RESUMEN
Archived formalin-fixed, paraffin embedded (FFPE) tissues constitute a vast, well-annotated, but underexploited resource for the molecular study of cancer progression, largely because degradation, chemical modification, and cross-linking, render FFPE RNA a suboptimal substrate for conventional analytical methods. We report here a modified protocol for RNA extraction from FFPE tissues which maximized the success rate (with 100% of samples) in the expression profiling of a set of 60 breast cancer samples on the WG-DASL platform; yielding data of sufficient quality such that in hierarchical clustering (a) 12/12 (100%) replicates correctly identified their respective counterparts, with a high self-correlation (r = 0.979), and (b) the overall sample set grouped with high specificity into ER+ (38/40; 95%) and ER- (18/20; 90%) subtypes. These results indicate that a large fraction of decade-old FFPE samples, of diverse institutional origins and processing histories, can yield RNA suitable for gene expression profiling experiments.
Asunto(s)
Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Perfilación de la Expresión Génica/métodos , Mama/patología , Análisis por Conglomerados , Estudios de Cohortes , Receptor alfa de Estrógeno/biosíntesis , Femenino , Formaldehído/farmacología , Humanos , Inmunohistoquímica/métodos , Adhesión en Parafina/métodos , ARN Neoplásico/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The A1 allele of the dopamine D2 receptor gene (DRD2) is associated with a reduced number of dopamine binding sites in the brain and with the increased likelihood of substance abuse and addictive behavior. In a study of smokers enrolled in an open-label, randomized effectiveness trial, we investigated whether variants in the DRD2 receptor gene are associated with smoking cessation outcomes following treatment with a combination of bupropion SR and behavioral counseling. Adherence to treatment and point-prevalent smoking status were assessed at 3 and 12 months, respectively, following a target quit date. Compared to women who carry both A2 alleles, women with at least one A1 allele were more likely to report having stopped taking bupropion due to medication side effects (odds ratio (OR)=1.91, 95% confidence interval (CI)=1.01-3.60; P<0.04) and at 12 months were somewhat more likely to report smoking (OR=0.76, 95% CI=0.56-1.03; P<0.076). Significant associations or trends were not observed in men. In women, individual variability in responsiveness to bupropion-based treatment may be partially due to differences in genetic variants influencing dopamine receptor function.
Asunto(s)
Bupropión/uso terapéutico , Receptores de Dopamina D2/genética , Cese del Hábito de Fumar , Fumar/tratamiento farmacológico , Fumar/genética , Adulto , Femenino , Estudios de Seguimiento , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Cese del Hábito de Fumar/métodos , Resultado del TratamientoRESUMEN
The Wilms' tumor gene, WT1, is expressed in few tissues, mainly the developing kidney, genitourinary system, and mesothelium, and in immature hematopoietic cells. To develop an understanding of the role of WT1 in development and tumorigenesis, we have identified transcriptional regulatory elements that function in transient reporter gene constructs transfected into kidney and hematopoietic cell lines. We found three transcription start sites of the WT1 gene and have identified an essential promoter region by deletion analysis. The WT1 promoter is a member of the GC-rich, TATA-less, and CCAAT-less class of polymerase II promoters. Whereas the WT1 promoter is similar to other tumor suppressor gene promoters, the WT1 expression pattern (unlike Rb and p53) is tissue-restricted. The WT1 GC-rich promoter is promiscuous, functioning in all cell lines tested, independent of WT1 expression. This finding suggests that the promoter is not tissue-specific, but that tissue-specific expression of WT1 is modulated by additional regulatory elements. Indeed, we have identified a transcriptional enhancer located 3' of the WT1 gene > 50 kilobases downstream from the promoter. This orientation-independent enhancer increases the basal transcription rate of the WT1 promoter in the human erythroleukemia cell line K562, but not in any of the other cell lines tested.
Asunto(s)
Proteínas de Unión al ADN/genética , Genes del Tumor de Wilms , Tumor de Wilms/genética , Secuencia de Bases , Sitios de Unión , Análisis Mutacional de ADN , Elementos de Facilitación Genéticos , Regulación Neoplásica de la Expresión Génica , Humanos , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , ARN Mensajero/genética , Mapeo Restrictivo , Eliminación de Secuencia , Factores de Transcripción/metabolismo , Transcripción Genética , Células Tumorales Cultivadas , Proteínas WT1RESUMEN
The PAX6 gene is expressed at high levels in the developing eye and cerebellum and is mutated in patients with autosomal dominant aniridia. We have tested the role of PAX6 mutations in three families with Gillespie syndrome, a rare autosomal recessive condition consisting of partial aniridia, cerebellar ataxia, and mental retardation. Single-strand conformational polymorphism analysis of affected individuals revealed no alteration of PAX6 sequences. In two families, the disease trait segregates independently from chromosome 11p markers flanking PAX6. We conclude that Gillespie syndrome is genetically distinct from autosomal dominant aniridia.
Asunto(s)
Aniridia/genética , Ataxia Cerebelosa/genética , Proteínas de Unión al ADN/genética , Genes , Proteínas de Homeodominio , Discapacidad Intelectual/genética , Alelos , Aniridia/clasificación , Secuencia de Bases , Brasil , Análisis Mutacional de ADN , Proteínas del Ojo , Femenino , Humanos , Masculino , Datos de Secuencia Molecular , Irlanda del Norte , Factor de Transcripción PAX6 , Factores de Transcripción Paired Box , Linaje , Polimorfismo Genético , Proteínas Represoras , SíndromeRESUMEN
Phenotypic parallels and genetic evidence from comparative mapping suggest that the murine Small eye (Sey) and human aniridia (AN) disorders are homologous. This report describes the isolation of a murine embryonic cDNA that is structurally homologous to the AN cDNA were recently cloned. The murine cDNA detects a 2.7-kb transcript in the adult mouse eye and cerebellum and in human glioblastomas, suggesting a neuroectodermal involvement in the etiology of Sey/AN. Sequence comparison between the murine and the human cDNAs revealed extensive homology in nucleotide sequence (greater than 92%) and virtual identity at the amino acid level. None of the differing amino acids was located within the paired box and homeobox DNA-binding domains. These results provide evidence for a common molecular basis underlying the two genetic disorders and suggest that the Sey system would be an authentic model for human AN.
Asunto(s)
Anomalías del Ojo/genética , Genes Letales , Ratones/genética , Secuencia de Aminoácidos , Animales , Aniridia/embriología , Aniridia/genética , Secuencia de Bases , Encéfalo/crecimiento & desarrollo , Encéfalo/metabolismo , Neoplasias del Sistema Nervioso Central/genética , Neoplasias del Sistema Nervioso Central/patología , Clonación Molecular , ADN/genética , Ojo/crecimiento & desarrollo , Ojo/metabolismo , Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Datos de Secuencia Molecular , Cresta Neural/metabolismo , Nariz/anomalías , Especificidad de Órganos , ARN Mensajero/biosíntesis , ARN Neoplásico/biosíntesis , Secuencias Reguladoras de Ácidos Nucleicos , Homología de Secuencia de Ácido Nucleico , Células Tumorales Cultivadas/metabolismoRESUMEN
Small eye (Sey) in mouse is a semidominant mutation which in the homozygous condition results in the complete lack of eyes and nasal primordia. On the basis of comparative mapping studies and on phenotypic similarities, Sey has been suggested to be homologous to congenital aniridia (lack of iris) in human. A candidate gene for the aniridia (AN) locus at 11p13 has been isolated by positional cloning and its sequence and that of the mouse homologue has been established (C.T., manuscript in preparation). This gene belongs to the paired-like class of developmental genes first described in Drosophila which contain two highly conserved motifs, the paired box and the homeobox. In vertebrates, genes which encode the single paired domain as well as those which express both motifs have been described as the Pax multigene family. A Pax gene recently described as Pax-6 is identical to the mouse homologue of the candidate aniridia gene. Here we report the analysis of three independent Sey alleles and show that indeed this gene is mutated and that the mutations would predictably interrupt gene function.
Asunto(s)
Genes Homeobox , Ratones Mutantes/genética , Mutación , Alelos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Southern Blotting , Deleción Cromosómica , Clonación Molecular , Cruzamientos Genéticos , Embrión de Mamíferos , Desarrollo Embrionario y Fetal , Ojo/embriología , Anomalías del Ojo/genética , Femenino , Heterocigoto , Masculino , Ratones , Ratones Endogámicos C57BL/genética , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Fenotipo , Reacción en Cadena de la Polimerasa/métodos , Mapeo RestrictivoRESUMEN
Based on the map location of the aniridia (AN) locus in human chromosomal band 11p13, we have cloned a candidate AN cDNA (D11S812E) that is completely or partially deleted in two patients with AN. The less than 70 kb smallest region of overlap between the two deletions encompasses the 3' coding region of the cDNA. This cDNA, which spans over 50 kb of genomic DNA, detects a 2.7 kb message specifically within all tissues affected in AN. The predicted polypeptide product possesses a paired domain, a homeodomain, and a serine/threonine-rich carboxy-terminal domain, structural motifs characteristic of certain transcription factors. The concordance between expression and pathology, map location, structure, and predicted function argues that the cDNA corresponds to the AN gene.
Asunto(s)
Aniridia/genética , Cromosomas Humanos Par 11 , Proteínas de Unión al ADN/genética , Genes Homeobox , Factores de Transcripción/genética , Secuencia de Aminoácidos , Secuencia de Bases , Northern Blotting , Southern Blotting , Deleción Cromosómica , Clonación Molecular , ADN/genética , Ojo/embriología , Expresión Génica , Humanos , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , ARN Mensajero , Mapeo Restrictivo , Alineación de SecuenciaRESUMEN
The development of Wilms tumor (WT) has been associated with the inactivation of a "tumor suppressor" locus in human chromosome 11 band p13. Several WTs that exhibit homozygous deletions of an 11p13 candidate WT gene in its entirety have been reported. We report here a partial deletion of the candidate gene which, upon comparison with other documented homozygous deletions, permitted a precise definition of the critical genomic target in Wilms tumor. The smallest region of overlap between these deletions is a 16-kb segment of DNA encompassing the 5' exon(s) of an 11p13 gene coding for a zinc finger protein, together with an associated CpG island. This finding supports the notion that the candidate gene in question corresponds to the 11p13 WT1 Wilms tumor locus.
Asunto(s)
Deleción Cromosómica , Cromosomas Humanos Par 11 , Tumor de Wilms/genética , Dedos de Zinc/genética , Preescolar , Femenino , Humanos , Masculino , Mapeo RestrictivoAsunto(s)
Proteínas Bacterianas , Cromosomas Humanos Par 11 , Antígenos HLA-B/genética , Antígenos HLA-DR/genética , Antígeno HLA-DR4/genética , Antígeno HLA-DR7/genética , Polimorfismo de Longitud del Fragmento de Restricción , Alelos , Bandeo Cromosómico , Sondas de ADN , Desoxirribonucleasas de Localización Especificada Tipo II , HumanosRESUMEN
The influence of ascorbate deficiency and megadosage on the metabolism of N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) was investigated in the guinea pig. After 21 days on a scorbutogenic diet, microsomal cytochrome P-450 and cytochrome b5 levels fell by 51 and 32%, respectively, while cytochrome c reductase activity remained constant. The activities of NDMA and NDEA dealkylase I were also depressed significantly. The Vmax of NDMA demethylase I and NDEA deethylase I was significantly depressed. Also, ascorbate deficiency significantly decreased the plasma clearance of both nitrosamines though the LD50 of neither were altered by ascorbate nutrition. Covalent binding of 14C from [14C]NDMA and [14C]NDEA to DNA obtained from liver slices was significantly lower in the deficient than in the control samples; megadosage appeared to have the opposite effect.