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Systematic efforts are underway to decipher the genetic changes associated with tumor initiation and progression. However, widespread clinical application of this information is hampered by an inability to identify critical genetic events across the spectrum of human tumors with adequate sensitivity and scalability. Here, we have adapted high-throughput genotyping to query 238 known oncogene mutations across 1,000 human tumor samples. This approach established robust mutation distributions spanning 17 cancer types. Of 17 oncogenes analyzed, we found 14 to be mutated at least once, and 298 (30%) samples carried at least one mutation. Moreover, we identified previously unrecognized oncogene mutations in several tumor types and observed an unexpectedly high number of co-occurring mutations. These results offer a new dimension in tumor genetics, where mutations involving multiple cancer genes may be interrogated simultaneously and in 'real time' to guide cancer classification and rational therapeutic intervention.
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Análisis Mutacional de ADN/métodos , Mutación , Neoplasias/genética , Oncogenes , Perfilación de la Expresión Génica , Genoma Humano , Genotipo , HumanosRESUMEN
The sensitivity of conventional DNA sequencing in tumor biopsies is limited by stromal contamination and by genetic heterogeneity within the cancer. Here, we show that microreactor-based pyrosequencing can detect rare cancer-associated sequence variations by independent and parallel sampling of multiple representatives of a given DNA fragment. This technology can thereby facilitate accurate molecular diagnosis of heterogeneous cancer specimens and enable patient selection for targeted cancer therapies.
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Mapeo Cromosómico/métodos , ADN de Neoplasias/genética , Mutación , Neoplasias/genética , Secuencia de Bases , Humanos , Datos de Secuencia Molecular , Neoplasias/diagnóstico , Sensibilidad y EspecificidadRESUMEN
Polycythemia vera (PV), essential thrombocythemia (ET), and myeloid metaplasia with myelofibrosis (MMM) are clonal disorders arising from hematopoietic progenitors. An internet-based protocol was used to collect clinical information and biological specimens from patients with these diseases. High-throughput DNA resequencing identified a recurrent somatic missense mutation JAK2V617F in granulocyte DNA samples of 121 of 164 PV patients, of which 41 had homozygous and 80 had heterozygous mutations. Molecular and cytogenetic analyses demonstrated that homozygous mutations were due to duplication of the mutant allele. JAK2V617F was also identified in granulocyte DNA samples from 37 of 115 ET and 16 of 46 MMM patients, but was not observed in 269 normal individuals. In vitro analysis demonstrated that JAK2V617F is a constitutively active tyrosine kinase.
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Mutación Missense/genética , Policitemia Vera/genética , Mielofibrosis Primaria/genética , Proteínas Tirosina Quinasas/genética , Proteínas Proto-Oncogénicas/genética , Trombocitemia Esencial/genética , Adulto , Anciano , Anciano de 80 o más Años , Línea Celular Tumoral , Activación Enzimática/genética , Femenino , Genotipo , Granulocitos/metabolismo , Heterocigoto , Homocigoto , Humanos , Janus Quinasa 2 , Masculino , Persona de Mediana Edad , Mitosis/genética , Modelos Moleculares , Mucosa Bucal/metabolismo , Fosforilación , Mielofibrosis Primaria/complicaciones , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Recombinación Genética/genética , TransfecciónRESUMEN
Comprehensive knowledge of the genomic alterations that underlie cancer is a critical foundation for diagnostics, prognostics, and targeted therapeutics. Systematic efforts to analyze cancer genomes are underway, but the analysis is hampered by the lack of a statistical framework to distinguish meaningful events from random background aberrations. Here we describe a systematic method, called Genomic Identification of Significant Targets in Cancer (GISTIC), designed for analyzing chromosomal aberrations in cancer. We use it to study chromosomal aberrations in 141 gliomas and compare the results with two prior studies. Traditional methods highlight hundreds of altered regions with little concordance between studies. The new approach reveals a highly concordant picture involving approximately 35 significant events, including 16-18 broad events near chromosome-arm size and 16-21 focal events. Approximately half of these events correspond to known cancer-related genes, only some of which have been previously tied to glioma. We also show that superimposed broad and focal events may have different biological consequences. Specifically, gliomas with broad amplification of chromosome 7 have properties different from those with overlapping focalEGFR amplification: the broad events act in part through effects on MET and its ligand HGF and correlate with MET dependence in vitro. Our results support the feasibility and utility of systematic characterization of the cancer genome.
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Aberraciones Cromosómicas/estadística & datos numéricos , Glioma/genética , Línea Celular Tumoral , Interpretación Estadística de Datos , Glioma/patología , Humanos , Polimorfismo de Nucleótido Simple , ProbabilidadRESUMEN
BACKGROUND: The epidermal growth factor receptor (EGFR) is frequently amplified, overexpressed, or mutated in glioblastomas, but only 10 to 20 percent of patients have a response to EGFR kinase inhibitors. The mechanism of responsiveness of glioblastomas to these inhibitors is unknown. METHODS: We sequenced kinase domains in the EGFR and human EGFR type 2 (Her2/neu) genes and analyzed the expression of EGFR, EGFR deletion mutant variant III (EGFRvIII), and the tumor-suppressor protein PTEN in recurrent malignant gliomas from patients who had received EGFR kinase inhibitors. We determined the molecular correlates of clinical response, validated them in an independent data set, and identified effects of the molecular abnormalities in vitro. RESULTS: Of 49 patients with recurrent malignant glioma who were treated with EGFR kinase inhibitors, 9 had tumor shrinkage of at least 25 percent. Pretreatment tissue was available for molecular analysis from 26 patients, 7 of whom had had a response and 19 of whom had rapid progression during therapy. No mutations in EGFR or Her2/neu kinase domains were detected in the tumors. Coexpression of EGFRvIII and PTEN was significantly associated with a clinical response (P<0.001; odds ratio, 51; 95 percent confidence interval, 4 to 669). These findings were validated in 33 patients who received similar treatment for glioblastoma at a different institution (P=0.001; odds ratio, 40; 95 percent confidence interval, 3 to 468). In vitro, coexpression of EGFRvIII and PTEN sensitized glioblastoma cells to erlotinib. CONCLUSIONS: Coexpression of EGFRvIII and PTEN by glioblastoma cells is associated with responsiveness to EGFR kinase inhibitors.
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Receptores ErbB/genética , Glioblastoma/genética , Fosfohidrolasa PTEN/metabolismo , Inhibidores de Proteínas Quinasas/uso terapéutico , Adulto , Anciano , Antineoplásicos/uso terapéutico , ADN de Neoplasias/análisis , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/metabolismo , Clorhidrato de Erlotinib , Femenino , Gefitinib , Amplificación de Genes , Eliminación de Gen , Expresión Génica , Genes erbB-1 , Genes erbB-2 , Glioblastoma/tratamiento farmacológico , Glioblastoma/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Mutación , Oligodendroglioma/tratamiento farmacológico , Oligodendroglioma/genética , Oligodendroglioma/metabolismo , Fosfohidrolasa PTEN/genética , Reacción en Cadena de la Polimerasa , Quinazolinas/uso terapéutico , Análisis de Secuencia de ADN , Transducción de SeñalRESUMEN
PURPOSE: Mutations in the epidermal growth factor receptor (EGFR) are associated with clinical and radiographic responses to EGFR tyrosine kinase inhibitors gefitinib and erlotinib. Currently available methods of EGFR mutation detection rely on direct DNA sequencing, which requires isolation of DNA from a relatively pure population of tumor cells, cannot be done on small diagnostic specimens, and lack sensitivity. Here we describe the use of a sensitive screening method that overcomes many of these limitations. EXPERIMENTAL DESIGN: We screened 178 non-small cell lung cancer specimens for mutations in exons 18 to 21 of EGFR using a DNA endonuclease, SURVEYOR, which cleaves mismatched heteroduplexed DNA. Samples were analyzed by high-performance liquid chromatography on the Transgenomic WAVE HS system. Selected specimens that produced digestion products using SURVEYOR were subsequently reanalyzed by size separation or under partially denaturing conditions, followed by fractionation and sequencing. The specimens included DNA isolated from frozen tumor specimens, dissected formalin-fixed, paraffin-embedded tumor specimens undergoing clinical sequencing, and undissected formalin-fixed, paraffin-embedded specimens. One hundred sixty specimens were independently analyzed using direct DNA sequencing in a blinded fashion. RESULTS: EGFR mutations were detected in 16 of 61 fresh frozen tumor specimens, 24 of 91 dissected formalin-fixed, paraffin-embedded tumor specimens, and 11 of 26 undissected formalin-fixed, paraffin-embedded tumor specimens. Compared with sequencing, the sensitivity and specificity of the present method were 100% and 87%. The positive and negative predictive values were 74% and 100%, respectively. SURVEYOR analysis detected 7 (4%) mutations that were not previously detected by direct sequencing. CONCLUSIONS: SURVEYOR analysis provides a rapid method for EGFR mutation screening with 100% sensitivity and negative predictive value. This unbiased scanning technique is superior to direct sequencing when used with undissected formalin-fixed, paraffin-embedded specimens.
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Carcinoma de Pulmón de Células no Pequeñas/genética , Desoxirribonucleasa I , Receptores ErbB/genética , Neoplasias Pulmonares/genética , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Línea Celular Tumoral , Cromatografía Líquida de Alta Presión , Análisis Mutacional de ADN/métodos , ADN de Neoplasias/química , ADN de Neoplasias/aislamiento & purificación , Exones , Femenino , Formaldehído/química , Gefitinib , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Masculino , Mutación , Adhesión en Parafina , Valor Predictivo de las Pruebas , Quinazolinas/uso terapéutico , Sensibilidad y EspecificidadRESUMEN
As the literature about epidermal growth factor receptor (EGFR) mutations grows and screening for mutations becomes increasingly integrated into clinical care, it is important to examine how best to do somatic mutational analyses and how best to use the test results in clinical decision making. We began offering mutation screening by comprehensive direct sequence analysis of exons 18 to 24 of the tyrosine kinase domain of EGFR in August 2004 as part of clinical cancer care and protocol therapy at our institutions. All identified potential mutations are confirmed with three to five independent PCRs of the original genomic DNA sample and, if not previously noted in the literature, are compared with the patient's germ-line DNA to ensure the finding is somatic. We formally analyzed the first 100 patients to undergo EGFR sequence analysis and found that testing was feasible and significantly affected the treatment of patients with non-small cell lung cancer (NSCLC). Patients harboring EGFR mutations were significantly more likely to receive recommendations for therapy with EGFR tyrosine kinase inhibitors (i.e., gefitinib or erlotinib) than patients without mutations. However, negative EGFR test results did not prevent physicians from administering these agents to selected patients. Ideally, a standardized technique for mutation testing could be developed, with demonstrated reproducibility and validity. Clinical trials incorporating molecular diagnostics are ongoing to assess the efficacy of EGFR tyrosine kinase inhibitors as first-line therapy for metastatic NSCLC and as adjuvant therapy for early-stage resected NSCLC. It is likely that mutation testing and other molecular analyses will be most useful in these two clinical situations.
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Carcinoma de Pulmón de Células no Pequeñas/genética , Genes erbB-1 , Neoplasias Pulmonares/genética , Mutación , Carcinoma de Pulmón de Células no Pequeñas/terapia , Análisis Mutacional de ADN/métodos , Humanos , Neoplasias Pulmonares/terapiaRESUMEN
Genome-wide copy number changes were analyzed in 70 primary human lung carcinoma specimens and 31 cell lines derived from human lung carcinomas, with high-density arrays representing approximately 115,000 single nucleotide polymorphism loci. In addition to previously characterized loci, two regions of homozygous deletion were found, one near the PTPRD locus on chromosome segment 9p23 in four samples representing both small cell lung carcinoma (SCLC) and non-small cell lung carcinoma (NSCLC) and the second on chromosome segment 3q25 in one sample each of NSCLC and SCLC. High-level amplifications were identified within chromosome segment 8q12-13 in two SCLC specimens, 12p11 in two NSCLC specimens and 22q11 in four NSCLC specimens. Systematic copy number analysis of tyrosine kinase genes identified high-level amplification of EGFR in three NSCLC specimens, FGFR1 in two specimens and ERBB2 and MET in one specimen each. EGFR amplification was shown to be independent of kinase domain mutational status.
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Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Células Pequeñas/genética , Aberraciones Cromosómicas , Neoplasias Pulmonares/genética , Línea Celular Tumoral , Deleción Cromosómica , Quinasa 4 Dependiente de la Ciclina , Quinasa 6 Dependiente de la Ciclina , Quinasas Ciclina-Dependientes/genética , ADN de Neoplasias/genética , Receptores ErbB/genética , Amplificación de Genes , Dosificación de Gen , Genoma Humano , Humanos , Polimorfismo de Nucleótido Simple , Proteínas Tirosina Quinasas/genética , Proteínas Proto-Oncogénicas/genéticaRESUMEN
PURPOSE: Effective and tolerable palliative treatments are needed for patients with incurable squamous cell carcinoma of the head and neck (SCCHN). Single-agent targeted therapies have limited activity in this setting. The feasibility of adding celecoxib to gefitinib for the treatment of incurable SCCHN is unknown. PATIENTS AND METHODS: Nineteen patients with unresectable recurrent locoregional and/or distant metastatic SCCHN with progressive disease after at least one prior chemotherapy or chemoradiotherapy regimen were enrolled onto this single-institution phase I study. Three dose levels were explored: (1) celecoxib 200 mg twice daily plus gefitinib 250 mg daily; (2) celecoxib 400 mg twice daily plus gefitinib 250 mg daily; and (3) celecoxib 400 mg twice daily plus gefitinib 500 mg daily. RESULTS: No dose-limiting toxicities were encountered at any dose level. The most common toxicities were acneiform rash, diarrhea, hand reaction, dyspepsia, and anemia. Four of 18 patients assessable for response (22%; 95% CI, 2% to 42%) achieved a confirmed partial response. CONCLUSION: The combination of gefitinib 500 mg daily plus celecoxib 400 mg twice daily is well-tolerated. The encouraging responses seen in this early study suggest further evaluation of epidermal growth factor receptor and cyclooxygenase-2 inhibitors in SCCHN is warranted.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Carcinoma de Células Escamosas/tratamiento farmacológico , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Administración Oral , Adulto , Anciano , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Celecoxib , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Femenino , Gefitinib , Humanos , Masculino , Persona de Mediana Edad , Metástasis de la Neoplasia , Cuidados Paliativos , Pirazoles/administración & dosificación , Quinazolinas/administración & dosificación , Sulfonamidas/administración & dosificaciónRESUMEN
BACKGROUND: Protein tyrosine kinases are important regulators of cellular homeostasis with tightly controlled catalytic activity. Mutations in kinase-encoding genes can relieve the autoinhibitory constraints on kinase activity, can promote malignant transformation, and appear to be a major determinant of response to kinase inhibitor therapy. Missense mutations in the EGFR kinase domain, for example, have recently been identified in patients who showed clinical responses to EGFR kinase inhibitor therapy. METHODS AND FINDINGS: Encouraged by the promising clinical activity of epidermal growth factor receptor (EGFR) kinase inhibitors in treating glioblastoma in humans, we have sequenced the complete EGFR coding sequence in glioma tumor samples and cell lines. We identified novel missense mutations in the extracellular domain of EGFR in 13.6% (18/132) of glioblastomas and 12.5% (1/8) of glioblastoma cell lines. These EGFR mutations were associated with increased EGFR gene dosage and conferred anchorage-independent growth and tumorigenicity to NIH-3T3 cells. Cells transformed by expression of these EGFR mutants were sensitive to small-molecule EGFR kinase inhibitors. CONCLUSIONS: Our results suggest extracellular missense mutations as a novel mechanism for oncogenic EGFR activation and may help identify patients who can benefit from EGFR kinase inhibitors for treatment of glioblastoma.
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Receptores ErbB/genética , Mutación Missense , Quinazolinas/farmacología , Animales , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Sitios de Unión/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Células Cultivadas , Receptores ErbB/química , Receptores ErbB/metabolismo , Clorhidrato de Erlotinib , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glioblastoma/genética , Glioblastoma/patología , Humanos , Ratones , Ratones Desnudos , Modelos Moleculares , Células 3T3 NIH , Neoplasias Experimentales/genética , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/patología , Fosforilación , Unión Proteica , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Estructura Terciaria de Proteína , Quinazolinas/química , Quinazolinas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , TransfecciónRESUMEN
We report a two-step postsynthetic functionalization reaction of zeolite HZSM-5 that proceeds with high selectivity at room temperature. In the first step the framework acid sites of the zeolite are reacted with phenylsilane to replace the acidic proton with a hydrosilyl (-SiH3) group covalently linked to the framework. This group readily couples to acetone in a second step to form a framework-bound hydrosilyl isopropyl ether that is thermally stable at 473 K, but decomposes in the presence of moisture. We characterized these reactions using 29Si, 1H, and 13C MAS NMR, as appropriate. Theoretical modeling using density functional theory and cluster models of the zeolite acid site confirmed that both steps were exothermic and provided theoretical chemical shift values in excellent agreement with experiment.
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Major efforts are underway to systematically define the somatic and germline genetic variations causally associated with disease. Genome-wide genetic analysis of actual clinical samples is, however, limited by the paucity of genomic DNA available. Here we have tested the fidelity and genome representation of phi29 polymerase-based genome amplification (phi29MDA) using direct sequencing and high density oligonucleotide arrays probing >10,000 SNP alleles. Genome representation was comprehensive and estimated to be 99.82% complete, although six regions encompassing a maximum of 5.62 Mb failed to amplify. There was no degradation in the accuracy of SNP genotyping and, in direct sequencing experiments sampling 500,000 bp, the estimated error rate (9.5 x 10(-6)) was the same as in paired unamplified samples. The detection of cancer-associated loss of heterozygosity and copy number changes, including homozygous deletion and gene amplification, were similarly robust. These results suggest that phi29MDA yields high fidelity, near-complete genome representation suitable for high resolution genetic analysis.
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Fagos de Bacillus/enzimología , ADN Polimerasa Dirigida por ADN/metabolismo , Genoma Humano , Genómica/métodos , Reacción en Cadena de la Polimerasa/métodos , Alelos , Línea Celular , Línea Celular Tumoral , Deleción Cromosómica , Dosificación de Gen , Genotipo , Homocigoto , Humanos , Pérdida de Heterocigocidad/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Polimorfismo de Nucleótido Simple/genética , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Análisis de Secuencia de ADNAsunto(s)
Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/genética , Antineoplásicos/uso terapéutico , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Receptores ErbB/genética , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Mutación , Quinazolinas/uso terapéutico , Anciano , Gefitinib , Humanos , Masculino , Proteínas Tirosina Quinasas/antagonistas & inhibidoresRESUMEN
Targeted cancer therapies impede cancer cell growth by inhibiting the function of activated oncogene products. Patients with non-small cell lung cancer and somatic mutations of EGFR can have a dramatic response to treatment with erlotinib and gefitinib; different somatic mutations are associated with different times to progression and survival. In this study, the relative and absolute potencies of two distinct EGFR tyrosine kinase inhibitors, erlotinib and an investigational irreversible inhibitor, HKI-272, were found to vary significantly in a panel of Ba/F3 cells transformed by representative EGFR somatic mutations. HKI-272 more potently inhibited the primary exon 20 insertion mutants, the secondary erlotinib-resistance mutants including T790M and many erlotinib-sensitive mutants including L858R. In contrast, erlotinib is a more potent inhibitor of the major exon 19 deletion mutants than is HKI-272. Analyses of EGFR autophosphorylation patterns confirmed the mutation-specific variation in relative potency of these tyrosine kinase inhibitors. Our finding that distinct EGFR inhibitors are more effective in vitro for different mutant forms of the protein suggests that tyrosine kinase inhibitor treatment could be tailored to specific EGFR mutations. More broadly, these results imply that the development and deployment of targeted therapies should focus on inhibition of specific cancer-causing mutations, not only on the mutated target.
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Alelos , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/genética , Neoplasias Pulmonares/genética , Inhibidores de Proteínas Quinasas/farmacología , Quinazolinas/farmacología , Quinolinas/farmacología , Sustitución de Aminoácidos , Animales , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Cartilla de ADN/química , Clorhidrato de Erlotinib , Humanos , Ratones , Fragmentos de Péptidos/química , Fosforilación , Mutación Puntual , Células Precursoras de Linfocitos B/efectos de los fármacos , Células Precursoras de Linfocitos B/metabolismo , Retroviridae/genética , Eliminación de SecuenciaRESUMEN
Phosphorus modification of a HZSM-5 (MFI) zeolite by wet impregnation has long been known to decrease aromatic formation in methanol conversion chemistry. We prepared and studied a catalyst modified by introducing trimethylphosphine under reaction conditions followed by oxidation. Magic-angle spinning (MAS) NMR shows that extensive dealumination occurs, resulting in a catalyst with a much higher framework SiO2/Al2O3 ratio, as well as extraframework aluminum and approximately 1.4 equiv of entrained phosphoric acid (under working conditions) per aluminum. Upon dehydration or regeneration, the phosphoric acid is converted, reversibly, to entrained P4O10. The aromatic selectivity of the modified catalyst is significantly lower than that of an unmodified zeolite with a similar, increased framework SiO2/Al2O3 ratio. By comparing the rates of H/D exchange in propene under conditions similar to those in methanol conversion chemistry, we determined that the acid site strength is indistinguishable on modified and unmodified zeolites, and this is consistent with theoretical modeling. On the phosphorus-modified zeolite, the rate of propene oligomerization is greatly suppressed, suggesting that entrained phosphate is an impediment to sterically demanding reactions.
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MOTIVATION: Predicting the outcome of specific experiments (such as the growth of a particular mutant strain in a particular medium) has the potential to allow researchers to devote resources to experiments with higher expected numbers of 'hits'. RESULTS: We use decision trees to predict phenotypes associated with Saccharomyces cerevisiae genes on the basis of Gene Ontology (GO) functional annotations from the Saccharomyces Genome Database (SGD) and other phenotypic annotations from the Yeast Phenotype Catalog at the Munich Information Center for Protein Sequences (MIPS). We assess the methodology in three ways: (1) we use cross-validation on the phenotypic annotations listed in MIPS, and show ROC curves indicating the tradeoff between true-positive rate and false-positive rate; (2) we do a literature-search for 100 of the predicted gene-phenotype associations that are not listed in MIPS, and find evidence for 43 of them; (3) we use deletion strains to experimentally assess 61 predicted gene-phenotype associations not listed in MIPS; significantly more of these deletion strains show abnormal growth than would be expected by chance.
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Algoritmos , Inteligencia Artificial , Documentación , Perfilación de la Expresión Génica/métodos , Fenotipo , Saccharomyces cerevisiae/genética , Saccharomycetales/genética , Bases de Datos Genéticas , Reconocimiento de Normas Patrones Automatizadas , Saccharomyces cerevisiae/metabolismo , Saccharomycetales/clasificación , Saccharomycetales/metabolismoRESUMEN
Lineally related multipotent neuroepithelial cells (NEP), neuronal restricted precursors (NRP), and glial restricted precursors (GRP) have been identified in the spinal cord. To determine the sequence of differentiation and identify lineage and stage-specific markers, we have examined the spatiotemporal expression of established glial markers during rodent embryonic development and within fetal cell culture. In this report, we show that proliferating stem cells in the developing neural tube do not express any glial markers at E10.5. By E11, however, glial precursors have begun to differentiate and at least two regions of the ventral neural tube containing glial precursor cells can be distinguished, an Nkx2.2/Neurogenin 3 (Ngn3) domain and a platelet-derived growth factor receptor alpha (PDGFRalpha)/Olig2/Sox10 domain. Radial glia, as identified by RC1 immunoreactivity, develop in concert with other glial precursors and can be distinguished by their morphology, spatial distribution, and antigen expression. Astrocytes as assessed by glial fibrillary acidic protein (GFAP) immunoreactivity are first detected at E16. A novel dorsal domain of CD44 immunoreactivity that can be distinguished from the more ventral glial precursor domains can be detected as early as E13.5.
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Astrocitos/metabolismo , Diferenciación Celular/fisiología , Sistema Nervioso Central/embriología , Sistema Nervioso Central/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica/genética , Oligodendroglía/metabolismo , Células Madre/metabolismo , Animales , Animales Recién Nacidos , Astrocitos/citología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , División Celular/genética , Células Cultivadas , Sistema Nervioso Central/citología , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Feto , Proteína Ácida Fibrilar de la Glía/genética , Proteína Ácida Fibrilar de la Glía/metabolismo , Proteínas del Grupo de Alta Movilidad/genética , Proteínas del Grupo de Alta Movilidad/metabolismo , Proteína Homeobox Nkx-2.2 , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Receptores de Hialuranos/genética , Receptores de Hialuranos/metabolismo , Inmunohistoquímica , Hibridación in Situ , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Oligodendroglía/citología , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/genética , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Factores de Transcripción SOXE , Células Madre/citología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas de Pez CebraRESUMEN
RNA interference (RNAi) mediated by short interfering RNAs (siRNAs) is a widely used method to analyze gene function. To use RNAi knockdown accurately to infer gene function, it is essential to determine the specificity of siRNA-mediated RNAi. We have assessed the specificity of 10 different siRNAs corresponding to the MEN1 gene by examining the expression of two additional genes, TP53 (p53) and CDKN1A (p21), which are considered functionally unrelated to menin but are sensitive markers of cell state. MEN1 RNA and corresponding protein levels were all reduced after siRNA transfection of HeLa cells, although the degree of inhibition mediated by individual siRNAs varied. Unexpectedly, we observed dramatic and significant changes in protein levels of p53 and p21 that were unrelated to silencing of the target gene. The modulations in p53 and p21 levels were not abolished on titration of the siRNAs, and similar results were obtained in three other cell lines; in none of the cell lines tested did we see an effect on the protein levels of actin. These data suggest that siRNAs can induce nonspecific effects on protein levels that are siRNA sequence dependent but that these effects may be difficult to detect until genes central to a pivotal cellular response, such as p53 and p21, are studied. We find no evidence that activation of the double-stranded RNA-triggered IFN-associated antiviral pathways accounts for these effects, but we speculate that partial complementary sequence matches to off-target genes may result in a micro-RNA-like inhibition of translation.
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Regulación de la Expresión Génica , Proteínas Proto-Oncogénicas/genética , ARN Interferente Pequeño/metabolismo , 2',5'-Oligoadenilato Sintetasa/genética , Animales , Western Blotting , Línea Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/genética , Ciclinas/metabolismo , Células HeLa , Humanos , Interferones/fisiología , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia , Especificidad por Sustrato , Transfección , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Receptor tyrosine kinase genes were sequenced in non-small cell lung cancer (NSCLC) and matched normal tissue. Somatic mutations of the epidermal growth factor receptor gene EGFR were found in 15of 58 unselected tumors from Japan and 1 of 61 from the United States. Treatment with the EGFR kinase inhibitor gefitinib (Iressa) causes tumor regression in some patients with NSCLC, more frequently in Japan. EGFR mutations were found in additional lung cancer samples from U.S. patients who responded to gefitinib therapy and in a lung adenocarcinoma cell line that was hypersensitive to growth inhibition by gefitinib, but not in gefitinib-insensitive tumors or cell lines. These results suggest that EGFR mutations may predict sensitivity to gefitinib.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/genética , Receptores ErbB/antagonistas & inhibidores , Genes erbB-1 , Neoplasias Pulmonares/genética , Mutación , Quinazolinas/uso terapéutico , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Línea Celular Tumoral , Ensayos Clínicos Controlados como Asunto , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Receptores ErbB/química , Receptores ErbB/genética , Receptores ErbB/metabolismo , Femenino , Gefitinib , Humanos , Japón , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/metabolismo , Masculino , Datos de Secuencia Molecular , Mutación Missense , Fosforilación , Conformación Proteica , Estructura Terciaria de Proteína , Quinazolinas/farmacología , Eliminación de Secuencia , Resultado del Tratamiento , Estados UnidosRESUMEN
The cellular function of the menin tumor suppressor protein, product of the MEN1 gene mutated in familial multiple endocrine neoplasia type 1, has not been defined. We now show that menin is associated with a histone methyltransferase complex containing two trithorax family proteins, MLL2 and Ash2L, and other homologs of the yeast Set1 assembly. This menin-associated complex methylates histone H3 on lysine 4. A subset of tumor-derived menin mutants lacks the associated histone methyltransferase activity. In addition, menin is associated with RNA polymerase II whose large subunit carboxyl-terminal domain is phosphorylated on Ser 5. Men1 knockout embryos and cells show decreased expression of the homeobox genes Hoxc6 and Hoxc8. Chromatin immunoprecipitation experiments reveal that menin is bound to the Hoxc8 locus. These results suggest that menin activates the transcription of differentiation-regulating genes by covalent histone modification, and that this activity is related to tumor suppression by MEN1.