RESUMEN
PURPOSE: The histone deacetylase inhibitor FK228 shows strong activity as a potent antitumor drug but its precise mechanism is still obscure. The purpose of this study is to reveal the effect of FK228 on gene expression in the cell and to determine the mechanism of the antitumor activity of FK228 for further clinical applications. EXPERIMENTAL DESIGN AND RESULTS: Microarray analysis was applied to verify the gene expression profiles of 4,608 genes after FK228 treatment using human esophageal squamous cell cancer cell lines T.Tn and TE2. Among them, peroxiredoxin 1 (Prdx1), a member of the peroxiredoxin family of antioxidant enzymes having cell growth suppression activity, as well as p21(WAF1), were significantly activated by FK288. In addition, FK228 strongly inhibited the cell growth of T.Tn and TE2 by the induction of apoptosis. Further, chromatin immunoprecipitation analysis revealed that FK228 induced the accumulation of acetylated histones H3 and H4 in Prdx1 promoter, including the Sp1-binding site. In mouse xenograft models of T.Tn and TE2 cells, FK228 injection resulted in significant tumor regression as well as activated Prdx1 expression in tumor tissues. Prdx1 suppression by RNA interference hindered the antitumor effect of FK228. CONCLUSION: Our results indicate that the antitumor effect of FK228 in esophageal cancer cells is shown at least in part through Prdx1 activation by modulating acetylation of histones in the promoter, resulting in tumor growth inhibition with apoptosis induction.
Asunto(s)
Antibióticos Antineoplásicos/farmacología , Apoptosis , Depsipéptidos/farmacología , Neoplasias Esofágicas/tratamiento farmacológico , Neoplasias Esofágicas/patología , Regulación Neoplásica de la Expresión Génica , Proteínas de Choque Térmico/metabolismo , Inhibidores de Histona Desacetilasas , Peroxidasas/metabolismo , Animales , Antineoplásicos , Ciclo Celular , Línea Celular Tumoral , Proliferación Celular , Inmunoprecipitación de Cromatina , ADN Complementario/metabolismo , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo , Esófago/patología , Exones , Silenciador del Gen , Histonas/química , Humanos , Etiquetado Corte-Fin in Situ , Intrones , Ratones , Trasplante de Neoplasias , Análisis de Secuencia por Matrices de Oligonucleótidos , Peroxirredoxinas , Reacción en Cadena de la Polimerasa , ARN/metabolismo , Interferencia de ARN , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo , Regulación hacia ArribaRESUMEN
NeuroD-related factor (NDRF)/NeuroD2 is a basic helix-loop-helix (bHLH) protein that plays important roles in neuronal development. To elucidate the NDRF transcription network, we used mouse cDNA microarray analysis combined with a tetracycline-regulatable expression system in P19 embryonal carcinoma cells. Five genes were identified to be up-regulated in the presence of NDRF protein. RNA hybridization analysis confirmed that brain-lipid-binding protein (BLBP) and inhibitor of differentiation 1 (Id1) genes were among the five genes that were rapidly and significantly up-regulated after induction of NDRF. When a dominant negative form of NDRF protein was expressed during retinoic acid-induced neuronal differentiation of P19 cells, the BLBP gene, but not the Id1 gene, was potently repressed. Immunohistochemical analysis revealed that both NDRF and Id1 immunoreactivities were observed in some granule cells of the cerebellum in the postnatal period. These results suggest that NDRF or its related bHLH proteins may act upstream of these genes in a subset of developing neurons.
Asunto(s)
Regulación de la Expresión Génica , Neuronas/metabolismo , Neuropéptidos/biosíntesis , Neuropéptidos/fisiología , Análisis de Secuencia por Matrices de Oligonucleótidos , Tetraciclina/farmacología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Northern Blotting , Western Blotting , Encéfalo/embriología , Diferenciación Celular , Línea Celular Tumoral , ADN Complementario/metabolismo , Doxiciclina/farmacología , Genes Dominantes , Inmunohistoquímica , Proteína 1 Inhibidora de la Diferenciación , Ratones , Neuronas/citología , Hibridación de Ácido Nucleico , Plásmidos/metabolismo , Estructura Terciaria de Proteína , ARN/metabolismo , ARN Mensajero/metabolismo , Proteínas Represoras/metabolismo , Factores de Tiempo , Factores de Transcripción/metabolismo , Transcripción Genética , Activación Transcripcional , Transfección , Tretinoina/farmacología , Regulación hacia ArribaRESUMEN
In the dystrophin-mutant mdx mouse, an animal model for Duchenne muscular dystrophy (DMD), damaged skeletal muscles are efficiently regenerated and thus the animals thrive. The phenotypic differences between DMD patients and the mdx mice suggest the existence of factors that modulate the muscle wasting in the mdx mice. To identify these factors, we searched for mRNAs affected by the mdx mutation by using cDNA microarrays with newly established skeletal muscle cell lines from mdx and normal mice. We found that in the mdx muscle cell line, 12 genes, including L-arginine:glycine amidinotransferase and thymosin beta4, are up-regulated, whereas 7 genes, including selenoprotein P and a novel regeneration-associated muscle protease (RAMP), are down-regulated. Northern blot analysis and in situ hybridization revealed that RAMP mRNA is predominantly expressed in normal skeletal muscle and brain, and its production is enhanced in the regenerating area of injured skeletal muscle in mice. RAMP expression was much lower in individual muscle cell lines derived from biopsies of six DMD patients compared to a normal muscle cell line. These results suggest that RAMP may play a role in the regeneration of skeletal muscle and that its down-regulation could be involved in the progression of DMD in humans.