RESUMEN
Dual oxidase 2 (DUOX2) generates H2O2 that plays a critical role in both host defense and chronic inflammation. Previously, we demonstrated that the proinflammatory mediators IFN-γ and LPS enhance expression of DUOX2 and its maturation factor DUOXA2 through STAT1- and NF-κBâmediated signaling in human pancreatic cancer cells. Using a panel of colon and pancreatic cancer cell lines, we now report the induction of DUOX2/DUOXA2 mRNA and protein expression by the TH2 cytokine IL-4. IL-4 activated STAT6 signaling that, when silenced, significantly decreased induction of DUOX2. Furthermore, the TH17 cytokine IL-17A combined synergistically with IL-4 to increase DUOX2 expression in both colon and pancreatic cancer cells mediated, at least in part, by signaling through NF-κB. The upregulation of DUOX2 was associated with a significant increase in the production of extracellular H2O2 and DNA damage-as indicated by the accumulation of 8-oxo-dG and γH2AX-which was suppressed by the NADPH oxidase inhibitor diphenylene iodonium and a DUOX2-specific small interfering RNA. The clinical relevance of these experiments is suggested by immunohistochemical, microarray, and quantitative RT-PCR studies of human colon and pancreatic tumors demonstrating significantly higher DUOX2, IL-4R, and IL-17RA expression in tumors than in adjacent normal tissues; in pancreatic adenocarcinoma, increased DUOX2 expression is adversely associated with overall patient survival. These data suggest a functional association between DUOX2-mediated H2O2 production and induced DNA damage in gastrointestinal malignancies.
Asunto(s)
Neoplasias del Colon/metabolismo , Daño del ADN , Oxidasas Duales/genética , Peróxido de Hidrógeno/metabolismo , Interleucina-17/farmacología , Interleucina-4/farmacología , Neoplasias Pancreáticas/metabolismo , Línea Celular Tumoral , Neoplasias del Colon/patología , Humanos , FN-kappa B/fisiología , Oxidación-Reducción , Neoplasias Pancreáticas/patología , Receptores de Interleucina-4/fisiología , Factor de Transcripción STAT6/fisiología , Transducción de Señal , Regulación hacia ArribaRESUMEN
Chromosomal abnormalities are a defining feature of solid tumors. Such cytogenetic alterations are mainly classified into structural chromosomal aberrations and copy number alterations, giving rise to aneuploid karyotypes. The increasing detection of these genetic changes allowed the description of specific tumor entities and the associated patterns of gene expression. In fact, tumor-specific landscapes of gross genomic copy number changes, including aneuploidies of entire chromosome arms and chromosomes result in a global deregulation of the transcriptome of cancer cells. Furthermore, the molecular characterization of cytogenetic abnormalities has provided insights into the mechanisms of tumorigenesis and has, in a few instances, led to the clinical implementation of effective diagnostic and prognostic tools, as well as treatment strategies that target a specific genetic abnormality.
Asunto(s)
Aberraciones Cromosómicas , Neoplasias/genética , Animales , Deleción Cromosómica , Amplificación de Genes , Dosificación de Gen , Humanos , Translocación GenéticaRESUMEN
DNA methyltransferase inhibitors (DNMTi), most commonly cytidine analogs, are compounds that decrease 5'-cytosine methylation. DNMTi are used clinically based on the hypothesis that cytosine demethylation will lead to re-expression of tumor suppressor genes. 5-Aza-4'-thio-2'-deoxycytidine (Aza-TdCyd or ATC) is a recently described thiol-substituted DNMTi that has been shown to have anti-tumor activity in solid tumor models. In this study, we investigated the therapeutic potential of ATC in a murine transplantation model of myelodysplastic syndrome. ATC treatment led to the transformation of transplanted wild-type bone marrow nucleated cells into lymphoid leukemia, and healthy mice treated with ATC also developed lymphoid leukemia. Whole-exome sequencing revealed 1,000 acquired mutations, almost all of which were C>G transversions in a specific 5'-NCG-3' context. These mutations involved dozens of genes involved in human lymphoid leukemia, such as Notch1, Pten, Pax5, Trp53, and Nf1. Human cells treated in vitro with ATC showed 1,000 acquired C>G transversions in a similar context. Deletion of Dck, the rate-limiting enzyme for the cytidine salvage pathway, eliminated C>G transversions. Taken together, these findings demonstrate a highly penetrant mutagenic and leukemogenic phenotype associated with ATC. Significance: Treatment with a DNA methyltransferase inhibitor generates a distinct mutation signature and triggers leukemic transformation, which has important implications for the research and clinical applications of these inhibitors.
Asunto(s)
Azacitidina , Leucemia-Linfoma Linfoblástico de Células Precursoras , Animales , Ratones , Humanos , Azacitidina/farmacología , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Metilación de ADN/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Decitabina/farmacología , Síndromes Mielodisplásicos/tratamiento farmacológico , Síndromes Mielodisplásicos/genética , Síndromes Mielodisplásicos/patología , Ratones Endogámicos C57BLRESUMEN
Human epithelial cancers are defined by a recurrent distribution of specific chromosomal aneuploidies, a trait less typical for murine cancer models induced by an oncogenic stimulus. After prolonged culture, mouse epithelial cells spontaneously immortalize, transform and become tumorigenic. We assessed genome and transcriptome alterations in cultures derived from bladder and kidney utilizing spectral karyotyping, array CGH, FISH and gene expression profiling. The results show widespread aneuploidy, yet a recurrent and tissue-specific distribution of genomic imbalances, just as in human cancers. Losses of chromosome 4 and gains of chromosome 15 are common and occur early during the transformation process. Global gene expression profiling revealed early and significant transcriptional deregulation. Chromosomal aneuploidy resulted in expression changes of resident genes and consequently in a massive deregulation of the cellular transcriptome. Pathway interrogation of expression changes during the sequential steps of transformation revealed enrichment of genes associated with DNA repair, centrosome regulation, stem cell characteristics and aneuploidy. Genes that modulate the epithelial to mesenchymal transition and genes that define the chromosomal instability phenotype played a dominant role and were changed in a directionality consistent with loss of cell adhesion, invasiveness and proliferation. Comparison with gene expression changes during human bladder and kidney tumorigenesis revealed remarkable overlap with changes observed in the spontaneously transformed murine cultures. Therefore, our novel mouse models faithfully recapitulate the sequence of genomic and transcriptomic events that define human tumorigenesis, hence validating them for both basic and preclinical research.
Asunto(s)
Carcinogénesis/genética , Células Epiteliales/fisiología , Transición Epitelial-Mesenquimal/genética , Amplificación de Genes , Oncogenes , Aneuploidia , Animales , Carcinogénesis/metabolismo , Inestabilidad Cromosómica , Aberraciones Cromosómicas , Células Epiteliales/metabolismo , Células Epiteliales/patología , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Riñón/citología , Masculino , Ratones , Ratones Endogámicos C57BL , Cariotipificación Espectral/métodos , Transcripción Genética , Transcriptoma , Vejiga Urinaria/citologíaRESUMEN
Chromosomal aneuploidies are a defining feature of carcinomas, i.e., tumors of epithelial origin. Such aneuploidies result in tumor specific genomic copy number alterations. The patterns of genomic imbalances are tumor specific, and to a certain extent specific for defined stages of tumor development. Genomic imbalances occur already in premalignant precursor lesions, i.e., before the transition to invasive disease, and their distribution is maintained in metastases, and in cell lines derived from primary tumors. These observations are consistent with the interpretation that tumor specific genomic imbalances are drivers of malignant transformation. Naturally, this precipitates the question of how such imbalances influence the expression of resident genes. A number of laboratories have systematically integrated copy number alterations with gene expression changes in primary tumors and metastases, cell lines, and experimental models of aneuploidy to address the question as to whether genomic imbalances deregulate the expression of one or few key genes, or rather affect the cancer transcriptome more globally. The majority of these studies showed that gene expression levels follow genomic copy number. Therefore, gross genomic copy number changes, including aneuploidies of entire chromosome arms and chromosomes, result in a massive deregulation of the transcriptome of cancer cells. This article is part of a Special Issue entitled: Chromatin in time and space.
Asunto(s)
Aneuploidia , Neoplasias/metabolismo , Transcriptoma , Animales , Línea Celular Tumoral , Cromosomas Humanos , Dosificación de Gen , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias/genéticaRESUMEN
The chromosomal instability syndromes Nijmegen breakage syndrome (NBS) and ataxia telangiectasia (AT) share many overlapping phenotypes, including cancer predisposition, radiation sensitivity, cell-cycle checkpoint defects, immunodeficiency, and gonadal dysfunction. The NBS protein Nbs1 is not only a downstream target of AT mutated (ATM) kinase but also acts upstream, promoting optimal ATM activation, ATM recruitment to breaks, and ATM accessibility to substrates. By reconstituting Nbs1 knockout mice with bacterial artificial chromosomes, we have assessed the contribution of distinct regions of Nbs1 to the ATM-dependent DNA damage response. We find that T cell and oocyte development, as well as DNA damage-induced G2/M and S phase checkpoint arrest and radiation survival are dependent on the N-terminal forkhead-associated domain, but not on the principal residues phosphorylated by ATM (S278 and S343) or on the evolutionarily conserved C-terminal region of Nbs1. However, the C-terminal region regulates irradiation-induced apoptosis. These studies provide insight into the complex interplay between Nbs1 and ATM in the DNA damage response.
Asunto(s)
Apoptosis/fisiología , Proteínas de Ciclo Celular/metabolismo , Inestabilidad Cromosómica/fisiología , Daño del ADN/fisiología , Proteínas Nucleares/metabolismo , Animales , Proteínas de la Ataxia Telangiectasia Mutada , Western Blotting , Proteínas de Ciclo Celular/genética , Cromosomas Artificiales Bacterianos , Cartilla de ADN , Proteínas de Unión al ADN/metabolismo , Técnica del Anticuerpo Fluorescente , Humanos , Inmunoprecipitación , Etiquetado Corte-Fin in Situ , Ratones , Ratones Noqueados , Proteínas Nucleares/genética , Oocitos/crecimiento & desarrollo , Proteínas Serina-Treonina Quinasas/metabolismo , Estructura Terciaria de Proteína , Linfocitos T/fisiología , Proteínas Supresoras de Tumor/metabolismoRESUMEN
BACKGROUND: Colorectal carcinomas (CRC) carry massive genetic and transcriptional alterations that influence multiple cellular pathways. The study of proteins whose loss-of-function (LOF) alters the growth of CRC cells can be used to further understand the cellular processes cancer cells depend upon for survival. RESULTS: A small-scale RNAi screen of ~400 genes conducted in SW480 CRC cells identified several candidate genes as required for the viability of CRC cells, most prominently CASP8AP2/FLASH. To understand the function of this gene in maintaining the viability of CRC cells in an unbiased manner, we generated gene specific expression profiles following RNAi. Silencing of CASP8AP2/FLASH resulted in altered expression of over 2500 genes enriched for genes associated with cellular growth and proliferation. Loss of CASP8AP2/FLASH function was significantly associated with altered transcription of the genes encoding the replication-dependent histone proteins as a result of the expression of the non-canonical polyA variants of these transcripts. Silencing of CASP8AP2/FLASH also mediated enrichment of changes in the expression of targets of the NFκB and MYC transcription factors. These findings were confirmed by whole transcriptome analysis of CASP8AP2/FLASH silenced cells at multiple time points. Finally, we identified and validated that CASP8AP2/FLASH LOF increases the expression of neurofilament heavy polypeptide (NEFH), a protein recently linked to regulation of the AKT1/ß-catenin pathway. CONCLUSIONS: We have used unbiased RNAi based approaches to identify and characterize the function of CASP8AP2/FLASH, a protein not previously reported as required for cell survival. This study further defines the role CASP8AP2/FLASH plays in the regulating expression of the replication-dependent histones and shows that its LOF results in broad and reproducible effects on the transcriptome of colorectal cancer cells including the induction of expression of the recently described tumor suppressor gene NEFH.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/genética , Proteínas de Unión al Calcio/genética , Neoplasias Colorrectales/genética , Regulación Neoplásica de la Expresión Génica , Histonas/genética , Interferencia de ARN , Transcripción Genética , Transcriptoma , Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas de Unión al Calcio/metabolismo , Línea Celular Tumoral , Supervivencia Celular/genética , Análisis por Conglomerados , Neoplasias Colorrectales/metabolismo , Biología Computacional/métodos , Replicación del ADN , Perfilación de la Expresión Génica , Silenciador del Gen , Humanos , Proteínas de Neurofilamentos/genéticaRESUMEN
DNA aneuploidy has been identified as a prognostic factor for epithelial malignancies. Further understanding of the translation of DNA aneuploidy into protein expression will help to define novel biomarkers to improve therapies and prognosis. DNA ploidy was assessed by image cytometry. Comparison of gel-electrophoresis-based protein expression patterns of three diploid and four aneuploid colorectal cancer cell lines detected 64 ploidy-associated proteins. Proteins were identified by mass spectrometry and subjected to Ingenuity Pathway Analysis resulting in two overlapping high-ranked networks maintaining Cellular Assembly and Organization, Cell Cycle, and Cellular Growth and Proliferation. CAPZA1, TXNL1, and HDAC2 were significantly validated by Western blotting in cell lines and the latter two showed expression differences also in clinical samples using a tissue microarray of normal mucosa (n=19), diploid (n=31), and aneuploid (n=47) carcinomas. The results suggest that distinct protein expression patterns, affecting TXNL1 and HDAC2, distinguish aneuploid with poor prognosis from diploid colorectal cancers.
Asunto(s)
Aneuploidia , Carcinoma/genética , Neoplasias Colorrectales/genética , Diploidia , Histona Desacetilasa 2/genética , Tiorredoxinas/genética , Western Blotting , Proteína CapZ/genética , Proteína CapZ/metabolismo , Proteína CapZ/fisiología , Carcinoma/diagnóstico , Carcinoma/patología , Línea Celular Tumoral , Estudios de Cohortes , Neoplasias Colorrectales/diagnóstico , Neoplasias Colorrectales/patología , ADN de Neoplasias/química , Inestabilidad Genómica , Histona Desacetilasa 2/metabolismo , Histona Desacetilasa 2/fisiología , Humanos , Pronóstico , Tiorredoxinas/metabolismo , Tiorredoxinas/fisiologíaRESUMEN
MicroRNAs (miRNAs) are small non-coding RNAs, which regulate gene expression by inhibiting translation or promoting degradation of specific target messenger RNAs (mRNAs). Alteration of the levels of a number of miRNAs is common in solid and hematological tumors. We have shown previously that miR-214 regulates Ezh2 in skeletal muscle and embryonic stem cells. The current study was aimed at examining the role of miR-214 in breast cancer where miR-214 levels are reduced but whether this phenomenon bears a functional relevance is unknown. MiR-214 expression was inversely correlated with Ezh2 mRNA and protein levels in breast cancer cell lines and at least one copy of the miR-214 alleles was found to be deleted in 24% (6/25) of primary breast tumors. Experimental increase of miR-214 in breast cancer cell lines correlated with reduction of Ezh2 protein levels, a known marker of invasion and aggressive breast cancer behavior. Supporting a direct targeting mechanism, miR-214 decreased luciferase activity from a construct containing the Ezh2 3' untranslated region. Expression of miR-214 specifically reduced cell proliferation of breast cancer cells and inhibited the invasive potential of a highly metastatic breast cancer cell line. These findings indicate that reduced miR-214 levels may contribute to breast tumorigenesis by allowing abnormally elevated Ezh2 accumulation and subsequent unchecked cell proliferation and invasion.
Asunto(s)
Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Movimiento Celular , Proliferación Celular , Proteínas de Unión al ADN/metabolismo , MicroARNs/genética , Factores de Transcripción/metabolismo , Western Blotting , Neoplasias de la Mama/enzimología , Adhesión Celular , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Proteína Potenciadora del Homólogo Zeste 2 , Femenino , Humanos , Técnicas para Inmunoenzimas , Luciferasas/metabolismo , Invasividad Neoplásica , Complejo Represivo Polycomb 2 , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/genética , Células Tumorales CultivadasRESUMEN
Genes that are highly overexpressed in tumor cells can be required for tumor cell survival and have the potential to be selective therapeutic targets. In an attempt to identify such targets, we combined a functional genomics and a systems biology approach to assess the consequences of RNAi-mediated silencing of overexpressed genes that were selected from 140 gene expression profiles from colorectal cancers (CRCs) and matched normal mucosa. In order to identify credible models for in-depth functional analysis, we first confirmed the overexpression of these genes in 25 different CRC cell lines. We then identified five candidate genes that profoundly reduced the viability of CRC cell lines when silenced with either siRNAs or short-hairpin RNAs (shRNAs), i.e., HMGA1, TACSTD2, RRM2, RPS2 and NOL5A. These genes were further studied by systematic analysis of comprehensive gene expression profiles generated following siRNA-mediated silencing. Exploration of these RNAi-specific gene expression signatures allowed the identification of the functional space in which the five genes operate and showed enrichment for cancer-specific signaling pathways, some known to be involved in CRC. By comparing the expression of the RNAi signature genes with their respective expression levels in an independent set of primary rectal carcinomas, we could recapitulate these defined RNAi signatures, therefore, establishing the biological relevance of our observations. This strategy identified the signaling pathways that are affected by the prominent oncogenes HMGA1 and TACSTD2, established a yet unknown link between RRM2 and PLK1 and identified RPS2 and NOL5A as promising potential therapeutic targets in CRC.
Asunto(s)
Neoplasias Colorrectales/genética , Genómica , Línea Celular Tumoral , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/terapia , Perfilación de la Expresión Génica , Silenciador del Gen , Humanos , Inmunohistoquímica , Interferencia de ARNRESUMEN
In defining the genetic profiles in cancer, cytogenetically aberrant cell lines derived from primary tumors are important tools for the study of carcinogenesis. Here, we present the results of a comprehensive investigation of 15 established colorectal cancer cell lines using spectral karyotyping (SKY), fluorescence in situ hybridization, and comparative genomic hybridization (CGH). Detailed karyotypic analysis by SKY on five of the lines (P53HCT116, T84, NCI-H508, NCI-H716, and SK-CO-1) is described here for the first time. The five lines with karyotypes in the diploid range and that are characterized by defects in DNA mismatch repair had a mean of 4.8 chromosomal abnormalities per line, whereas the 10 aneuploid lines exhibited complex karyotypes and a mean of 30 chromosomal abnormalities. Of the 150 clonal translocations, only eight were balanced and none were recurrent among the lines. We also reviewed the karyotypes of 345 cases of adenocarcinoma of the large intestine listed in the Mitelman Database of Chromosome Aberrations in Cancer. The types of abnormalities observed in the cell lines reflected those seen in primary tumors: there were no recurrent translocations in either tumors or cell lines; isochromosomes were the most common recurrent abnormalities; and breakpoints occurred most frequently at the centromeric/pericentromeric and telomere regions. Of the genomic imbalances detected by array CGH, 87% correlated with chromosome aberrations observed in the SKY studies. The fact that chromosome abnormalities predominantly result in copy number changes rather than specific chromosome or gene fusions suggests that this may be the major mechanism leading to carcinogenesis in colorectal cancer.
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Neoplasias Colorrectales/genética , Análisis Citogenético/métodos , Línea Celular Tumoral , Aberraciones Cromosómicas/estadística & datos numéricos , Bandeo Cromosómico , Hibridación Genómica Comparativa/métodos , Reparación de la Incompatibilidad de ADN , Humanos , Hibridación Fluorescente in Situ , Cariotipificación , Translocación GenéticaRESUMEN
BACKGROUND: Many carcinomas have recurrent chromosomal aneuploidies specific to the tissue of tumor origin. The reason for this specificity is not completely understood. METHODS: In this study, we looked at the frequency of chromosomal arm gains and losses in different cancer types from the The Cancer Genome Atlas (TCGA) and compared them to the mean gene expression of each chromosome arm in corresponding normal tissues of origin from the Genotype-Tissue Expression (GTEx) database, in addition to the distribution of tissue-specific oncogenes and tumor suppressors on different chromosome arms. RESULTS: This analysis revealed a complex picture of factors driving tumor karyotype evolution in which some recurrent chromosomal copy number reflect the chromosome arm-wide gene expression levels of the their normal tissue of tumor origin. CONCLUSIONS: We conclude that the cancer type-specific distribution of chromosomal arm gains and losses is potentially "hardwiring" gene expression levels characteristic of the normal tissue of tumor origin, in addition to broadly modulating the expression of tissue-specific tumor driver genes.
Asunto(s)
Aneuploidia , Biomarcadores de Tumor , Mapeo Cromosómico , Regulación Neoplásica de la Expresión Génica , Neoplasias/genética , Algoritmos , Análisis por Conglomerados , Biología Computacional/métodos , Metilación de ADN , Bases de Datos Genéticas , Perfilación de la Expresión Génica , Humanos , Mutación , Oncogenes , Especificidad de Órganos/genéticaRESUMEN
Nonreciprocal translocations and gene amplifications are commonly found in human tumors. Although little is known about the mechanisms leading to such aberrations, tissue culture models predict that they can arise from DNA breakage, followed by cycles of chromatid fusion, asymmetric mitotic breakage, and replication. Mice deficient in both a nonhomologous end joining (NHEJ) DNA repair protein and the p53 tumor suppressor develop lymphomas at an early age harboring amplification of an IgH/c-myc fusion. Here we report that these chromosomal rearrangements are initiated by a recombination activating gene (RAG)-induced DNA cleavage. Subsequent DNA repair events juxtaposing IgH and c-myc are mediated by a break-induced replication pathway. Cycles of breakage-fusion-bridge result in amplification of IgH/c-myc while chromosome stabilization occurs through telomere capture. Thus, mice deficient in NHEJ provide excellent models to study the etiology of unbalanced translocations and amplification events during tumorigenesis.
Asunto(s)
Antígenos Nucleares , Daño del ADN , ADN Helicasas , Reparación del ADN , Replicación del ADN , Proteínas de Unión al ADN/fisiología , Cadenas Pesadas de Inmunoglobulina/genética , Linfoma de Células B/genética , Proteínas Proto-Oncogénicas c-myc/genética , Neoplasias del Timo/genética , Translocación Genética , Animales , Secuencia de Bases , Proteínas de Unión al ADN/genética , Amplificación de Genes , Reordenamiento Génico , Autoantígeno Ku , Ratones , Ratones Noqueados , Ratones SCID , Datos de Secuencia Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/fisiología , Oncogenes , Telómero , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/fisiologíaRESUMEN
To evaluate the mechanisms and consequences of chromosomal aberrations in colorectal cancer (CRC), we used a combination of spectral karyotyping, array comparative genomic hybridization (aCGH), and array-based global gene expression profiling on 31 primary carcinomas and 15 established cell lines. Importantly, aCGH showed that the genomic profiles of primary tumors are recapitulated in the cell lines. We revealed a preponderance of chromosome breakpoints at sites of copy number variants (CNVs) in the CRC cell lines, a novel mechanism of DNA breakage in cancer. The integration of gene expression and aCGH led to the identification of 157 genes localized within high-level copy number changes whose transcriptional deregulation was significantly affected across all of the samples, thereby suggesting that these genes play a functional role in CRC. Genomic amplification at 8q24 was the most recurrent event and led to the overexpression of MYC and FAM84B. Copy number dependent gene expression resulted in deregulation of known cancer genes such as APC, FGFR2, and ERBB2. The identification of only 36 genes whose localization near a breakpoint could account for their observed deregulated expression demonstrates that the major mechanism for transcriptional deregulation in CRC is genomic copy number changes resulting from chromosomal aberrations.
Asunto(s)
Neoplasias Colorrectales/genética , Variaciones en el Número de Copia de ADN , Regulación Neoplásica de la Expresión Génica , Línea Celular Tumoral , Puntos de Rotura del Cromosoma , Cromosomas Humanos Par 8 , Neoplasias Colorrectales/metabolismo , Hibridación Genómica Comparativa/métodos , Eliminación de Gen , Perfilación de la Expresión Génica/métodos , Redes Reguladoras de Genes , Humanos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Receptores Virales/genética , Receptores Virales/metabolismo , Cariotipificación Espectral/métodos , Transcripción GenéticaRESUMEN
BACKGROUND: While attempting to reanalyze published data from Agilent 4 x 44 human expression chips, we found that some of the 60-mer olignucleotide features could not be interpreted as representing single human genes. For example, some of the oligonucleotides align with the transcripts of more than one gene. We decided to check the annotations for all autosomes and the X chromosome systematically using bioinformatics methods. RESULTS: Out of 42683 reporters, we found that 25505 (60%) passed all our tests and are considered "fully valid". 9964 (23%) reporters did not have a meaningful identifier, mapped to the wrong chromosome, or did not pass basic alignment tests preventing us from correlating the expression values of these reporters with a unique annotated human gene. The remaining 7214 (17%) reporters could be associated with either a unique gene or a unique intergenic location, but could not be mapped to a transcript in RefSeq. The 7214 reporters are further partitioned into three different levels of validity. CONCLUSION: Expression array studies should evaluate the annotations of reporters and remove those reporters that have suspect annotations. This evaluation can be done systematically and semi-automatically, but one must recognize that data sources are frequently updated leading to slightly changing validation results over time.
Asunto(s)
Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Secuencia de Bases , Biología Computacional , Bases de Datos Genéticas , Humanos , Internet , Datos de Secuencia Molecular , ARN Mensajero/genética , Reproducibilidad de los Resultados , Programas InformáticosRESUMEN
The nonrandom positioning of chromosome territories in eukaryotic cells is largely correlated with gene density and is conserved throughout evolution. Gene-rich chromosomes are predominantly central, while gene-poor chromosomes are peripherally localized in interphase nuclei. We previously demonstrated that artificially introduced human chromosomes assume a position equivalent to their endogenous homologues in the diploid colon cancer cell line DLD-1. These chromosomal aneuploidies result in a significant increase in transcript levels, suggesting a relationship between genomic copy number, gene expression, and chromosome position. We previously proposed that each chromosome is marked by a "zip code" that determines its nonrandom position in the nucleus. In this paper, we investigated (1) whether mouse nuclei recognize such determinants of nuclear position on human chromosomes to facilitate their distinct partitioning and (2) if chromosome positioning and transcriptional activity remain coupled under these trans-species conditions. Using three-dimensional fluorescence in situ hybridization, confocal microscopy, and gene expression profiling, we show (1) that gene-poor and gene-rich human chromosomes maintain their divergent but conserved positions in mouse-human hybrid nuclei and (2) that a foreign human chromosome is actively transcribed in mouse nuclei. Our results suggest a species-independent conserved mechanism for the nonrandom positioning of chromosomes in the three-dimensional interphase nucleus.
Asunto(s)
Núcleo Celular/fisiología , Posicionamiento de Cromosoma , Cromosomas Humanos/fisiología , Expresión Génica/fisiología , Transcripción Genética/fisiología , Animales , Línea Celular , Núcleo Celular/genética , Núcleo Celular/ultraestructura , Cromosomas Humanos/genética , Cromosomas Humanos/ultraestructura , Expresión Génica/genética , Genoma/genética , Genoma/fisiología , Humanos , RatonesRESUMEN
To characterize patterns of global transcriptional deregulation in primary colon carcinomas, we did gene expression profiling of 73 tumors [Unio Internationale Contra Cancrum stage II (n = 33) and stage III (n = 40)] using oligonucleotide microarrays. For 30 of the tumors, expression profiles were compared with those from matched normal mucosa samples. We identified a set of 1,950 genes with highly significant deregulation between tumors and mucosa samples (P < 1e-7). A significant proportion of these genes mapped to chromosome 20 (P = 0.01). Seventeen genes had a >5-fold average expression difference between normal colon mucosa and carcinomas, including up-regulation of MYC and of HMGA1, a putative oncogene. Furthermore, we identified 68 genes that were significantly differentially expressed between lymph node-negative and lymph node-positive tumors (P < 0.001), the functional annotation of which revealed a preponderance of genes that play a role in cellular immune response and surveillance. The microarray-derived gene expression levels of 20 deregulated genes were validated using quantitative real-time reverse transcription-PCR in >40 tumor and normal mucosa samples with good concordance between the techniques. Finally, we established a relationship between specific genomic imbalances, which were mapped for 32 of the analyzed colon tumors by comparative genomic hybridization, and alterations of global transcriptional activity. Previously, we had conducted a similar analysis of primary rectal carcinomas. The systematic comparison of colon and rectal carcinomas revealed a significant overlap of genomic imbalances and transcriptional deregulation, including activation of the Wnt/beta-catenin signaling cascade, suggesting similar pathogenic pathways.
Asunto(s)
Adenocarcinoma/genética , Adenocarcinoma/patología , Aneuploidia , Neoplasias del Colon/genética , Neoplasias del Colon/patología , Ganglios Linfáticos/patología , Adenocarcinoma/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Colon/metabolismo , Colon/fisiología , Neoplasias del Colon/metabolismo , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Genoma Humano , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/fisiología , Metástasis Linfática , Masculino , Persona de Mediana Edad , Familia de Multigenes , Estadificación de Neoplasias , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
To identify genetic alterations underlying rectal carcinogenesis, we used global gene expression profiling of a series of 17 locally advanced rectal adenocarcinomas and 20 normal rectal mucosa biopsies on oligonucleotide arrays. A total of 351 genes were differentially expressed (P < 1.0e-7) between normal rectal mucosa and rectal carcinomas, 77 genes had a >5-fold difference, and 85 genes always had at least a 2-fold change in all of the matched samples. Twelve genes satisfied all three of these criteria. Altered expression of genes such as PTGS2 (COX-2), WNT1, TGFB1, VEGF, and MYC was confirmed, whereas our data for other genes, like PPARD and LEF1, were inconsistent with previous reports. In addition, we found deregulated expression of many genes whose involvement in rectal carcinogenesis has not been reported. By mapping the genomic imbalances in the tumors using comparative genomic hybridization, we could show that DNA copy number gains of recurrently aneuploid chromosome arms 7p, 8q, 13q, 18q, 20p, and 20q correlated significantly with their average chromosome arm expression profile. Taken together, our results show that both the high-level, significant transcriptional deregulation of specific genes and general modification of the average transcriptional activity of genes residing on aneuploid chromosomes coexist in rectal adenocarcinomas.
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Adenocarcinoma/genética , Aneuploidia , Neoplasias del Recto/genética , Proteína wnt2/fisiología , beta Catenina/fisiología , Adenocarcinoma/metabolismo , Dosificación de Gen , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/fisiología , Familia de Multigenes , Hibridación de Ácido Nucleico , Neoplasias del Recto/metabolismo , Transducción de SeñalRESUMEN
A systems approach is being applied in many areas of the biological sciences, particularly in cancer research. The coordinated, simultaneous extraction of DNA, RNA, and proteins from a single sample is crucial for accurate correlations between genomic aberrations and their consequences on the transcriptome and proteome. We present an approach to extract and completely solubilize up to 98% of the total protein recovered from archived samples following TRIzoL isolation of RNA and DNA. We also demonstrate using polyacrylamide gel electrophoresis (PAGE) and Western blot analysis that the proteins, representing both a wide molecular weight range and some posttranslational modifications, such as protein phosphorylation, remain stable in phenol-ethanol for up to 3 years at -20 degrees C.
Asunto(s)
Biotecnología/métodos , Proteínas/aislamiento & purificación , ADN/aislamiento & purificación , Guanidinas/química , Células HCT116 , Humanos , Fenoles/química , ARN/aislamiento & purificación , Manejo de EspecímenesRESUMEN
Hepatitis B virus (HBV) includes an X gene (HBx gene) that plays a critical role in liver carcinogenesis. Because centrosome abnormalities are associated with genomic instability in most human cancer cells, we examined the effect of HBx on centrosomes. We found that HBx induced supernumerary centrosomes and multipolar spindles. This effect was independent of mutations in the p21 gene. Furthermore, the ability of HBV to induce supernumerary centrosomes was dependent on the presence of physiological HBx expression. We recently showed that HBx induces cytoplasmic sequestration of Crm1, a nuclear export receptor that binds to Ran GTPase, thereby inducing nuclear localization of NF-kappaB. Consistently, supernumerary centrosomes were observed in cells treated with a Crm1-specific inhibitor but not with an HBx mutant that lacked the ability to sequester Crm1 in the cytoplasm. Moreover, a fraction of Crm1 was found to be localized at the centrosomes. Immunocytochemical and ultrastructural examination of these supernumerary centrosomes revealed that inactivation of Crm1 was associated with abnormal centrioles. The presence of more than two centrosomes led to an increased frequency of defective mitoses and chromosome transmission errors. Based on this evidence, we suggest that Crm1 is actively involved in maintaining centrosome integrity and that HBx disrupts this process by inactivating Crm1 and thus contributes to HBV-mediated carcinogenesis.