RESUMEN
The loss of anti-proliferative responsiveness in prostate cancer cell lines toward ligands for vitamin D receptor, retinoic acid receptors/retinoid X receptors and peroxisome proliferator activated receptor (PPAR)alpha/gamma may entail underlying epigenetic events, as ligand insensitivity reflects significantly altered messenger RNA expression of corepressors and histone-modifying enzymes. Expression patterns were dependent on phases of the cell cycle and associated with repressed basal gene expression of vitamin D receptor and PPARalpha/gamma target genes, for example CDKN1A [encodes p21((waf1/cip1))]. Elevated nuclear corepressor 1 (NCOR1) and nuclear corepressor 2/silencing mediator of retinoic acid and thyroid hormone receptor protein levels were detected in prostate cancer cell lines compared with non-malignant counterparts. Knockdown of the corepressor NCOR1 significantly elevated basal expression of a cohort of target genes, including CDKN1A. Both chemical [histone deacetylases inhibitor (HDACi)] and NCOR1 knockdown targeting enhanced anti-proliferative sensitivity toward PPARalpha/gamma ligands in prostate cancer cell lines. Pursuing PPARalpha/gamma signaling, microarray approaches were undertaken to identify pathways and genes regulated uniquely by a combination of PPARalpha/gamma activation and HDAC inhibition. Again, HDACi and knockdown approaches demonstrated that elevated NCOR1 expression and activity distorted PPARalpha/gamma gene targets centered on, for example cell cycle control, including CDKN1A and TGFBRAP1. Quantitative real time polymerase chain reaction validation and chromatin immunoprecipitation assays both confirmed that elevated NCOR1 disrupted the ability of PPARalpha/gamma to regulate key target genes (CDKN1A and TGFBRAP1). Interrogation of these relationships in prostate cancer samples using principal component and partial correlation analyses established significant interdependent relationships between NCOR1-PPARalpha/gamma and representative target genes, independently of androgen receptor expression. Therefore, we conclude that elevated NCOR1 distorts the actions of PPARalpha/gamma selectively and generates a potential epigenetic lesion with diagnostic and prognostic significance.
Asunto(s)
Biomarcadores de Tumor/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Epigénesis Genética , Péptidos y Proteínas de Señalización Intracelular/genética , Co-Represor 1 de Receptor Nuclear/metabolismo , PPAR alfa/metabolismo , PPAR gamma/metabolismo , Neoplasias de la Próstata/metabolismo , Apoptosis , Biomarcadores de Tumor/metabolismo , Western Blotting , Ciclo Celular , Proliferación Celular , Inmunoprecipitación de Cromatina , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Perfilación de la Expresión Génica , Inhibidores de Histona Desacetilasas/farmacología , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Masculino , Co-Represor 1 de Receptor Nuclear/antagonistas & inhibidores , Co-Represor 1 de Receptor Nuclear/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Próstata/metabolismo , Próstata/patología , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , ARN Mensajero/genética , Receptores de Calcitriol/genética , Receptores de Calcitriol/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de SeñalRESUMEN
As the goal to eradicate wild polio virus (WPV) is approached, outbreaks associated with vaccine derived polioviruses (VDPV) with neurovirulent properties have emerged. The relevance for the spread of infection by nonparalytic cVDPV cases, with mutations associated with neurovirulence, is discussed with reference to the molecular analysis of a VDPV isolated from a Jamaican child who presented with aseptic meningitis. Potential risks to the Jamaican community resulting from circulation of cVDPV and critical factors defined by the World Health Organization (WHO) in the global eradication of Polio are analyzed in the context of immunization coverage, and the need to stop all Oral Polio Vaccine (OPV) use once wild polioviruses (WPVs) have been eradicated.
Asunto(s)
Poliomielitis/epidemiología , Poliomielitis/prevención & control , Vacuna Antipolio Oral/efectos adversos , Poliovirus , Vacunación/efectos adversos , Preescolar , Humanos , Programas de Inmunización , Jamaica , Masculino , Poliovirus/patogenicidad , Vacuna Antipolio Oral/administración & dosificación , Factores de RiesgoRESUMEN
Two versions of an inexpensive mixing device capable of measuring reaction kinetics with millisecond resolution are described. The instruments deliver reactants via microsyringes controlled by nitrogen pressure at time intervals that are either mechanically or software controlled. In addition, an optical system for calibration of the time required to deliver the reactants as well as the intervals between syringe firing is given. Data illustrating the effect of variations in volume and syringe size on the accuracy and reproducibility of these time measurements as well as the volume of fluid delivered are presented. Examples of kinetic measurements of several reactions illustrating the capabilities of the instrument are also presented.