RESUMEN
MOTIVATION: Long-read transcriptome sequencing (LRTS) has the potential to enhance our understanding of alternative splicing and the complexity of this process requires the use of versatile computational tools, with the ability to accommodate various stages of the workflow with maximum flexibility. RESULTS: We introduce IsoTools, a Python-based LRTS analysis framework that offers a wide range of functionality for transcriptome reconstruction and quantification of transcripts. Furthermore, we integrate a graph-based method for identifying alternative splicing events and a statistical approach based on the beta-binomial distribution for detecting differential events. To demonstrate the effectiveness of our methods, we applied IsoTools to PacBio LRTS data of human hepatocytes treated with the histone deacetylase inhibitor valproic acid. Our results indicate that LRTS can provide valuable insights into alternative splicing, particularly in terms of complex and differential splicing patterns, in comparison to short-read RNA-seq. AVAILABILITY AND IMPLEMENTATION: IsoTools is available on GitHub and PyPI, and its documentation, including tutorials, CLI, and API references, can be found at https://isotools.readthedocs.io/.
Asunto(s)
Empalme Alternativo , Transcriptoma , Humanos , Flujo de Trabajo , Perfilación de la Expresión Génica , Empalme del ARN , Secuenciación de Nucleótidos de Alto Rendimiento , Análisis de Secuencia de ARN/métodosRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
Cellular adaptation is important to cope with various stresses induced by altered environmental conditions. By controlling mRNA translation rates cells may adapt to stress to promote survival. Phosphorylation of eIF2α at serine 51 is one of the pathways controlling mRNA translation. Here we investigated the role of phosphorylated eIF2α during exposure to the environmental carcinogen benzo(a)pyrene (BaP). For our study we used mouse embryonic fibroblasts with a wild type eIF2α (MEF WT) and mouse embryonic fibroblasts with an eIF2α S51A knock-in mutation that cannot be phosphorylated. Here, we show that eIF2α phosphorylation occurs in MEF WT cells but not in MEF S51A cells. Survival of MEF S51A cells is profoundly reduced compared to MEF WT controls after BaP exposure. No differences in DNA damage or ROS production were observed between MEF WT and S51A cells. Disruption of eIF2α phosphorylation caused increased levels of apoptosis in response to BaP. This work demonstrates that eIF2α phosphorylation is important for reducing apoptosis and promoting cell survival in order to adapt to BaP exposure.
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Benzo(a)pireno/toxicidad , Carcinógenos/toxicidad , Contaminantes Ambientales/toxicidad , Factor 2 Eucariótico de Iniciación/metabolismo , Animales , Línea Celular , Supervivencia Celular/efectos de los fármacos , Fibroblastos , Ratones , Fosforilación/efectos de los fármacosRESUMEN
Translational control is a cellular response mechanism which initiates adaptation during various stress situations. Here, we investigated the role of translational control after benzo[a]pyrene (BaP) exposure in primary mouse hepatocytes. Translated mRNAs were separated and captured based on the number of associated ribosomes using sucrose gradients and subjected to RNA sequencing (RNAseq) to investigate translational changes. Furthermore, unseparated RNA (total RNA) was used for RNAseq to determine the transcriptional alterations. We showed that, after 24â¯h of exposure to 10 µM BaP, the number of genes altered by changes in mRNA translation was substantially higher compared with the number of genes altered by transcription. Although part of the BaP regulated genes were regulated by both transcription and translation, we identified genes that were uniquely regulated by mRNA translation. These mRNA transcripts encode proteins that are involved in biological processes that are not affected by transcriptional regulation. Al together this work identified a new layer of gene expression regulation that might contribute to BaP-induced carcinogenesis.
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Benzo(a)pireno/toxicidad , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Hepatocitos/efectos de los fármacos , Biosíntesis de Proteínas/efectos de los fármacos , Ribosomas/efectos de los fármacos , Animales , Células Cultivadas , Enfermedad Hepática Inducida por Sustancias y Drogas/genética , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Hepatocitos/metabolismo , Ratones , Cultivo Primario de Células , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ribosomas/metabolismo , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , Transcripción Genética/efectos de los fármacosRESUMEN
Regulation of DNA methylation plays a crucial role in biological processes and carcinogenesis. The formation of 5-hydroxymethylcytosine (5hmC) by oxidation of 5-methylcytosine (5mC) has been proposed as an intermediate of active demethylation. However, whether and how active demethylation is regulated by oxidative stress-related processes is not well understood. Here we investigated whether free oxygen radicals are capable of directly forming 5hmC and how this enhanced whole genome gene expression. We applied LC-MS/MS technology for the analysis of 5mC, 5hmC, 5-formylcytosine (5fC) and 5-hydroxymethyluracyl (5hmU) in HepG2 cells exposed to hydroxyl- and methyl radicals, formed by tert-butyl hydroperoxide (TBH) at multiple time points. We observed that TBH is able to induce a significant increase in 5hmC. A detailed evaluation of the hydroxymethylome using a combination of 5hmC-immunoprecipitation and microarrays resulted in the identification of highly dynamic modifications that appear to increase during prolonged oxidant exposure. Analyses of temporal gene expression changes in combination with network analysis revealed different subnetworks containing differentially expressed genes (DEGs) with differentially hydroxyl-methylated regions (DhMRs) in different regulatory kinases enriched with serine-threonine kinases. These serine-threonine kinases compromises MAPK14, RPSK6KA1, RIPK1, and PLK3 and were all previously identified as key-regulators in hepatocarcinogenesis and subject of study for chemotherapeutic interventions.
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5-Metilcitosina/análogos & derivados , Regulación de la Expresión Génica/efectos de los fármacos , Genómica/métodos , Estrés Oxidativo/efectos de los fármacos , Proteínas Quinasas/metabolismo , terc-Butilhidroperóxido/toxicidad , 5-Metilcitosina/toxicidad , Animales , Redes Reguladoras de Genes/efectos de los fármacos , Células Hep G2 , Humanos , Neoplasias Hepáticas Experimentales/inducido químicamente , Neoplasias Hepáticas Experimentales/genética , Neoplasias Hepáticas Experimentales/patología , Metaboloma , Análisis por Micromatrices , Proteínas Quinasas/genética , ARN/genética , ARN/aislamiento & purificaciónRESUMEN
DNA damage mediates widespread changes in transcription through activation or repression of transcription factors (TFs). However, the consequences of regulating specific TFs for the outcome of the DNA repair process remain incompletely understood. Here, we combined transcriptomics and TF binding prediction with functional genomics to identify TFs essential for adequate DNA repair in HepG2 liver cells after a non-cytotoxic dose of carcinogens benzo(a)pyrene (BaP) (2µM) and aflatoxin B1 (AFB1) (5µM). BaP and AFB1 induced a largely common transcriptional response, mediated by similar TFs. A lentiviral shRNA screen knocking down the top31 identified TFs, was performed to determine their effect on DNA repair by assessing phosphorylation of H2AX (γ-H2AX). In addition to the top candidate p53, we identified several other interesting TFs that modulated γ-H2AX after BaP and AFB1 treatment. Validation studies confirmed the role of p53 in reducing γ-H2AX formation and DNA breaks measured by COMET assay after BaP and AFB1 exposure. Expression of the cell cycle inhibitor p21 was profoundly impaired upon p53 knock-down. In addition, the expression of 2 genes involved in nucleotide exchange repair, DDB2 and XPC was significantly reduced in p53 knock-down cells. Although p63 knock-down affected DNA damage upon BaP treatment this was not associated with altered expression of DDB2 or XPC. Finally, knock-down of ARNT reduced γ-H2AX in response to BaP, which was associated with reduced CYP1A1 expression. Importantly, our results suggest a new role for ARNT and its dimerization partner AHR in the occurrence of H2AX phosphorylation after AFB1 treatment. These data show that modulation of TF activity impacts on the repair of BaP- and AFB1-induced DNA damage. Our study also demonstrates the potential of combining functional genomics with genome-wide expression analysis to identify yet unknown causal relationships, thereby aiding in the interpretation of complex biological systems.
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Aflatoxina B1/toxicidad , Benzo(a)pireno/toxicidad , Daño del ADN , Reparación del ADN/efectos de los fármacos , Perfilación de la Expresión Génica/métodos , Hepatocitos/efectos de los fármacos , Proteómica/métodos , Factores de Transcripción/metabolismo , Transcriptoma , Translocador Nuclear del Receptor de Aril Hidrocarburo/genética , Translocador Nuclear del Receptor de Aril Hidrocarburo/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Ensayo Cometa , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Células Hep G2 , Hepatocitos/metabolismo , Hepatocitos/patología , Histonas/metabolismo , Humanos , Fosforilación , Interferencia de ARN , Receptores de Hidrocarburo de Aril/genética , Receptores de Hidrocarburo de Aril/metabolismo , Factores de Tiempo , Factores de Transcripción/genética , Transfección , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
BACKGROUND: A comprehensive assessment of the epigenetic dynamics in cancer cells is the key to understanding the molecular mechanisms underlying cancer and to improving cancer diagnostics, prognostics and treatment. By combining genome-wide ChIP-seq epigenomics and microarray transcriptomics, we studied the effects of oxygen deprivation and subsequent reoxygenation on histone 3 trimethylation of lysine 4 (H3K4me3) and lysine 27 (H3K27me3) in a breast cancer cell line, serving as a model for abnormal oxygenation in solid tumors. A priori, epigenetic markings and gene expression levels not only are expected to vary greatly between hypoxic and normoxic conditions, but also display a large degree of heterogeneity across the cell population. Where traditionally ChIP-seq data are often treated as dichotomous data, the model and experiment here necessitate a quantitative, data-driven analysis of both datasets. RESULTS: We first identified genomic regions with sustained epigenetic markings, which provided a sample-specific reference enabling quantitative ChIP-seq data analysis. Sustained H3K27me3 marking was located around centromeres and intergenic regions, while sustained H3K4me3 marking is associated with genes involved in RNA binding, translation and protein transport and localization. Dynamic marking with both H3K4me3 and H3K27me3 (hypoxia-induced bivalency) was found in CpG-rich regions at loci encoding factors that control developmental processes, congruent with observations in embryonic stem cells. CONCLUSIONS: In silico-identified epigenetically sustained and dynamic genomic regions were confirmed through ChIP-PCR in vitro, and obtained results are corroborated by published data and current insights regarding epigenetic regulation.
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Hipoxia de la Célula , Histonas/metabolismo , Línea Celular Tumoral , Inmunoprecipitación de Cromatina , Islas de CpG , Ciclina A2/genética , Ciclina A2/metabolismo , Epigenómica , Secuenciación de Nucleótidos de Alto Rendimiento , Histonas/genética , Humanos , Células MCF-7 , Metilación , Análisis de Secuencia por Matrices de Oligonucleótidos , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/genética , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Análisis de Secuencia de ARNRESUMEN
In recent years, it has been shown that free radicals not only react directly with DNA but also regulate epigenetic processes such as DNA methylation, which may be relevant within the context of, for example, tumorigenesis. However, how these free radicals impact the epigenome remains unclear. We therefore investigated whether methyl and hydroxyl radicals, formed by tert-butyl hydroperoxide (TBH), change temporal DNA methylation patterns and how this interferes with genome-wide gene expression. At three time points, TBH-induced radicals in HepG2 cells were identified by electron spin resonance spectroscopy. Total 5-methylcytosine (5mC) levels were determined by liquid chromatography and tandem mass spectrometry and genome-wide changes in 5mC and gene expression by microarrays. Induced methylome changes rather represent an adaptive response to the oxidative stress-related reactions observed in the transcriptome. More specifically, we found that methyl radicals did not induce DNA methylation directly. An initial oxidative and alkylating stress-related response of the transcriptome during the early phase of TBH treatment was followed by an epigenetic response associated with cell survival signaling. Also, we identified genes of which the expression seems directly regulated by DNA methylation. This work suggests an important role of the methylome in counter-regulating primary oxidative and alkylating stress responses in the transcriptome to restore normal cell function. Altogether, the methylome may play an important role in counter-regulating primary oxidative and alkylating stress responses in the transcriptome presumably to restore normal cell function.
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Metilación de ADN , Estrés Oxidativo/genética , Estrés Fisiológico/genética , Transcriptoma/genética , Alquilación , Cromatografía Liquida , Radicales Libres/química , Células Hep G2 , Humanos , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
Matrix metalloproteinase-2 (MMP2) is important in tumorigenesis, angiogenesis and tumor invasion. In this study, we investigated if the Cy5-tagged small immuno protein targeting the catalytic domain of human MMP2 (aMMP2-SIP) detects MMP2 in tumors non-invasively. For this purpose, we generated MMP2 expressing (empty vector EV) and knock-down (KD) HT1080, U373 and U87 cells, which were injected subcutaneously in the lateral flank of NMRI-nu mice. Optical imaging (Optix MX2) performed at 0.5, 2, 4, 8, 24 and 48 hour post injection (h.p.i.) of Cy5 tagged aMMP2-SIP, indicated significantly lower tumor to background ratios at both 24 (P = 0.0090) and 48 h.p.i. (P < 0.0001) for the U87 MMP2-KD compared to control tumors. No differences were found for HT1080 and U373 models. U87 MMP2-KD tumors had significantly lower MMP2 activity (P < 0.0001) than EV tumors as determined by gelatin zymography in tumor sections and lysates, while no differences were observed between EV and MMP2-KD in HT1080 and U373. In line with these data, only U87 MMP2-KD tumors had a reduced tumor growth compared to control tumors (P = 0.0053). aMMP2-SIP uptake correlates with MMP2 activity and might therefore be a potential non-invasive imaging biomarker for the evaluation of MMP2 activity in tumors.
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Anticuerpos , Metaloproteinasa 2 de la Matriz/metabolismo , Imagen Molecular/métodos , Neoplasias/metabolismo , Imagen Óptica/métodos , Animales , Anticuerpos/inmunología , Línea Celular Tumoral , Modelos Animales de Enfermedad , Activación Enzimática , Expresión Génica , Xenoinjertos , Integrina alfaVbeta3/genética , Integrina alfaVbeta3/metabolismo , Metaloproteinasa 2 de la Matriz/genética , Metaloproteinasa 2 de la Matriz/inmunología , Ratones , Ratones Noqueados , Neoplasias/diagnóstico , Neoplasias/genética , Reproducibilidad de los Resultados , Microambiente TumoralRESUMEN
BACKGROUND: (Pre)clinical studies indicate that autophagy inhibition increases response to anti-cancer therapies. Although promising, due to contradicting reports, it remains unclear if radiation therapy changes autophagy activity and if autophagy inhibition changes the cellular intrinsic radiosensitivity. Discrepancies may result from different assays and models through off-target effects and influencing other signaling routes. In this study, we directly compared the effects of genetic and pharmacological inhibition of autophagy after irradiation in human cancer cell lines. MATERIALS AND METHODS: Changes in autophagy activity after ionizing radiation (IR) were assessed by flux analysis in eight cell lines. Clonogenic survival, DNA damage (COMET-assay) and H2AX phosphorylation were assessed after chloroquine or 3-methyladenine pretreatment and after ATG7 or LC3b knockdown. RESULTS: IR failed to induce autophagy and chloroquine failed to change intrinsic radiosensitivity of cells. Interestingly, 3-methyladenine and ATG7- or LC3b-deficiency sensitized cancer cells to irradiation. Surprisingly, the radiosensitizing effect of 3-methyladenine was also observed in ATG7 and LC3b deficient cells and was associated with attenuated γ-H2AX formation and DNA damage repair. CONCLUSION: Our data demonstrate that the anti-tumor effects of chloroquine are independent of changes in intrinsic radioresistance. Furthermore, ATG7 and LC3b support radioresistance independent of canonical autophagy that involves lysosomal degradation.
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Autofagia , Adenina/análogos & derivados , Adenina/farmacología , Autofagia/efectos de los fármacos , Línea Celular Tumoral , Cloroquina/farmacología , Reparación del ADN/efectos de los fármacos , Humanos , Fosforilación , Tolerancia a Radiación/genética , Radiación Ionizante , Fármacos Sensibilizantes a Radiaciones/farmacología , Transducción de Señal/efectos de los fármacosRESUMEN
Cancer-related deregulation of miRNA biogenesis has been suggested, but the underlying mechanisms remain elusive. Here we report a previously unrecognized effect of hypoxia in the downregulation of Drosha and Dicer in cancer cells that leads to dysregulation of miRNA biogenesis and increased tumour progression. We show that hypoxia-mediated downregulation of Drosha is dependent on ETS1/ELK1 transcription factors. Moreover, mature miRNA array and deep sequencing studies reveal altered miRNA maturation in cells under hypoxic conditions. At a functional level, this phenomenon results in increased cancer progression in vitro and in vivo, and data from patient samples are suggestive of miRNA biogenesis downregulation in hypoxic tumours. Rescue of Drosha by siRNAs targeting ETS1/ELK1 in vivo results in significant tumour regression. These findings provide a new link in the mechanistic understanding of global miRNA downregulation in the tumour microenvironment.
Asunto(s)
Progresión de la Enfermedad , Regulación hacia Abajo/genética , MicroARNs/biosíntesis , Neoplasias/genética , Neoplasias/patología , Animales , Hipoxia de la Célula/genética , Línea Celular Tumoral , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Transición Epitelial-Mesenquimal/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones Desnudos , MicroARNs/genética , Modelos Biológicos , Neoplasias/tratamiento farmacológico , Proteína Proto-Oncogénica c-ets-1/genética , Proteína Proto-Oncogénica c-ets-1/metabolismo , Ribonucleasa III/genética , Ribonucleasa III/metabolismo , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
MicroRNAs are small regulatory RNAs that post transcriptionally control gene expression. Reduced expression of DICER, the enzyme involved in microRNA processing, is frequently observed in cancer and is associated with poor clinical outcome in various malignancies. Yet, the underlying mechanisms are not well understood. Here we identify tumour hypoxia as a regulator of DICER expression in large cohorts of breast cancer patients. We show that DICER expression is suppressed by hypoxia through an epigenetic mechanism that involves inhibition of oxygen-dependent H3K27me3 demethylases KDM6A/B and results in silencing of the DICER promoter. Subsequently, reduced miRNA processing leads to derepression of the miR-200 target ZEB1, stimulates the epithelial to mesenchymal transition and ultimately results in the acquisition of stem cell phenotypes in human mammary epithelial cells. Our study uncovers a previously unknown relationship between oxygen-sensitive epigenetic regulators, miRNA biogenesis and tumour stem cell phenotypes that may underlie poor outcome in breast cancer.
Asunto(s)
ARN Helicasas DEAD-box/genética , Epigénesis Genética , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Ribonucleasa III/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Hipoxia de la Célula/genética , ARN Helicasas DEAD-box/metabolismo , Transición Epitelial-Mesenquimal/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Silenciador del Gen , Humanos , Fenotipo , Pronóstico , Regiones Promotoras Genéticas/genética , Procesamiento Postranscripcional del ARN/genética , Ribonucleasa III/metabolismoRESUMEN
In COPD, matrix remodeling contributes to airflow limitation. Recent evidence suggests that next to fibroblasts, the process of epithelial-mesenchymal transition can contribute to matrix remodeling. CSE has been shown to induce EMT in lung epithelial cells, but the signaling mechanisms involved are largely unknown and subject of this study. EMT was assessed in A549 and BEAS2B cells stimulated with CSE by qPCR, Western blotting and immunofluorescence for epithelial and mesenchymal markers, as were collagen production, cell adhesion and barrier integrity as functional endpoints. Involvement of TGF-ß and HIF1α signaling pathways were investigated. In addition, mouse models were used to examine the effects of CS on hypoxia signaling and of hypoxia per se on mesenchymal expression. CSE induced EMT characteristics in A549 and BEAS2B cells, evidenced by decreased expression of epithelial markers and a concomitant increase in mesenchymal marker expression after CSE exposure. Furthermore cells that underwent EMT showed increased production of collagen, decreased adhesion and disrupted barrier integrity. The induction of EMT was found to be independent of TGF-ß signaling. On the contrary, CS was able to induce hypoxic signaling in A549 and BEAS2B cells as well as in mice lung tissue. Importantly, HIF1α knock-down prevented induction of mesenchymal markers, increased collagen production and decreased adhesion after CSE exposure, data that are in line with the observed induction of mesenchymal marker expression by hypoxia in vitro and in vivo. Together these data provide evidence that both bronchial and alveolar epithelial cells undergo a functional phenotypic shift in response to CSE exposure which can contribute to increased collagen deposition in COPD lungs. Moreover, HIF1α signaling appears to play an important role in this process.
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Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Transición Epitelial-Mesenquimal/efectos de los fármacos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Nicotiana/química , Humo/efectos adversos , Animales , Biomarcadores/metabolismo , Bronquios/citología , Hipoxia de la Célula/efectos de los fármacos , Línea Celular , Humanos , Ratones , Fenotipo , Alveolos Pulmonares/citología , Transducción de Señal/efectos de los fármacos , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Cell surface proteins have a wide range of biological functions, and are often used as lineage-specific markers. Antibodies that recognize cell surface antigens are widely used as research tools, diagnostic markers, and even therapeutic agents. The ability to obtain broad cell surface protein profiles would thus be of great value in a wide range of fields. There are however currently few available methods for high-throughput analysis of large numbers of cell surface proteins. We describe here a high-throughput flow cytometry (HT-FC) platform for rapid analysis of 363 cell surface antigens. Here we demonstrate that HT-FC provides reproducible results, and use the platform to identify cell surface antigens that are influenced by common cell preparation methods. We show that multiple populations within complex samples such as primary tumors can be simultaneously analyzed by co-staining of cells with lineage-specific antibodies, allowing unprecedented depth of analysis of heterogeneous cell populations. Furthermore, standard informatics methods can be used to visualize, cluster and downsample HT-FC data to reveal novel signatures and biomarkers. We show that the cell surface profile provides sufficient molecular information to classify samples from different cancers and tissue types into biologically relevant clusters using unsupervised hierarchical clustering. Finally, we describe the identification of a candidate lineage marker and its subsequent validation. In summary, HT-FC combines the advantages of a high-throughput screen with a detection method that is sensitive, quantitative, highly reproducible, and allows in-depth analysis of heterogeneous samples. The use of commercially available antibodies means that high quality reagents are immediately available for follow-up studies. HT-FC has a wide range of applications, including biomarker discovery, molecular classification of cancers, or identification of novel lineage specific or stem cell markers.
Asunto(s)
Antígenos de Superficie/análisis , Citometría de Flujo/métodos , Proteoma/análisis , Proteómica/métodos , Biomarcadores/análisis , Línea Celular Tumoral , Células Cultivadas , Análisis por Conglomerados , Humanos , Células Jurkat , Células MCF-7 , Microscopía Fluorescente , Proteoma/clasificación , Proteoma/inmunología , Reproducibilidad de los ResultadosRESUMEN
The cellular response to DNA double strand breaks (DSBs) involves the ordered assembly of repair proteins at or near sites of damage. This process is mediated through post-translational protein modifications that include both phosphorylation and ubiquitylation. Recent data have demonstrated that recruitment of the repair proteins BRCA1, 53BP1, and RAD18 to ionizing irradiation (IR) induced DSBs is dependent on formation of non-canonical K63-linked polyubiquitin chains by the RNF8 and RNF168 ubiquitin ligases. Here we report a novel role for K63-ubiquitylation in response to replication-associated DSBs that contributes to both cell survival and maintenance of genome stability. Suppression of K63-ubiquitylation markedly increases large-scale mutations and chromosomal aberrations in response to endogenous or exogenous replication-associated DSBs. These effects are associated with an S-phase specific defect in DNA repair as revealed by an increase in residual 53BP1 foci. Use of both knockdown and knockout cell lines indicates that unlike the case for IR-induced DSBs, the requirement for K63-ubiquitylation for the repair of replication associated DSBs was found to be RNF8-independent. Our findings reveal the existence of a novel K63-ubiquitylation dependent repair pathway that contributes to the maintenance of genome integrity in response to replication-associated DSBs.
Asunto(s)
Roturas del ADN de Doble Cadena/efectos de la radiación , Reparación del ADN , Replicación del ADN , Inestabilidad Genómica , Procesamiento Proteico-Postraduccional , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Línea Celular Tumoral , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Células Epiteliales/citología , Células Epiteliales/metabolismo , Células Epiteliales/efectos de la radiación , Regulación de la Expresión Génica , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Lisina/metabolismo , Fosforilación , Radiación Ionizante , Transducción de Señal , Proteína 1 de Unión al Supresor Tumoral P53 , Ubiquitina-Proteína Ligasas , UbiquitinaciónRESUMEN
Most proteins destined for the extracellular space require disulfide bonds for folding and stability. Disulfide bonds are introduced co- and post-translationally in endoplasmic reticulum (ER) cargo in a redox relay that requires a terminal electron acceptor. Oxygen can serve as the electron acceptor in vitro, but its role in vivo remains unknown. Hypoxia causes ER stress, suggesting a role for oxygen in protein folding. Here we demonstrate the existence of two phases of disulfide bond formation in living mammalian cells, with differential requirements for oxygen. Disulfide bonds introduced rapidly during protein synthesis can occur without oxygen, whereas those introduced during post-translational folding or isomerization are oxygen dependent. Other protein maturation processes in the secretory pathway, including ER-localized N-linked glycosylation, glycan trimming, Golgi-localized complex glycosylation, and protein transport, occur independently of oxygen availability. These results suggest that an alternative electron acceptor is available transiently during an initial phase of disulfide bond formation and that post-translational oxygen-dependent disulfide bond formation causes hypoxia-induced ER stress.
Asunto(s)
Disulfuros/metabolismo , Oxígeno/farmacología , Hipoxia de la Célula/efectos de los fármacos , Hipoxia de la Célula/genética , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Células HCT116 , Células HeLa , Humanos , Isomerismo , Modelos Biológicos , Pliegue de Proteína/efectos de los fármacos , Procesamiento Proteico-Postraduccional/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transcripción Genética/efectos de los fármacosRESUMEN
Hypoxia is a common feature of tumors and an important contributor to malignancy and treatment resistance. The ability of tumor cells to survive hypoxic stress is mediated in part by hypoxia-inducible factor (HIF)-dependent transcriptional responses. More severe hypoxia activates endoplasmatic reticulum stress responses, including the double-stranded RNA-activated protein kinase (PKR)-like endoplasmic reticulum kinase (PERK)/eukaryotic initiation factor 2α (eIF2α)-dependent arm of the unfolded protein response (UPR). Although several studies implicate important roles for HIF and UPR in adaption to hypoxia, their importance for hypoxic cells responsible for therapy resistance in tumors is unknown. By using isogenic models, we find that HIF and eIF2α signaling contribute to the survival of hypoxic cells in vitro and in vivo. However, the eIF2α-dependent arm of the UPR is uniquely required for the survival of a subset of hypoxic cells that determine tumor radioresistance. We demonstrate that eIF2α signaling induces uptake of cysteine, glutathione synthesis, and protection against reactive oxygen species produced during periods of cycling hypoxia. Together these data imply that eIF2α signaling is a critical contributor to the tolerance of therapy-resistant cells that arise as a consequence of transient changes in oxygenation in solid tumors and thus a therapeutic target in curative treatments for solid cancers.
Asunto(s)
Factor 2 Eucariótico de Iniciación/metabolismo , Glutatión/biosíntesis , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Respuesta de Proteína Desplegada , eIF-2 Quinasa/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Hipoxia de la Célula/genética , Línea Celular Tumoral , Factor 2 Eucariótico de Iniciación/genética , Glutatión/genética , Humanos , Ratones , Ratones Desnudos , Proteínas de Neoplasias/genética , Trasplante de Neoplasias , Neoplasias/genética , Neoplasias/terapia , Transducción de Señal/genética , Trasplante Heterólogo , eIF-2 Quinasa/genéticaRESUMEN
Tribbles homolog 3 (TRIB3) is a scaffold protein activated under hypoxic conditions and involved in several cell survival and proliferation pathways. Recently, we reported opposite associations of TRIB3 mRNA and protein with breast cancer prognosis. In this study, we investigated this discrepancy between TRIB3 mRNA and protein in human breast cancer. We provide several lines of evidence demonstrating that TRIB3 is a stabile protein which levels are not controlled by rapid protein breakdown. Interestingly, we were able to show that during anoxia TRIB3 mRNA translation was profoundly inhibited. Hypoxia induced micro RNA 24 was not responsible for the translational repression of TRIB3. Furthermore miRNA-24 expression levels in breast cancer patient specimens showed no correlation with TRIB3 mRNA or TRIB3 protein levels, or with prognosis. Thus, the expression of miRNA-24 does not explain the difference between mRNA and protein expression of TRIB3 in this cohort of breast cancer patients. In conclusion, TRIB3 protein is a stable protein which levels are predominantly regulated by translational control of TRIB3 mRNA transcript.
Asunto(s)
Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Proteínas de Ciclo Celular/biosíntesis , Regulación Neoplásica de la Expresión Génica , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Represoras/biosíntesis , Adulto , Anciano , Línea Celular Tumoral , Progresión de la Enfermedad , Femenino , Humanos , Hipoxia , MicroARNs/metabolismo , Persona de Mediana Edad , Pronóstico , Proteínas Serina-Treonina Quinasas/biosíntesis , Proteínas/química , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Factores de TiempoRESUMEN
BACKGROUND AND PURPOSE: Hypoxia is a common feature of the microenvironment of solid tumors which has been shown to promote malignancy and poor patient outcome through multiple mechanisms. The association of hypoxia with more aggressive disease may be due in part to recently identified links between hypoxia and genetic instability. For example, hypoxia has been demonstrated to impede DNA repair by down-regulating the homologous recombination protein RAD51. Here we investigated hypoxic regulation of UBE2T, a ubiquitin ligase required in the Fanconi anemia (FA) DNA repair pathway. MATERIALS AND METHODS: We analysed UBE2T expression by microarray, quantitative PCR and western blot analysis in a panel of cancer cell lines as a function of oxygen concentration. The importance of this regulation was assessed by measuring cell survival in response to DNA damaging agents under normoxia or hypoxia. Finally, HIF dependency was determined using knockdown cell lines and RCC4 cells which constitutively express HIF1α. RESULTS: Hypoxia results in rapid and potent reductions in mRNA levels of UBE2T in a panel of cancer cell lines. Reduced UBE2T mRNA expression is HIF independent and was not due to changes in mRNA or protein stability, but rather reflected reduced promoter activity. Exposure of tumor cells to hypoxia greatly increased their sensitivity to treatment with the interstrand crosslinking (ICL) agent mitomycin C. CONCLUSIONS: Exposure to hypoxic conditions down-regulates UBE2T expression which correlates with an increased sensitivity to crosslinking agents consistent with a defective Fanconi anemia pathway. This pathway can potentially be exploited to target hypoxic cells in tumors.
Asunto(s)
Anemia de Fanconi/tratamiento farmacológico , Anemia de Fanconi/metabolismo , Hipoxia/metabolismo , Transducción de Señal/efectos de los fármacos , Enzimas Ubiquitina-Conjugadoras/metabolismo , Antineoplásicos/farmacología , Western Blotting , Supervivencia Celular , Reparación del ADN , Regulación hacia Abajo , Anemia de Fanconi/etiología , Regulación Neoplásica de la Expresión Génica , Humanos , Hipoxia/complicaciones , Neoplasias/irrigación sanguínea , Neoplasias/complicaciones , Neoplasias/tratamiento farmacológico , ARN Mensajero/metabolismo , Valores de Referencia , Transducción de Señal/genética , Células Tumorales CultivadasRESUMEN
BACKGROUND AND PURPOSE: Hypoxia is a common feature of solid tumors that is associated with an aggressive phenotype, resistance to therapy and poor prognosis. Major contributors to these adverse effects are the transcriptional program activated by the HIF family of transcription factors as well as the translational response mediated by PERK-dependent phosphorylation of eIF2α and inhibition of mTORC1 activity. In this study we determined the relative contribution of both transcriptional and translational responses to changes in hypoxia induced gene expression. MATERIAL AND METHODS: Total and efficiently translated (polysomal) mRNA was isolated from DU145 prostate carcinoma cells that were exposed for up to 24 h of hypoxia (<0.02% O(2)). Changes in transcription and translation were assessed using affymetrix microarray technology. RESULTS: Our data reveal an unexpectedly large contribution of translation control on both induced and repressed gene expression at all hypoxic time points, particularly during acute hypoxia (2-4 h). Gene ontology analysis revealed that gene classes like transcription and signal transduction are stimulated by translational control whereas expression of genes involved in cell growth and protein metabolism are repressed during hypoxic conditions by translational control. CONCLUSIONS: Our data indicate that translation influences gene expression during hypoxia on a scale comparable to that of transcription.