RESUMO
Mutations in KEAP1 and NFE2L2 (encoding the protein Nrf2) are prevalent in both adeno and squamous subtypes of non-small cell lung cancer, as well as additional tumor indications. The consequence of these mutations is stabilized Nrf2 and chronic induction of a battery of Nrf2 target genes. We show that knockdown of Nrf2 caused modest growth inhibition of cells growing in two-dimension, which was more pronounced in cell lines expressing mutant KEAP1. In contrast, Nrf2 knockdown caused almost complete regression of established KEAP1-mutant tumors in mice, with little effect on wild-type (WT) KEAP1 tumors. The strong dependency on Nrf2 could be recapitulated in certain anchorage-independent growth environments and was not prevented by excess extracellular glutathione. A CRISPR screen was used to investigate the mechanism(s) underlying this dependence. We identified alternative pathways critical for Nrf2-dependent growth in KEAP1-mutant cell lines, including the redox proteins thioredoxin and peroxiredoxin, as well as the growth factor receptors IGF1R and ERBB3. IGF1R inhibition was effective in KEAP1-mutant cells compared with WT, especially under conditions of anchorage-independent growth. These results point to addiction of KEAP1-mutant tumor cells to Nrf2 and suggest that inhibition of Nrf2 or discrete druggable Nrf2 target genes such as IGF1R could be an effective therapeutic strategy for disabling these tumors. SIGNIFICANCE: This study identifies pathways activated by Nrf2 that are important for the proliferation and tumorigenicity of KEAP1-mutant non-small cell lung cancer.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Transdução de Sinais/fisiologia , Animais , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Xenoenxertos , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Neoplasias Pulmonares/genética , Camundongos , Mutação , Fator 2 Relacionado a NF-E2/metabolismo , Receptor ErbB-3/metabolismo , Receptor IGF Tipo 1/metabolismoRESUMO
Gasdermin-D (GSDMD) is cleaved by caspase-1, caspase-4, and caspase-11 in response to canonical and noncanonical inflammasome activation. Upon cleavage, GSDMD oligomerizes and forms plasma membrane pores, resulting in interleukin-1ß (IL-1ß) secretion, pyroptotic cell death, and inflammatory pathologies, including periodic fever syndromes and septic shock-a plague on modern medicine. Here, we showed that IRF2, a member of the interferon regulatory factor (IRF) family of transcription factors, was essential for the transcriptional activation of GSDMD. A forward genetic screen with N-ethyl-N-nitrosourea (ENU)-mutagenized mice linked IRF2 to inflammasome signaling. GSDMD expression was substantially attenuated in IRF2-deficient macrophages, endothelial cells, and multiple tissues, which corresponded with reduced IL-1ß secretion and inhibited pyroptosis. Mechanistically, IRF2 bound to a previously uncharacterized but unique site within the GSDMD promoter to directly drive GSDMD transcription for the execution of pyroptosis. Disruption of this single IRF2-binding site abolished signaling by both the canonical and noncanonical inflammasomes. Together, our data illuminate a key transcriptional mechanism for expression of the gene encoding GSDMD, a critical mediator of inflammatory pathologies.
Assuntos
Fator Regulador 2 de Interferon/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Ligação a Fosfato/genética , Piroptose/genética , Transcrição Gênica/genética , Animais , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Inflamassomos/genética , Inflamassomos/metabolismo , Fator Regulador 2 de Interferon/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas de Ligação a Fosfato/metabolismo , Transdução de Sinais/genética , Ativação Transcricional/genéticaRESUMO
Mutant KRAS represents one of the most frequently observed oncogenes in NSCLC, yet no therapies are approved for tumors that express activated KRAS variants. While there is strong rationale for the use of MEK inhibitors to treat tumors with activated RAS/MAPK signaling, these have proven ineffective clinically. We therefore implemented a CRISPR screening approach to identify novel agents to sensitize KRAS mutant NSCLC cells to MEK inhibitor treatment. This approach identified multiple components of the canonical RAS/MAPK pathway consistent with previous studies. In addition, we identified MAPK7 as a novel, strong hit and validated this finding using multiple orthogonal approaches including knockdown and pharmacological inhibition. We show that MAPK7 inhibition attenuates the re-activation of MAPK signaling occurring following long-term MEK inhibition, thereby illustrating that MAPK7 mediates pathway reactivation in the face of MEK inhibition. Finally, genetic knockdown of MAPK7 combined with the MEK inhibitor cobimetinib in a mutant KRAS NSCLC xenograft model to mediate improved tumor growth inhibition. These data highlight that MAPK7 represents a promising target for combination treatment with MEK inhibition in KRAS mutant NSCLC.
Assuntos
Biomarcadores Tumorais/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Proteína Quinase 7 Ativada por Mitógeno/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Células A549 , Animais , Carcinoma Pulmonar de Células não Pequenas/patologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Humanos , Sistema de Sinalização das MAP Quinases/genética , Camundongos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Despite widespread use of CRISPR, comprehensive data on the frequency and impact of Cas9-mediated off-targets in modified rodents are limited. Here we present deep-sequencing data from 81 genome-editing projects on mouse and rat genomes at 1,423 predicted off-target sites, 32 of which were confirmed, and show that high-fidelity Cas9 versions reduced off-target mutation rates in vivo. Using whole-genome sequencing data from ten mouse embryos, treated with a single guide RNA (sgRNA), and from their genetic parents, we found 43 off-targets, 30 of which were predicted by an adapted version of GUIDE-seq.
Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Engenharia Genética , Genômica/métodos , Animais , Linhagem Celular , Feminino , Masculino , Camundongos , Reação em Cadeia da Polimerase Multiplex/métodos , RNA/genética , Ratos , Sequenciamento Completo do Genoma/métodosRESUMO
The NLRC4 inflammasome recognizes bacterial flagellin and components of the type III secretion apparatus. NLRC4 stimulation leads to caspase-1 activation followed by a rapid lytic cell death known as pyroptosis. NLRC4 is linked to pathogen-free auto-inflammatory diseases, suggesting a role for NLRC4 in sterile inflammation. Here, we show that NLRC4 activates an alternative cell death program morphologically similar to apoptosis in caspase-1-deficient BMDMs. By performing an unbiased genome-wide CRISPR/Cas9 screen with subsequent validation studies in gene-targeted mice, we highlight a critical role for caspase-8 and ASC adaptor in an alternative apoptotic pathway downstream of NLRC4. Furthermore, caspase-1 catalytically dead knock-in (Casp1 C284A KI) BMDMs genetically segregate pyroptosis and apoptosis, and confirm that caspase-1 does not functionally compete with ASC for NLRC4 interactions. We show that NLRC4/caspase-8-mediated apoptotic cells eventually undergo plasma cell membrane damage in vitro, suggesting that this pathway can lead to secondary necrosis. Unexpectedly, we found that DFNA5/GSDME, a member of the pore-forming gasdermin family, is dispensable for the secondary necrosis that follows NLRC4-mediated apoptosis in macrophages. Together, our data confirm the existence of an alternative caspase-8 activation pathway diverging from the NLRC4 inflammasome in primary macrophages.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Apoptose , Proteínas Adaptadoras de Sinalização CARD/fisiologia , Proteínas de Ligação ao Cálcio/metabolismo , Caspase 1/fisiologia , Caspase 8/fisiologia , Inflamassomos/metabolismo , Macrófagos/patologia , Animais , Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Proteínas Reguladoras de Apoptose/genética , Sistemas CRISPR-Cas , Proteínas de Ligação ao Cálcio/antagonistas & inibidores , Proteínas de Ligação ao Cálcio/genética , Genoma , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
The necroptotic cell death pathway is a key component of human pathogen defense that can become aberrantly derepressed during tissue homeostasis to contribute to multiple types of tissue damage and disease. While formation of the necrosome kinase signaling complex containing RIPK1, RIPK3, and MLKL has been extensively characterized, additional mechanisms of its regulation and effector functions likely remain to be discovered. We screened 19,883 mouse protein-coding genes by CRISPR/Cas9-mediated gene knockout for resistance to cytokine-induced necroptosis and identified 112 regulators and mediators of necroptosis, including 59 new candidate pathway components with minimal or no effect on cell growth in the absence of necroptosis induction. Among these, we further characterized the function of PTBP1, an RNA binding protein whose activity is required to maintain RIPK1 protein abundance by regulating alternative splice-site selection.
Assuntos
Processamento Alternativo , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Fibroblastos/enzimologia , Marcação de Genes/métodos , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Necroptose , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Animais , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Fibroblastos/patologia , Regulação Enzimológica da Expressão Gênica , Células HEK293 , Células HT29 , Ribonucleoproteínas Nucleares Heterogêneas/genética , Humanos , Camundongos , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Transdução de SinaisRESUMO
A propensity for rewiring genetic and epigenetic regulatory networks, thus enabling sustained cell proliferation, suppression of apoptosis, and the ability to evade the immune system, is vital to cancer cell propagation. An increased understanding of how this is achieved is critical for identifying or improving therapeutic interventions. In this study, using acute myeloid leukemia (AML) human cell lines and a custom CRISPR/Cas9 screening platform, we identify the H3K9 methyltransferase SETDB1 as a novel, negative regulator of innate immunity. SETDB1 is overexpressed in many cancers, and loss of this gene in AML cells triggers desilencing of retrotransposable elements that leads to the production of double-stranded RNAs (dsRNAs). This is coincident with induction of a type I interferon response and apoptosis through the dsRNA-sensing pathway. Collectively, our findings establish a unique gene regulatory axis that cancer cells can exploit to circumvent the immune system.
Assuntos
Inativação Gênica , Interferon Tipo I/metabolismo , Leucemia Mieloide Aguda/enzimologia , Proteínas Metiltransferases/metabolismo , Retroelementos , Apoptose , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Sobrevivência Celular , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Histona-Lisina N-Metiltransferase , Humanos , Imunidade Inata , Interferon Tipo I/imunologia , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/imunologia , Leucemia Mieloide Aguda/patologia , Proteínas Metiltransferases/genética , Interferência de RNA , RNA de Cadeia Dupla/biossíntese , RNA de Cadeia Dupla/genética , Transdução de Sinais , Fatores de Tempo , Transfecção , Evasão TumoralRESUMO
Colon tumors arise in a stepwise fashion from either discrete genetic perturbations or epigenetic dysregulation. To uncover the key epigenetic regulators that drive colon cancer growth, we used a CRISPR loss-of-function screen and identified a number of essential genes, including the bromodomain and extraterminal (BET) protein BRD4. We found that BRD4 is critical for colon cancer proliferation, and its knockdown led to differentiation effects in vivo. JQ1, a BET inhibitor, preferentially reduced growth in a subset of epigenetically dysregulated colon cancers characterized by the CpG island methylator phenotype (CIMP). Integrated transcriptomic and genomic analyses defined a distinct superenhancer in CIMP+ colon cancers that regulates cMYC transcription. We found that the long noncoding RNA colon cancer-associated transcript 1 (CCAT1) is transcribed from this superenhancer and is exquisitely sensitive to BET inhibition. Concordantly, cMYC transcription and cell growth were tightly correlated with the presence of CCAT1 RNA in a variety of tumor types. Taken together, we propose that CCAT1 is a clinically tractable biomarker for identifying patients who are likely to benefit from BET inhibitors.
Assuntos
Biomarcadores Tumorais/metabolismo , Proliferação de Células , Proteínas Nucleares/metabolismo , RNA Longo não Codificante/metabolismo , RNA Neoplásico/metabolismo , Fatores de Transcrição/metabolismo , Animais , Azepinas/farmacologia , Biomarcadores Tumorais/genética , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Neoplasias Colorretais , Ilhas de CpG , Metilação de DNA/efeitos dos fármacos , DNA de Neoplasias/genética , DNA de Neoplasias/metabolismo , Feminino , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Camundongos Nus , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Longo não Codificante/genética , RNA Neoplásico/genética , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Triazóis/farmacologiaRESUMO
Incorporating miRNA-like features into vector-based hairpin scaffolds has been shown to augment small RNA processing and RNAi efficiency. Therefore, defining an optimal, native hairpin context may obviate a need for hairpin-specific targeting design schemes, which confound the movement of functional siRNAs into shRNA/artificial miRNA backbones, or large-scale screens to identify efficacious sequences. Thus, we used quantitative cell-based assays to compare separate third generation artificial miRNA systems, miR-E (based on miR-30a) and miR-3G (based on miR-16-2 and first described in this study) to widely-adopted, first and second generation formats in both Pol-II and Pol-III expression vector contexts. Despite their unique structures and strandedness, and in contrast to first and second-generation RNAi triggers, the third generation formats operated with remarkable similarity to one another, and strong silencing was observed with a significant fraction of the evaluated target sequences within either promoter context. By pairing an established siRNA design algorithm with the third generation vectors we could readily identify targeting sequences that matched or exceeded the potency of those discovered through large-scale sensor-based assays. We find that third generation hairpin systems enable the maximal level of siRNA function, likely through enhanced processing and accumulation of precisely-defined guide RNAs. Therefore, we predict future gains in RNAi potency will come from improved hairpin expression and identification of optimal siRNA-intrinsic silencing properties rather than further modification of these scaffolds. Consequently, third generation systems should be the primary format for vector-based RNAi studies; miR-3G is advantageous due to its small expression cassette and simplified, cost-efficient cloning scheme.
Assuntos
Vetores Genéticos/genética , MicroRNAs/genética , Interferência de RNA , RNA Interferente Pequeno/análise , Algoritmos , Animais , Células HEK293 , Humanos , Camundongos , RNA Polimerase II/metabolismo , RNA Polimerase III/metabolismo , RNA de Cadeia Dupla , RNA Interferente Pequeno/químicaRESUMO
Allele-specific gene expression, ASE, is an important aspect of gene regulation. We developed a novel method MBASED, meta-analysis based allele-specific expression detection for ASE detection using RNA-seq data that aggregates information across multiple single nucleotide variation loci to obtain a gene-level measure of ASE, even when prior phasing information is unavailable. MBASED is capable of one-sample and two-sample analyses and performs well in simulations. We applied MBASED to a panel of cancer cell lines and paired tumor-normal tissue samples, and observed extensive ASE in cancer, but not normal, samples, mainly driven by genomic copy number alterations.
Assuntos
Biologia Computacional/métodos , Regulação Neoplásica da Expressão Gênica , Neoplasias/genética , Alelos , Distribuição Binomial , Humanos , Polimorfismo de Nucleotídeo Único , Análise de Sequência de RNA , Software , Células Tumorais CultivadasRESUMO
Lung cancer is the leading cause of cancer-related mortality worldwide, with non-small-cell lung carcinomas in smokers being the predominant form of the disease. Although previous studies have identified important common somatic mutations in lung cancers, they have primarily focused on a limited set of genes and have thus provided a constrained view of the mutational spectrum. Recent cancer sequencing efforts have used next-generation sequencing technologies to provide a genome-wide view of mutations in leukaemia, breast cancer and cancer cell lines. Here we present the complete sequences of a primary lung tumour (60x coverage) and adjacent normal tissue (46x). Comparing the two genomes, we identify a wide variety of somatic variations, including >50,000 high-confidence single nucleotide variants. We validated 530 somatic single nucleotide variants in this tumour, including one in the KRAS proto-oncogene and 391 others in coding regions, as well as 43 large-scale structural variations. These constitute a large set of new somatic mutations and yield an estimated 17.7 per megabase genome-wide somatic mutation rate. Notably, we observe a distinct pattern of selection against mutations within expressed genes compared to non-expressed genes and in promoter regions up to 5 kilobases upstream of all protein-coding genes. Furthermore, we observe a higher rate of amino acid-changing mutations in kinase genes. We present a comprehensive view of somatic alterations in a single lung tumour, and provide the first evidence, to our knowledge, of distinct selective pressures present within the tumour environment.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Genoma Humano/genética , Neoplasias Pulmonares/genética , Mutação Puntual/genética , Análise Mutacional de DNA , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Biológicos , Proto-Oncogene Mas , Seleção Genética/genéticaRESUMO
Various cancer genome projects are underway to identify novel mutations that drive tumorigenesis. While these screens will generate large data sets, the majority of identified missense changes are likely to be innocuous passenger mutations or polymorphisms. As a result, it has become increasingly important to develop computational methods for distinguishing functionally relevant mutations from other variations. We previously developed an algorithm, and now present the web application, CanPredict (http://www.canpredict.org/ or http://www.cgl.ucsf.edu/Research/genentech/canpredict/), to allow users to determine if particular changes are likely to be cancer-associated. The impact of each change is measured using two known methods: Sorting Intolerant From Tolerant (SIFT) and the Pfam-based LogR.E-value metric. A third method, the Gene Ontology Similarity Score (GOSS), provides an indication of how closely the gene in which the variant resides resembles other known cancer-causing genes. Scores from these three algorithms are analyzed by a random forest classifier which then predicts whether a change is likely to be cancer-associated. CanPredict fills an important need in cancer biology and will enable a large audience of biologists to determine which mutations are the most relevant for further study.
Assuntos
Predisposição Genética para Doença , Mutação de Sentido Incorreto , Neoplasias/genética , Polimorfismo de Nucleotídeo Único , Algoritmos , Alelos , Aminoácidos/química , Linhagem Celular Tumoral , Biologia Computacional/métodos , DNA de Neoplasias/genética , Etiquetas de Sequências Expressas , Humanos , Internet , Interface Usuário-ComputadorRESUMO
MOTIVATION: We introduce GMAP, a standalone program for mapping and aligning cDNA sequences to a genome. The program maps and aligns a single sequence with minimal startup time and memory requirements, and provides fast batch processing of large sequence sets. The program generates accurate gene structures, even in the presence of substantial polymorphisms and sequence errors, without using probabilistic splice site models. Methodology underlying the program includes a minimal sampling strategy for genomic mapping, oligomer chaining for approximate alignment, sandwich DP for splice site detection, and microexon identification with statistical significance testing. RESULTS: On a set of human messenger RNAs with random mutations at a 1 and 3% rate, GMAP identified all splice sites accurately in over 99.3% of the sequences, which was one-tenth the error rate of existing programs. On a large set of human expressed sequence tags, GMAP provided higher-quality alignments more often than blat did. On a set of Arabidopsis cDNAs, GMAP performed comparably with GeneSeqer. In these experiments, GMAP demonstrated a several-fold increase in speed over existing programs. AVAILABILITY: Source code for gmap and associated programs is available at http://www.gene.com/share/gmap SUPPLEMENTARY INFORMATION: http://www.gene.com/share/gmap.
Assuntos
Algoritmos , Mapeamento Cromossômico/métodos , Etiquetas de Sequências Expressas , RNA Mensageiro/genética , Alinhamento de Sequência/métodos , Análise de Sequência de DNA/métodos , Análise de Sequência de RNA/métodos , Software , Genoma Humano , Humanos , Sítios de Splice de RNA/genética , Homologia de Sequência do Ácido NucleicoRESUMO
AIMS: Paclitaxel is a potent and effective inhibitor of neointimal proliferation after coronary stenting. The Conor stent loaded with Paclitaxel can be programmed with multi-parameter matrix of dose, temporal release profiles and release pathways. The aim of this study was to determine the most efficacious dose and release pattern of Paclitaxel in a porcine model and parallels the PISCES trial. METHODS: 32 farm pigs were implanted with Conor stents loaded with 10 or 30 microg of Paclitaxel with, 10 or 30 day and mural or bidirectional release patterns. Angiographic and histomorphometric analysis was -performed at 30 and 90 days. RESULTS: All doses of Paclitaxel were angiographically superior to control (P < 0.01). At 30 days, intimal thickness was similar between Pisces D4 (30 microg/10 days, bidirectional release), D5 (10 microg/30 day mural) and D6 (30 microg/30 day, mural) with D4 having the lowest intimal thickness (167+/-59 microm). There was a significant increase in the mural injury associated with D4 in comparison to all other doses (P < 0.00001). At 90 days D4 was significantly worse in comparison to Pisces D5 and D6; (P < 0.01) and Pisces D5 and D6 were similar to controls. CONCLUSIONS: 10 day release of Paclitaxel may be too short a period to inhibit neointimal proliferation after coronary stenting, or the rapid release of Paclitaxel may induce chemical injury causing secondary insult to the artery resulting in a rebound increase in intimal thickness at 90 days. These data parallel clinical findings in the PISCES trial.
RESUMO
The availability of cardiac ultrasound is limited in developing countries. We evaluated the feasibility and diagnostic capability of a hand-carried cardiac ultrasound device in 126 patients (age 44 +/- 24 years) referred for consultation to a cardiology clinic in rural Mexico. The hand-carried cardiac ultrasound device identified 86 cardiac findings and obviated the need for further comprehensive echocardiographic evaluation in 90% of patients (113 of 126).
Assuntos
Países em Desenvolvimento , Ecocardiografia Doppler em Cores/instrumentação , Ecocardiografia/instrumentação , Cardiopatias/diagnóstico por imagem , Sistemas Automatizados de Assistência Junto ao Leito , População Rural , Adulto , Instituições de Assistência Ambulatorial , California , Criança , Desenho de Equipamento , Humanos , Missões Médicas , México , Encaminhamento e Consulta , VoluntáriosRESUMO
MOTIVATION: Expression profiling in diverse tissues is fundamental to understanding gene function as well as therapeutic target identification. The vast collection of expressed sequence tags (ESTs) and the associated tissue source information provides an attractive opportunity for studying gene expression. RESULTS: To facilitate EST-based expression analysis, we developed GEPIS (gene expression profiling in silico), a tool that integrates EST and tissue source information to compute gene expression patterns in a large panel of normal and tumor samples. We found EST-based expression patterns to be consistent with published papers as well as our own experimental results. We also built a GEPIS Regional Atlas that depicts expression characteristics of all genes in a selected genomic region. This program can be adapted for large-scale screening for genes with desirable expression patterns, as illustrated by our large-scale mining for tissue- and tumor-specific genes. AVAILABILITY: The email server version of the GEPIS application is freely available at http://share.gene.com/share/gepis. An interactive version of GEPIS will soon be freely available at http://www.cgl.ucsf.edu/Research/genentech/gepis/. The source code, modules, data and gene lists can be downloaded at http://share.gene.com/share/gepis.
Assuntos
Algoritmos , Mapeamento Cromossômico/métodos , Perfilação da Expressão Gênica/métodos , Neoplasias/genética , Análise de Sequência de DNA/métodos , Software , Interface Usuário-Computador , Biomarcadores Tumorais/genética , Etiquetas de Sequências Expressas , Testes Genéticos/métodos , Humanos , Internet , Neoplasias/diagnóstico , Sistemas On-Line , Alinhamento de Sequência/métodosRESUMO
Genes up-regulated in tumor cells provide attractive anticancer therapeutic targets. Although the general underlying mechanism for the increased expression in tumors is unknown, tumor-specific up-regulation of some genes can be attributed to aberrant DNA amplification, a phenomenon common to many tumors. Using a computational method, we constructed a general transcriptome map with the human genomic sequences and expressed sequence tags in the public database. The transcriptome map revealed nonrandom chromosomal regions (termed region of increased tumor expression) where clusters of genes exhibited increased expression in the 10 tumor tissue types tested. These genomic regions often correspond to experimentally verified tumor amplicons. Our large-scale transcriptome analysis led to identification of many additional chromosomal regions with increased tumor expression, regions that represent potential tumor amplicons.
Assuntos
Etiquetas de Sequências Expressas , Neoplasias/genética , Cromossomos Humanos/genética , Bases de Dados Genéticas , Regulação Neoplásica da Expressão Gênica , Genoma Humano , Humanos , Família Multigênica , Neoplasias/metabolismo , Hibridização de Ácido Nucleico , Transcrição Gênica , Regulação para CimaRESUMO
A large-scale effort, termed the Secreted Protein Discovery Initiative (SPDI), was undertaken to identify novel secreted and transmembrane proteins. In the first of several approaches, a biological signal sequence trap in yeast cells was utilized to identify cDNA clones encoding putative secreted proteins. A second strategy utilized various algorithms that recognize features such as the hydrophobic properties of signal sequences to identify putative proteins encoded by expressed sequence tags (ESTs) from human cDNA libraries. A third approach surveyed ESTs for protein sequence similarity to a set of known receptors and their ligands with the BLAST algorithm. Finally, both signal-sequence prediction algorithms and BLAST were used to identify single exons of potential genes from within human genomic sequence. The isolation of full-length cDNA clones for each of these candidate genes resulted in the identification of >1000 novel proteins. A total of 256 of these cDNAs are still novel, including variants and novel genes, per the most recent GenBank release version. The success of this large-scale effort was assessed by a bioinformatics analysis of the proteins through predictions of protein domains, subcellular localizations, and possible functional roles. The SPDI collection should facilitate efforts to better understand intercellular communication, may lead to new understandings of human diseases, and provides potential opportunities for the development of therapeutics.
Assuntos
Moléculas de Adesão Celular Neuronais , Biologia Computacional/métodos , Proteínas de Membrana/genética , Proteínas/genética , Proteínas/metabolismo , Proteínas Ligadas por GPI , Biblioteca Gênica , Humanos , Dados de Sequência Molecular , Valor Preditivo dos Testes , Sinais Direcionadores de Proteínas/genéticaRESUMO
Peptide mass fingerprinting (PMF) grew from a need for a faster, more efficient method to identify frequently observed proteins in electrophoresis gels. We describe the genesis of the idea in 1989, and show the first demonstration with fast atom bombardment mass spectrometry. Despite its promise, the method was seldom used until 1992, with the coming of significantly more sensitive commercial instrumentation based on MALDI-TOF-MS. We recount the evolution of the method and its dependence on a number of technical breakthroughs, both in mass spectrometry and in other areas. We show how it laid the foundation for high-throughput, high-sensitivity methods of protein analysis, now known as proteomics. We conclude with recommendations for further improvements, and speculation of the role of PMF in the future.