurales/fisiología , Complejo Represivo Polycomb 1/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Represoras/metabolismo , Animales , Neoplasias Encefálicas/metabolismo , Diferenciación Celular , Proliferación Celular , Corteza Cerebral/citología , Cromatina/metabolismo , Daño del ADN , Metilación de ADN , Epigénesis Genética , Perfilación de la Expresión Génica , Glioma/genética , Proteínas Inhibidoras de la Apoptosis/genética , Histona Demetilasas con Dominio de Jumonji/metabolismo , Ratones , Neurogénesis , Estrés Oxidativo , Regiones Promotoras Genéticas , Proteínas Represoras/genética , Survivin , UbiquitinaciónRESUMEN
BACKGROUND: Both bone morphogenetic proteins (BMPs) and histone deacetylases (HDACs) have previously been established to play a role in the development of the three major cell types of the central nervous system: neurons, astrocytes, and oligodendrocytes. We have previously established a connection between these two protein families, showing that HDACs suppress BMP-promoted astrogliogenesis in the embryonic striatum. Since HDACs act in the nucleus to effect changes in transcription, an unbiased analysis of their transcriptional targets could shed light on their downstream effects on BMP-signaling. RESULTS: Using neurospheres from the embryonic striatum as an in vitro system to analyze this phenomenon, we have performed microarray expression profiling on BMP2- and TSA-treated cultures, followed by validation of the findings with quantitative RT-PCR and protein analysis. In BMP-treated cultures we first observed an upregulation of genes involved in cell-cell communication and developmental processes such as members of BMP and canonical Wnt signaling pathways. In contrast, in TSA-treated cultures we first observed an upregulation of genes involved in chromatin modification and transcription. Interestingly, we could not record direct changes in the protein levels of canonical members of BMP2 signaling, but we did observe an upregulation of both the transcription factor STAT3 and its active isoform phospho-STAT3 at the protein level. CONCLUSIONS: STAT3 and SMAD1/5/8 interact synergistically to promote astrogliogenesis, and thus we show for the first time that HDACs act to suppress BMP-promoted astrogliogenesis by suppression of the crucial partner STAT3.
Asunto(s)
Proteínas Morfogenéticas Óseas/metabolismo , Histona Desacetilasas/metabolismo , Prosencéfalo/citología , Prosencéfalo/embriología , Animales , Proteínas Morfogenéticas Óseas/genética , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Células Cultivadas , Redes Reguladoras de Genes/efectos de los fármacos , Redes Reguladoras de Genes/genética , Redes Reguladoras de Genes/fisiología , Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/genética , Ácidos Hidroxámicos/farmacología , Ratones , Prosencéfalo/metabolismo , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismo , Proteína Smad1/genética , Proteína Smad1/metabolismo , Proteína Smad5/genética , Proteína Smad5/metabolismo , Proteína Smad8/genética , Proteína Smad8/metabolismoRESUMEN
High grade gliomas (HGGs) are characterized by resistance to radiotherapy and chemotherapy. Targeting Rad51-dependent homologous recombination repair may be an effective target for chemo- and radiosensitization. In this study we assessed the role of Rad51-dependent repair on sensitivity to radiation and temozolomide (TMZ) as single agents or in combination. Repair protein levels in established glioma cell lines, early passage glioblastoma multiforme (GBM) cell lines, and normal human astrocytes (NHAs) were measured using western blot. Viability and clonogenic survival assays were used to measure the effects of Rad51 knockdown with radiation (XR) and TMZ. Immunocytochemistry was used to evaluate kinetics of Rad51 and γ-H2AX repair foci. Immunohistochemistry was used to assess Rad51 protein levels in glioma specimens. Repair proteins including Rad51 are upregulated in HGG cells compared with NHA. Established glioma cell lines show a dose-dependent increase in Rad51 foci formation after XR and TMZ. Rad51 levels are inversely correlated with radiosensitivity, and downregulation markedly increases the cytotoxicity of TMZ. Rad51 knockdown also promotes more residual γ-H2AX foci 24 h after combined treatment. Newly established GBM cell lines also have high Rad51 levels and are extremely sensitive to Rad51 knockdown. Clinical samples from recently resected gliomas of varying grades demonstrate that Rad51 levels do not correlate with tumor grade. Rad51-dependent repair makes a significant contribution to DNA repair in glioma cells and contributes to resistance to both XR and TMZ. Agents targeting Rad51-dependent repair would be effective adjuvants in standard combination regimens.