RESUMO
Heterochromatic repetitive satellite RNAs are extensively transcribed in a variety of human cancers, including BRCA1 mutant breast cancer. Aberrant expression of satellite RNAs in cultured cells induces the DNA damage response, activates cell cycle checkpoints, and causes defects in chromosome segregation. However, the mechanism by which satellite RNA expression leads to genomic instability is not well understood. Here we provide evidence that increased levels of satellite RNAs in mammary glands induce tumor formation in mice. Using mass spectrometry, we further show that genomic instability induced by satellite RNAs occurs through interactions with BRCA1-associated protein networks required for the stabilization of DNA replication forks. Additionally, de-stabilized replication forks likely promote the formation of RNA-DNA hybrids in cells expressing satellite RNAs. These studies lay the foundation for developing novel therapeutic strategies that block the effects of non-coding satellite RNAs in cancer cells.
Assuntos
Proteína BRCA1/genética , Neoplasias da Mama/genética , Transformação Celular Neoplásica/genética , Dano ao DNA , Instabilidade Genômica , Heterocromatina/genética , RNA Neoplásico/genética , RNA Satélite/genética , Animais , Proteína BRCA1/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proliferação de Células , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Heterocromatina/metabolismo , Humanos , Células MCF-7 , Camundongos , Ligação Proteica , RNA Neoplásico/metabolismo , RNA Satélite/metabolismo , Carga TumoralRESUMO
Lifeguard (LFG) is an inhibitor of Fas-mediated cell death and is highly expressed in the cerebellum. We investigated the biological role of LFG in the cerebellum in vivo, using mice with reduced LFG expression generated by shRNA lentiviral transgenesis (shLFG mice) as well as LFG null mice. We found that LFG plays a role in cerebellar development by affecting cerebellar size, internal granular layer (IGL) thickness, and Purkinje cell (PC) development. All these features are more severe in early developmental stages and show substantial recovery overtime, providing a remarkable example of cerebellar plasticity. In adult mice, LFG plays a role in PC maintenance shown by reduced cellular density and abnormal morphology with increased active caspase 8 and caspase 3 immunostaining in shLFG and knockout (KO) PCs. We studied the mechanism of action of LFG as an inhibitor of the Fas pathway and provided evidence of the neuroprotective role of LFG in cerebellar granule neurons (CGNs) and PCs in an organotypic cerebellar culture system. Biochemical analysis of the Fas pathway revealed that LFG inhibits Fas-mediated cell death by interfering with caspase 8 activation. This result is supported by the increased number of active caspase 8-positive PCs in adult mice lacking LFG. These data demonstrate that LFG is required for proper development and survival of granular and Purkinje cells and suggest LFG may play a role in cerebellar disorders.
Assuntos
Proteínas Reguladoras de Apoptose/fisiologia , Cerebelo/citologia , Cerebelo/fisiologia , Proteínas de Membrana/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Células de Purkinje/citologia , Células de Purkinje/fisiologia , Animais , Apoptose/fisiologia , Proteínas Reguladoras de Apoptose/genética , Sequência de Bases , Caspase 3/metabolismo , Caspase 8/metabolismo , Diferenciação Celular/fisiologia , Sobrevivência Celular/fisiologia , Cerebelo/anormalidades , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , RNA Interferente Pequeno/genética , Receptor fas/metabolismoRESUMO
We have engineered a lentivirus-based gene transfer system to achieve ecdysone-regulated transgene expression. The method combines the wide tropism of lentiviral vectors and the possibility of gene regulation by a small molecule with an excellent pharmacological profile. Using the hematopoietic tissue as a model, we transduced mouse progenitors with an ecdysone-regulated GFP expression cassette. The ecdysone gene switch efficiently turned GFP on and off in transplanted animals, showing low basal activity. This system allows the delivery of inducible transcriptional units in vitro and ex vivo and may be a useful tool for gene transfer purposes. Moreover, our work provides hints on the design of lentiviral vectors containing multiple expression cassettes with multiple promoters.