RESUMEN
Acute respiratory distress syndrome (ARDS) is a poorly understood condition with greater than 30% mortality. Massive recruitment of neutrophils to the lung occurs in the initial stages of the ARDS. Significant variability in the severity and duration of ARDS-associated pulmonary inflammation could be linked to heterogeneity in the inflammatory capacity of neutrophils. Interferon-stimulated genes (ISGs) are a broad gene family induced by Type I interferons. While ISGs are central to anti-viral immunity, the potential exists for these genes to evoke extensive modification in cellular response in other clinical settings. In this prospective study, we sought to determine if ISG expression in circulating neutrophils from ARDS patients is associated with changes in neutrophil function. Circulating neutrophil RNA was isolated, and hierarchical clustering ranked patients' expression of three ISGs. Neutrophil response to pathogenic bacteria was compared between normal and high ISG-expressing neutrophils. High neutrophil ISG expression was found in 25 of 95 (26%) of ARDS patients and was associated with reduced migration toward interleukin-8, and altered responses to Staphylococcus aureus, but not Pseudomonas aeruginosa, which included decreased p38 MAP kinase phosphorylation, superoxide anion release, interleukin-8 release, and a shift from necrotic to apoptotic cell death. These alterations in response were reflected in a decreased capacity to kill S. aureus, but not P. aeruginosa. Therefore, the ISG expression signature is associated with an altered circulating neutrophil response phenotype in ARDS that may predispose a large subgroup of patients to increased risk of specific bacterial infections.
Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Interferones/farmacología , Neutrófilos/patología , Síndrome de Dificultad Respiratoria/genética , Síndrome de Dificultad Respiratoria/microbiología , Staphylococcus aureus/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Muerte Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Separación Celular , Estudios de Cohortes , Femenino , Humanos , Interleucina-8/metabolismo , Masculino , Viabilidad Microbiana/efectos de los fármacos , Persona de Mediana Edad , Neutrófilos/efectos de los fármacos , Neutrófilos/enzimología , Síndrome de Dificultad Respiratoria/fisiopatología , Síndrome de Dificultad Respiratoria/virología , Especificidad de la Especie , Staphylococcus aureus/efectos de los fármacos , Superóxidos/metabolismo , Virus/efectos de los fármacos , Virus/inmunología , Adulto Joven , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
The epithelial to mesenchymal transition (EMT) is a developmental process enabling epithelial cells to gain a migratory mesenchymal phenotype. In cancer, this process contributes to metastases; however the regulatory signals and mechanistic details are not fully elucidated. Here, we sought to identify the subset of genes regulated in lung cancer by ZEB1, an E-box transcriptional repressor known to induce EMT. Using an Affymetrix-based expression database of 38 non-small cell lung cancer (NSCLC) cell lines, we identified 324 genes that correlated negatively with ZEB1 and 142 that were positively correlated. A mesenchymal gene pattern (low E-cadherin, high Vimentin or N-cadherin) was significantly associated with ZEB1 and ZEB2, but not with Snail, Slug, Twist1 or Twist2. Among eight genes selected for validation, seven were confirmed to correlate with ZEB1 by quantitative real-time RT-PCR in a series of 22 NSCLC cell lines, either negatively (CDS1, EpCAM, ESRP1, ESRP2, ST14) or positively (FGFR1, Vimentin). In addition, over-expression or knockdown of ZEB1 led to corresponding changes in gene expression, demonstrating that these genes are also regulated by ZEB1, either directly or indirectly. Of note, the combined knockdown of ZEB1 and ZEB2 led to apparent synergistic responses in gene expression. Furthermore, these responses were not restricted to artificial settings, since most genes were similarly regulated during a physiologic induction of EMT by TGF-ß plus EGF. Finally, the absence of ST14 (matriptase) was linked to ZEB1 positivity in lung cancer tissue microarrays, implying that the regulation observed in vitro applies to the human disease. In summary, this study identifies a new set of ZEB-regulated genes in human lung cancer cells and supports the hypothesis that ZEB1 and ZEB2 are key regulators of the EMT process in this disease.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/patología , Proteínas de Homeodominio/fisiología , Neoplasias Pulmonares/patología , Factores de Transcripción/fisiología , Western Blotting , Cadherinas/genética , Carcinoma de Pulmón de Células no Pequeñas/genética , Línea Celular Tumoral , Transición Epitelial-Mesenquimal , Proteínas de Homeodominio/análisis , Proteínas de Homeodominio/genética , Humanos , Neoplasias Pulmonares/genética , Proteínas Represoras/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Serina Endopeptidasas/análisis , Factores de Transcripción/análisis , Factores de Transcripción/genética , Caja Homeótica 2 de Unión a E-Box con Dedos de Zinc , Homeobox 1 de Unión a la E-Box con Dedos de ZincRESUMEN
Congenital heart defects comprise the most common form of major birth defects, affecting 0.7% of all newborn infants. Jacobsen syndrome (11q-) is a rare chromosomal disorder caused by deletions in distal 11q. We have previously determined that a wide spectrum of the most common congenital heart defects occur in 11q-, including an unprecedented high frequency of hypoplastic left heart syndrome (HLHS). We identified an approximately 7 Mb 'cardiac critical region' in distal 11q that contains a putative causative gene(s) for congenital heart disease. In this study, we utilized chromosomal microarray mapping to characterize three patients with 11q- and congenital heart defects that carry interstitial deletions overlapping the 7 Mb cardiac critical region. We propose that this 1.2 Mb region of overlap harbors a gene(s) that causes at least a subset of the congenital heart defects that occur in 11q-. We demonstrate that one gene in this region, ETS-1 (a member of the ETS family of transcription factors), is expressed in the endocardium and neural crest during early mouse heart development. Gene-targeted deletion of ETS-1 in mice in a C57/B6 background causes, with high penetrance, large membranous ventricular septal defects and a bifid cardiac apex, and less frequently a non-apex-forming left ventricle (one of the hallmarks of HLHS). Our results implicate an important role for the ETS-1 transcription factor in mammalian heart development and should provide important insights into some of the most common forms of congenital heart disease.
Asunto(s)
Eliminación de Gen , Defectos del Tabique Interventricular/genética , Ventrículos Cardíacos/anomalías , Síndrome de Deleción Distal 11q de Jacobsen/genética , Proteína Proto-Oncogénica c-ets-1/genética , Animales , Deleción Cromosómica , Mapeo Cromosómico , Cromosomas Humanos Par 11/genética , Defectos del Tabique Interventricular/embriología , Defectos del Tabique Interventricular/metabolismo , Ventrículos Cardíacos/embriología , Ventrículos Cardíacos/metabolismo , Humanos , Síndrome de Deleción Distal 11q de Jacobsen/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína Proto-Oncogénica c-ets-1/metabolismoRESUMEN
PURPOSE: Recognition that the epidermal growth factor receptor (EGFR) was a therapeutic target in non-small cell lung cancer (NSCLC) and other cancers led to development of the small-molecule receptor tyrosine kinase inhibitors gefitinib and erlotinib. Clinical trials established that EGFR tyrosine kinase inhibitors produced objective responses in a minority of NSCLC patients. We examined the sensitivity of 23 NSCLC lines with wild-type or mutated EGFR to gefitinib to determine genes/proteins related to sensitivity, including EGFR and HER2 cell surface expression, phosphorylated EGFR expression, EGFR gene copy number, and EGFR mutational status. Downstream cell cycle and signaling events were compared with growth-inhibitory effects. EXPERIMENTAL DESIGN: We determined gefitinib sensitivity by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays, EGFR expression by fluorescence-activated cell sorting and immunohistochemistry, phosphorylated EGFR by Western blotting, EGFR gene copy number by fluorescence in situ hybridization, and EGFR mutation by sequencing. The cellular effects of gefitinib on cell cycle were determined by flow cytometry and the molecular effects of gefitinib EGFR inhibition on downstream signal proteins by Western blotting. Gefitinib in vivo effects were evaluated in athymic nude mice bearing sensitive and resistant NSCLC xenografts. RESULTS: There was a significant correlation between EGFR gene copy number, EGFR gene mutations, and gefitinib sensitivity. EGFR protein was necessary but not sufficient for predicting sensitivity. Gefitinib-sensitive lines showed a G(1) cell cycle arrest and inactivation of downstream signaling proteins; resistant cell lines had no changes. The in vivo effects mirrored the in vitro effects. CONCLUSIONS: This panel of NSCLC lines characterized for gefitinib response was used to identify predictive molecular markers of response to gefitinib. Several of these have subsequently been shown to identify NSCLC patients likely to benefit from gefitinib therapy.