Cellular and molecular characterization of the effects of retinoic acid on ST1 rat glioma cells

Os gliomas são os tumores mais fatais do sistema nervoso central, para os quais ainda não há tratamento eficaz. Para analisar as bases celulares e moleculares da ação do agente diferenciador e antitumoral ácido retinóico (ATRA) sobre gliomas, foi utilizado, como modelo, a linhagem STl de glioma de rato. Propôs-se: a) analisar os efeitos de ATRA sobre a morfologia, proliferação e morte celular; b) isolar, identificar e caracterizar genes induzidos por ATRA em células STl. Verificou-se que o tratamento com ATRA promove achatamento celular e inibição da síntese de DNA e do crescimento em suspensão de agarose, caracterizando uma completa reversão fenotípica tumoral-normal, a qual não é acompanhada de indução de apoptose. Os genes induzidos por ATRA foram isolados através da construção de bibliotecas subtraídas de cDNA por: RDA (\"Representational Diference Analysis\") e SSH (\"Suppressive Subtractive Hybridization\") acoplados a rastreamento em \"macroarrays\". Foram identificados 10 genes regulados por ATRA durante a reversão fenotípica das células STl: p450rai2, spi3, vegf, cdv-3a, okl38, eya2, gem, retSDR1, aldose redutase-like, e um gene novo, com 61 % de identidade com uma fosfatase de galinha. Este estudo permitiu a caracterização dos efeitos de ATRA sobre STl e identificou novos alvos para futuro desenvolvimento de novas drogas e terapia gênica.Gliomas are the most fatal central nervous system tumors, for which efficient treatment is still not available. To analyze the cellular and molecular bases for the action of the differentiating and anti-tumor agent retinoic acid (ATRA) in gliomas, the rat glioma STl cell line was used as a model. We proposed: a) to analyze the effects of ATRA in STl cells morphology, growth and apoptosis; b) to isolate, identify and characterize the ATRA-induced genes in STl cells. We demonstrated that ATRA promotes cellular flattening and inhibition of DNA synthesis and growth in agarose suspension, characterizing a complete tumoral to normal phenotypic reversion, which is not accompanied by apoptosis. Subtracted cDNA libraries were generated, using 2 different methodologies: RDA (Representational Difference Analysis) and SSH (Suppressive Subtractive Hybridization) followed by macroarray screening. This allowed identification of 10 ATRA induced genes which are up regulated by ATRA during STl cells phenotypic reversion, namely: p450rai2, spi3, vegf, cdv-3a, okl38, eya2, gem, retSDR1, aldose redutase-like and a new gene with 61 % identity with chicken phosphatase. This study characterized the cellular and molecular effects of ATRA upon STl cells and allowed identification of new targets for future development of new drugs and gene therapy

Cellular and molecular characterization of the effects of retinoic acid on ST1 rat glioma cells

Os gliomas são os tumores mais fatais do sistema nervoso central, para os quais ainda não há tratamento eficaz. Para analisar as bases celulares e moleculares da ação do agente diferenciador e antitumoral ácido retinóico (ATRA) sobre gliomas, foi utilizado, como modelo, a linhagem STl de glioma de rato. Propôs-se: a) analisar os efeitos de ATRA sobre a morfologia, proliferação e morte celular; b) isolar, identificar e caracterizar genes induzidos por ATRA em células STl. Verificou-se que o tratamento com ATRA promove achatamento celular e inibição da síntese de DNA e do crescimento em suspensão de agarose, caracterizando uma completa reversão fenotípica tumoral-normal, a qual não é acompanhada de indução de apoptose. Os genes induzidos por ATRA foram isolados através da construção de bibliotecas subtraídas de cDNA por: RDA (\"Representational Diference Analysis\") e SSH (\"Suppressive Subtractive Hybridization\") acoplados a rastreamento em \"macroarrays\". Foram identificados 10 genes regulados por ATRA durante a reversão fenotípica das células STl: p450rai2, spi3, vegf, cdv-3a, okl38, eya2, gem, retSDR1, aldose redutase-like, e um gene novo, com 61 % de identidade com uma fosfatase de galinha. Este estudo permitiu a caracterização dos efeitos de ATRA sobre STl e identificou novos alvos para futuro desenvolvimento de novas drogas e terapia gênica.Gliomas are the most fatal central nervous system tumors, for which efficient treatment is still not available. To analyze the cellular and molecular bases for the action of the differentiating and anti-tumor agent retinoic acid (ATRA) in gliomas, the rat glioma STl cell line was used as a model. We proposed: a) to analyze the effects of ATRA in STl cells morphology, growth and apoptosis; b) to isolate, identify and characterize the ATRA-induced genes in STl cells. We demonstrated that ATRA promotes cellular flattening and inhibition of DNA synthesis and growth in agarose suspension, characterizing a complete tumoral to normal phenotypic reversion, which is not accompanied by apoptosis. Subtracted cDNA libraries were generated, using 2 different methodologies: RDA (Representational Difference Analysis) and SSH (Suppressive Subtractive Hybridization) followed by macroarray screening. This allowed identification of 10 ATRA induced genes which are up regulated by ATRA during STl cells phenotypic reversion, namely: p450rai2, spi3, vegf, cdv-3a, okl38, eya2, gem, retSDR1, aldose redutase-like and a new gene with 61 % identity with chicken phosphatase. This study characterized the cellular and molecular effects of ATRA upon STl cells and allowed identification of new targets for future development of new drugs and gene therapy

Genome-wide translation control analysis of developing human neurons.

During neuronal differentiation, neuroprogenitor cells become polarized, change shape, extend axons, and form complex dendritic trees. While growing, axons are guided by molecular cues to their final destination, where they establish synaptic connections with other neuronal cells. Several layers of regulation are integrated to control neuronal development properly. Although control of mRNA translation plays an essential role in mammalian gene expression, how it contributes temporarily to the modulation of later stages of neuronal differentiation remains poorly understood. Here, we investigated how translation control affects pathways and processes essential for neuronal maturation, using H9-derived human neuro progenitor cells differentiated into neurons as a model. Through Ribosome Profiling (Riboseq) combined with RNA sequencing (RNAseq) analysis, we found that translation control regulates the expression of critical hub genes. Fundamental synaptic vesicle secretion genes belonging to SNARE complex, Rab family members, and vesicle acidification ATPases are strongly translationally regulated in developing neurons. Translational control also participates in neuronal metabolism modulation, particularly affecting genes involved in the TCA cycle and glutamate synthesis/catabolism. Importantly, we found translation regulation of several critical genes with fundamental roles regulating actin and microtubule cytoskeleton pathways, critical to neurite generation, spine formation, axon guidance, and circuit formation. Our results show that translational control dynamically integrates important signals in neurons, regulating several aspects of its development and biology