RESUMEN
Background: A major limitation of circulating tumor DNA (ctDNA) for somatic mutation detection has been the low level of ctDNA found in a subset of cancer patients. We investigated whether using a combined isolation of exosomal RNA (exoRNA) and cell-free DNA (cfDNA) could improve blood-based liquid biopsy for EGFR mutation detection in non-small-cell lung cancer (NSCLC) patients. Patients and methods: Matched pretreatment tumor and plasma were collected from 84 patients enrolled in TIGER-X (NCT01526928), a phase 1/2 study of rociletinib in mutant EGFR NSCLC patients. The combined isolated exoRNA and cfDNA (exoNA) was analyzed blinded for mutations using a targeted next-generation sequencing panel (EXO1000) and compared with existing data from the same samples using analysis of ctDNA by BEAMing. Results: For exoNA, the sensitivity was 98% for detection of activating EGFR mutations and 90% for EGFR T790M. The corresponding sensitivities for ctDNA by BEAMing were 82% for activating mutations and 84% for T790M. In a subgroup of patients with intrathoracic metastatic disease (M0/M1a; n = 21), the sensitivity increased from 26% to 74% for activating mutations (P = 0.003) and from 19% to 31% for T790M (P = 0.5) when using exoNA for detection. Conclusions: Combining exoRNA and ctDNA increased the sensitivity for EGFR mutation detection in plasma, with the largest improvement seen in the subgroup of M0/M1a disease patients known to have low levels of ctDNA and poses challenges for mutation detection on ctDNA alone. Clinical Trials: NCT01526928.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/sangre , ADN Tumoral Circulante/sangre , Análisis Mutacional de ADN/métodos , Neoplasias Pulmonares/sangre , ARN/sangre , Acrilamidas/uso terapéutico , Adulto , Anciano , Antineoplásicos/uso terapéutico , Biomarcadores de Tumor/sangre , Biomarcadores de Tumor/genética , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Receptores ErbB/genética , Exosomas , Femenino , Genes erbB-1 , Humanos , Biopsia Líquida/métodos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Masculino , Persona de Mediana Edad , Pirimidinas/uso terapéutico , Sensibilidad y EspecificidadRESUMEN
The Huntington's disease (HD) gene encodes a protein, huntingtin, with no known function and no detectable sequence similarity to other proteins in current databases. To gain insight into the normal biological role of huntingtin, we isolated and sequenced a cDNA encoding a protein that is a likely homolog of the HD gene product in Drosophila melanogaster. We also determined the complete sequence of 43 125 contiguous base pairs of genomic DNA that encompass the Drosophila HD gene, allowing the intron-exon structure and 5'- and 3'-flanking regions to be delineated. The predicted Drosophila huntingtin protein has 3583 amino acids, which is several hundred amino acids larger than any other previously characterized member of the HD family. Analysis of the genomic and cDNA sequences indicates that Drosophila HD has 29 exons, compared with the 67 exons present in vertebrate HD genes, and that Drosophila huntingtin lacks the polyglutamine and polyproline stretches present in its mammalian counterparts. The Drosophila HD mRNA is expressed in a broad range of developmental stages and in the adult, a temporal pattern of expression similar to that observed for mammalian HD transcripts. We can discern five regions of high similarity from multiple sequence alignments between Drosophila and vertebrate huntingtins. These regions may define functionally important domains within the protein.
Asunto(s)
Drosophila melanogaster/genética , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/genética , Secuencia de Aminoácidos , Animales , Mapeo Cromosómico , Clonación Molecular , Secuencia Conservada , Exones , Regulación del Desarrollo de la Expresión Génica , Genes de Insecto , Humanos , Proteína Huntingtina , Enfermedad de Huntington/genética , Intrones , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/metabolismo , ARN Mensajero/metabolismo , Alineación de SecuenciaRESUMEN
The Huntington's disease (HD) gene contains a trinucleotide repeat that is expanded and unstable in patients with the disease (HDCRG, 1993). As the first step toward investigating a potential role for this gene in early vertebrate development, we isolated the homolog of the Huntington's disease (ZHD) cDNA in zebrafish. This cDNA encodes a predicted protein product of 3121 amino acids with 70% identity to human huntingtin. The first exon is predicted to encode four glutamines, followed by only one proline, demonstrating that the polymorphic polyproline stretch found in mammalian HD sequences is absent in the fish. We sequenced approximately 900bp upstream from the predicted start codon and found that it lacks a TATA box, CCAAT box, or Sp1 binding sites. Western blot analysis revealed that the protein is expressed at a high level in late embryonic development and at moderate levels in the adult head.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Enfermedad de Huntington/genética , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/genética , Repeticiones de Trinucleótidos , Pez Cebra/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cartilla de ADN , ADN Complementario , Embrión no Mamífero/fisiología , Peces , Biblioteca de Genes , Humanos , Proteína Huntingtina , Ratones , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/química , Proteínas Nucleares/biosíntesis , Proteínas Nucleares/química , Ratas , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/química , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Pez Cebra/embriologíaRESUMEN
The lozenge (lz) gene encodes a transcription factor involved in prepatterning photoreceptor precursors in the developing Drosophila eye. The central region of the predicted Lz protein product is homologous to AML1, a transcription factor associated with human leukemias, and to the Drosophila protein Runt. We show here that Lz plays a crucial role in governing the fate of two groups of cells that are born in a single round of mitosis in the larval eye disc. Lz helps define a subset of these cells as an equipotential group that is competent to respond to the Sevenless developmental signal. This is achieved by negative regulation of seven-up, a member of the steroid hormone receptor superfamily in these cells. In contrast, in a second group of cells, the Lz protein confers proper photoreceptor identity by positively regulating the homeo box gene Bar. Additionally, our genetic analysis suggests that Lz interacts with the Ras pathway to determine photoreceptor cell fate. This study suggests that the strategies involved in cell fate determination in the Drosophila eye are remarkably similar to those utilized during vertebrate hematopoietic development and require the coordinate action of growth factor and AML1-like pathways.
Asunto(s)
Proteínas de Drosophila , Drosophila/enzimología , Proteínas Proto-Oncogénicas , Factores de Transcripción/metabolismo , Alelos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , División Celular , Subunidad alfa 2 del Factor de Unión al Sitio Principal , ADN , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Ojo/embriología , Proteínas del Ojo/genética , Femenino , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio , Masculino , Datos de Secuencia Molecular , Proteínas de Neoplasias/química , Proteínas Nucleares , Receptores de Esteroides/genética , Homología de Secuencia de Aminoácido , Factores de Transcripción/química , Factores de Transcripción/genéticaRESUMEN
Activation of p21ras by receptor tyrosine kinases is thought to result from recruitment of guanine nucleotide exchange factors such as Son-of-sevenless (Sos) to plasma membrane receptor substrates via adaptor proteins such as Grb2. This hypothesis was tested in the present studies by evaluating the ability of truncation and deletion mutants of Drosophila (d)Sos to enhance [32P]GTP loading of p21ras when expressed in 32P-labeled COS or 293 cells. The dSos catalytic domain (residues 758-1125), expressed without the dSos NH2-terminal (residues 1-757) or adaptor-binding COOH-terminal (residues 1126-1596) regions, exhibits intrinsic exchange activity as evidenced by its rescue of mutant Saccharomyces cerevisiae deficient in endogenous GTP/GDP exchange activity. Here we show that this dSos catalytic domain fails to affect GTP p21ras levels when expressed in cultured mammalian cells unless the NH2-terminal domain is also present. Surprisingly, the COOH-terminal, adaptor binding domain of dSos was not sufficient to confer p21ras exchange activity to the Sos catalytic domain in these cells in the absence of the NH2-terminal domain. This function of promoting catalytic domain activity could be localized by mutational analysis to the pleckstrin and Dbl homology sequences located just NH2-terminal to the catalytic domain. The results demonstrate a functional role for these pleckstrin and Dbl domains within the dSos protein, and suggest the presence of unidentified cellular elements that interact with these domains and participate in the regulation of p21ras.
Asunto(s)
Proteínas Sanguíneas , Proteínas de la Membrana/metabolismo , Fosfoproteínas , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteínas Proto-Oncogénicas , Homología de Secuencia de Aminoácido , Secuencia de Aminoácidos , Animales , Células Cultivadas , Análisis Mutacional de ADN , Drosophila , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Proteínas de la Membrana/genética , Modelos Biológicos , Datos de Secuencia Molecular , Proteínas Recombinantes/metabolismo , Transducción de Señal , Proteínas Son Of Sevenless , Relación Estructura-ActividadRESUMEN
The Son of sevenless (Sos) protein functions as a guanine nucleotide transfer factor for Ras and interacts with the receptor tyrosine kinase Sevenless through the protein Drk, a homolog of mammalian Grb2. In vivo structure-function analysis revealed that the amino terminus of Sos was essential for its function in flies. A molecule lacking the amino terminus was a potent dominant negative. In contrast, a Sos fragment lacking the Drk binding sites was functional and its activity was dependent on the presence of the Sevenless receptor. Furthermore, membrane localization of Sos was independent of Drk. A possible role for Drk as an activator of Sos is discussed and a Drk-independent interaction between Sos and Sevenless is proposed that is likely mediated by the pleckstrin homology domain within the amino terminus.
Asunto(s)
Proteínas de Drosophila , Proteínas del Ojo/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Animales , Sitios de Unión , Membrana Celular/metabolismo , Drosophila , Factores de Intercambio de Guanina Nucleótido , Hormonas de Insectos/fisiología , Proteínas de la Membrana/química , Células Fotorreceptoras de Invertebrados/citología , Células Fotorreceptoras de Invertebrados/metabolismo , Transducción de Señal , Proteínas Son Of Sevenless , Factores de Intercambio de Guanina Nucleótido rasRESUMEN
The Son of sevenless (Sos) gene functions in signaling pathways initiated by the sevenless and epidermal growth factor receptor tyrosine kinases. The Sos gene has now been isolated and sequenced. Its product is a 1595-amino acid protein similar to the CDC25 protein in Saccharomyces cerevisiae, a guanine nucleotide exchange factor that activates Ras. These results imply a role for the ras pathway in Drosophila neuronal development.
Asunto(s)
Proteínas de Ciclo Celular , Drosophila/genética , Genes ras , Proteínas de la Membrana/genética , ras-GRF1 , Secuencia de Aminoácidos , Animales , Proteínas Fúngicas/genética , Biblioteca de Genes , Genotipo , Datos de Secuencia Molecular , Neuronas/fisiología , Mapeo Restrictivo , Saccharomyces cerevisiae/genética , Homología de Secuencia de Ácido Nucleico , Proteínas Son Of SevenlessRESUMEN
We have isolated a dominant mutation in a gene called Son of sevenless (Sos) that is an allele-specific suppressor of the sevenless phenotype. This suppressor function is autonomously required in R7 and is sensitive to the dosage of the Sos and bride of sevenless genes. Loss-of-function alleles of Sos are recessive lethals, but in the eye Sos has a role in R cell development. Mutations in Sos also interact with the Ellipse allele of the Drosophila EGF receptor. We propose a model suggesting that the Sos product is downstream of sevenless and the EGF receptor, and that the dominant suppression results from the overexpression or increased activity of the gene product.