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1.
J Extracell Vesicles ; 7(1): 1438727, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29511462

RESUMO

Sample amount is often a limiting factor for multi-parametric analyses that encompass at least three areas of '-omics' research: genomics, transcriptomics and proteomics. Limited sample amounts are also an important consideration when these multi-parametric analyses are performed on extracellular vesicles (EVs), as the amount of EVs (and EV cargo) that can be isolated is often very low. It is well understood that a monophasic solution of phenol and guanidine isothiocyanate (i.e. TRIzol©) can simultaneously isolate DNA, RNA and proteins from biological samples; however, it is most commonly used for the extraction of RNA. Validation of this reagent for the isolation of multiple classes of biological molecules from EVs would provide a widely applicable method for performing multi-parametric analyses of EV material. In this report, we describe a comparison of proteins identified from EVs processed with either TRIzol© or the conventional Laemmli buffer protein-extraction reagents. EVs were isolated from 3 mL of cell-culture supernatant derived from MCF-10A, MCF-7 and MDA-MB-231 cells using the Vn96 EV capture technology. For the TRIzol© extraction protocol, proteins were precipitated with acetone from the organic phase and then re-solubilized in a mixture of 8M urea, 0.2% SDS and 1 M Tris-HCl pH 6.8, followed by dilution in 5× loading buffer prior to fractionation with 1D SDS-PAGE. NanoLC-MS/MS of the trypsin-digested proteins was used to generate proteomic profiles from EV protein samples extracted with each method. Of the identified proteins, 57.7%, 69.2% and 57.0% were common to both extraction methods for EVs from MCF-10A, MCF-7 and MDA-MB-231, respectively. Our results suggest that TRIzol© extraction of proteins from EVs has significant equivalence to the traditional Laemmli method. The advantage of using TRIzol

2.
Sci Rep ; 7(1): 8642, 2017 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-28819186

RESUMO

The CD24 cell surface receptor promotes apoptosis in developing B cells, and we recently found that it induces B cells to release plasma membrane-derived, CD24-bearing microvesicles (MVs). Here we have performed a systematic characterization of B cell MVs released from WEHI-231 B lymphoma cells in response to CD24 stimulation. We found that B cells constitutively release MVs of approximately 120 nm, and that CD24 induces an increase in phosphatidylserine-positive MV release. RNA cargo is predominantly comprised of 5S rRNA, regardless of stimulation; however, CD24 causes a decrease in the incorporation of protein coding transcripts. The MV proteome is enriched with mitochondrial and metabolism-related proteins after CD24 stimulation; however, these changes were variable and could not be fully validated by Western blotting. CD24-bearing MVs carry Siglec-2, CD63, IgM, and, unexpectedly, Ter119, but not Siglec-G or MHC-II despite their presence on the cell surface. CD24 stimulation also induces changes in CD63 and IgM expression on MVs that is not mirrored by the changes in cell surface expression. Overall, the composition of these MVs suggests that they may be involved in releasing mitochondrial components in response to pro-apoptotic stress with changes to the surface receptors potentially altering the cell type(s) that interact with the MVs.


Assuntos
Antígeno CD24/metabolismo , Micropartículas Derivadas de Células/metabolismo , Proteínas/metabolismo , RNA/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Linfócitos B/metabolismo , Transporte Biológico , Linhagem Celular , Membrana Celular/metabolismo , Células Cultivadas , Biologia Computacional/métodos , Humanos , Espectrometria de Massas
3.
Int J Cancer ; 141(4): 778-790, 2017 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-28486780

RESUMO

Inactivation of the tumor suppressor gene, von Hippel-Lindau (VHL), is known to play an important role in the development of sporadic clear cell renal cell carcinomas (ccRCCs). Even if available targeted therapies for metastatic RCCs (mRCCs) have helped to improve progression-free survival rates, they have no durable clinical response. We have previously shown the feasibility of specifically targeting the loss of VHL with the identification of a small molecule, STF-62247. Understanding its functionality is crucial for developing durable personalized therapeutic agents differing from those available targeting hypoxia inducible factor (HIF-) pathways. By using SILAC proteomics, we identified 755 deregulated proteins in response to STF-62247 that were further analyzed by ingenuity pathway analysis (IPA). Bioinformatics analyses predicted alterations in 37 signaling pathways in VHL-null cells in response to treatment. Validation of some altered pathways shows that STF-62247's selectivity is linked to an important inhibition of mTORC1 activation in VHL-null cells leading to protein synthesis arrest, a mechanism differing from two allosteric inhibitors Rapamycin and Everolimus. Altogether, our study identified signaling cascades driving STF-62247 response and brings further knowledge for this molecule that shows selectivity for the loss of VHL. The use of a global SILAC approach was successful in identifying novel affected signaling pathways that could be exploited for the development of new personalized therapeutic strategies to target VHL-inactivated RCCs.


Assuntos
Carcinoma de Células Renais/metabolismo , Neoplasias Renais/metabolismo , Proteoma/efeitos dos fármacos , Piridinas/metabolismo , Tiazóis/metabolismo , Carcinoma de Células Renais/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Redes Reguladoras de Genes/efeitos dos fármacos , Humanos , Marcação por Isótopo , Neoplasias Renais/genética , Proteômica/métodos , Transdução de Sinais/efeitos dos fármacos , Proteína Supressora de Tumor Von Hippel-Lindau/genética
4.
Sci Rep ; 7: 45038, 2017 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-28332630

RESUMO

The promyelocytic leukemia (PML) protein is an essential component of PML nuclear bodies (PML NBs) frequently lost in cancer. PML NBs coordinate chromosomal regions via modification of nuclear proteins that in turn may regulate genes in the vicinity of these bodies. However, few PML NB-associated genes have been identified. PML and PML NBs can also regulate mTOR and cell fate decisions in response to cellular stresses. We now demonstrate that PML depletion in U2OS cells or TERT-immortalized normal human diploid fibroblasts results in decreased expression of the mTOR inhibitor DDIT4 (REDD1). DNA and RNA immuno-FISH reveal that PML NBs are closely associated with actively transcribed DDIT4 loci, implicating these bodies in regulation of basal DDIT4 expression. Although PML silencing did reduce the sensitivity of U2OS cells to metabolic stress induced by metformin, PML loss did not inhibit the upregulation of DDIT4 in response to metformin, hypoxia-like (CoCl2) or genotoxic stress. Analysis of publicly available cancer data also revealed a significant correlation between PML and DDIT4 expression in several cancer types (e.g. lung, breast, prostate). Thus, these findings uncover a novel mechanism by which PML loss may contribute to mTOR activation and cancer progression via dysregulation of basal DDIT4 gene expression.


Assuntos
Regulação da Expressão Gênica , Proteína da Leucemia Promielocítica/metabolismo , Serina-Treonina Quinases TOR/antagonistas & inibidores , Fatores de Transcrição/genética , Linhagem Celular Tumoral , Cobalto/farmacologia , Fibroblastos/metabolismo , Técnicas de Inativação de Genes , Inativação Gênica , Loci Gênicos , Humanos , Hipóxia/genética , Hipóxia/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Ligação Proteica , Biossíntese de Proteínas , Radiação Ionizante , Fatores de Transcrição/metabolismo , Transcrição Gênica
5.
PLoS One ; 9(10): e110443, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25329303

RESUMO

Recent studies indicate that extracellular vesicles are an important source material for many clinical applications, including minimally-invasive disease diagnosis. However, challenges for rapid and simple extracellular vesicle collection have hindered their application. We have developed and validated a novel class of peptides (which we named venceremin, or Vn) that exhibit nucleotide-independent specific affinity for canonical heat shock proteins. The Vn peptides were validated to specifically and efficiently capture HSP-containing extracellular vesicles from cell culture growth media, plasma, and urine by electron microscopy, atomic force microscopy, sequencing of nucleic acid cargo, proteomic profiling, immunoblotting, and nanoparticle tracking analysis. All of these analyses confirmed the material captured by the Vn peptides was comparable to those purified by the standard ultracentrifugation method. We show that the Vn peptides are a useful tool for the rapid isolation of extracellular vesicles using standard laboratory equipment. Moreover, the Vn peptides are adaptable to diverse platforms and therefore represent an excellent solution to the challenge of extracellular vesicle isolation for research and clinical applications.


Assuntos
Proteínas de Choque Térmico/metabolismo , Peptídeos/metabolismo , Vesículas Transportadoras/metabolismo , Western Blotting , Linhagem Celular Tumoral , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Espectrometria de Massas , Microscopia de Força Atômica , Microscopia Eletrônica , Proteômica , Ultracentrifugação
6.
Br J Haematol ; 167(1): 48-61, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24989799

RESUMO

Systemic mastocytosis (SM) is a rare myeloproliferative disease without curative therapy. Despite clinical variability, the majority of patients harbour a KIT-D816V mutation, but efforts to inhibit mutant KIT with tyrosine kinase inhibitors have been unsatisfactory, indicating a need for new preclinical approaches to identify alternative targets and novel therapies in this disease. Murine models to date have been limited and do not fully recapitulate the most aggressive forms of SM. We describe the generation of a transgenic zebrafish model expressing the human KIT-D816V mutation. Adult fish demonstrate a myeloproliferative disease phenotype, including features of aggressive SM in haematopoeitic tissues and high expression levels of endopeptidases, consistent with SM patients. Transgenic embryos demonstrate a cell-cycle phenotype with corresponding expression changes in genes associated with DNA maintenance and repair, such as reduced dnmt1. In addition, epcam was consistently downregulated in both transgenic adults and embryos. Decreased embryonic epcam expression was associated with reduced neuromast numbers, providing a robust in vivo phenotypic readout for chemical screening in KIT-D816V-induced disease. This study represents the first zebrafish model of a mast cell disease with an aggressive adult phenotype and embryonic markers that could be exploited to screen for novel agents in SM.


Assuntos
Expressão Gênica , Mastocitose Sistêmica/genética , Mutação , Proteínas Proto-Oncogênicas c-kit/genética , Animais , Animais Geneticamente Modificados , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Apoptose/genética , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Ciclo Celular/genética , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Modelos Animais de Doenças , Embrião não Mamífero/metabolismo , Molécula de Adesão da Célula Epitelial , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Ordem dos Genes , Vetores Genéticos , Hematopoese/genética , Humanos , Rim/patologia , Mastócitos/enzimologia , Mastocitose , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Fenótipo , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
7.
Mol Ther Nucleic Acids ; 3: e178, 2014 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-25072692

RESUMO

Antisense-based molecules targeting HIV-1 RNA have the potential to be used as part of gene or drug therapy to treat HIV-1 infection. In this study, HIV-1 RNA was screened to identify more conserved and accessible target sites for ribozymes based on the hepatitis delta virus motif. Using a quantitative screen for effects on HIV-1 production, we identified a ribozyme targeting a highly conserved site in the Gag coding sequence with improved inhibitory potential compared to our previously described candidates targeting the overlapping Tat/Rev coding sequence. We also demonstrate that this target site is highly accessible to short hairpin directed RNA interference, suggesting that it may be available for the binding of antisense RNAs with different modes of action. We provide evidence that this target site is structurally conserved in diverse viral strains and that it is sufficiently different from the human transcriptome to limit off-target effects from antisense therapies. We also show that the modified hepatitis delta virus ribozyme is more sensitive to a mismatch in its target site compared to the short hairpin RNA. Overall, our results validate the potential of a new target site in HIV-1 RNA to be used for the development of antisense therapies.

8.
Mar Biotechnol (NY) ; 13(4): 733-50, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21127932

RESUMO

The collapse of Atlantic cod (Gadus morhua) wild populations strongly impacted the Atlantic cod fishery and led to the development of cod aquaculture. In order to improve aquaculture and broodstock quality, we need to gain knowledge of genes and pathways involved in Atlantic cod responses to pathogens and other stressors. The Atlantic Cod Genomics and Broodstock Development Project has generated over 150,000 expressed sequence tags from 42 cDNA libraries representing various tissues, developmental stages, and stimuli. We used this resource to develop an Atlantic cod oligonucleotide microarray containing 20,000 unique probes. Selection of sequences from the full range of cDNA libraries enables application of the microarray for a broad spectrum of Atlantic cod functional genomics studies. We included sequences that were highly abundant in suppression subtractive hybridization (SSH) libraries, which were enriched for transcripts responsive to pathogens or other stressors. These sequences represent genes that potentially play an important role in stress and/or immune responses, making the microarray particularly useful for studies of Atlantic cod gene expression responses to immune stimuli and other stressors. To demonstrate its value, we used the microarray to analyze the Atlantic cod spleen response to stimulation with formalin-killed, atypical Aeromonas salmonicida, resulting in a gene expression profile that indicates a strong innate immune response. These results were further validated by quantitative PCR analysis and comparison to results from previous analysis of an SSH library. This study shows that the Atlantic cod 20K oligonucleotide microarray is a valuable new tool for Atlantic cod functional genomics research.


Assuntos
Etiquetas de Sequências Expressas , Gadus morhua/genética , Imunidade Inata/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Aeromonas salmonicida/imunologia , Animais , Primers do DNA/genética , Gadus morhua/imunologia , Perfilação da Expressão Gênica , Biblioteca Gênica , Genômica , Espectrometria de Massas , Nodaviridae/genética , Sondas de Oligonucleotídeos/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Baço/imunologia
9.
Magn Reson Chem ; 47 Suppl 1: S96-104, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19731396

RESUMO

The global analysis of metabolites can be used to define the phenotypes of cells, tissues or organisms. Classifying groups of samples based on their metabolic profile is one of the main topics of metabolomics research. Crisp clustering methods assign each feature to one cluster, thereby omitting information about the multiplicity of sample subtypes. Here, we present the application of fuzzy K-means clustering method for the classification of samples based on metabolomics 1D (1)H NMR fingerprints. The sample classification was performed on NMR spectra of cancer cell line extracts and of urine samples of type 2 diabetes patients and animal models. The cell line dataset included NMR spectra of lipophilic cell extracts for two normal and three cancer cell lines with cancer cell lines including two invasive and one non-invasive cancers. The second dataset included previously published NMR spectra of urine samples of human type 2 diabetics and healthy controls, mouse wild type and diabetes model and rat obese and lean phenotypes. The fuzzy K-means clustering method allowed more accurate sample classification in both datasets relative to the other tested methods including principal component analysis (PCA), hierarchical clustering (HCL) and K-means clustering. In the cell line samples, fuzzy clustering provided a clear separation of individual cell lines, groups of cancer and normal cell lines as well as non-invasive and invasive tumour cell lines. In the diabetes dataset, clear separation of healthy controls and diabetics in all three models was possible only by using the fuzzy clustering method.


Assuntos
Algoritmos , Metabolômica , Urina/química , Animais , Linhagem Celular , Linhagem Celular Tumoral , Análise por Conglomerados , Lógica Fuzzy , Humanos , Espectroscopia de Ressonância Magnética , Camundongos , Análise de Componente Principal , Ratos
10.
Nat Genet ; 33(1): 61-5, 2003 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-12496760

RESUMO

Transcriptional silencing by CpG island methylation is a prevalent mechanism of tumor-suppressor gene suppression in cancers. Genetic experiments have defined the importance of the DNA methyltransferase Dnmt1 for the maintenance of methylation in mouse cells and its role in neoplasia. In human bladder cancer cells, selective depletion of DNMT1 with antisense inhibitors has been shown to induce demethylation and reactivation of the silenced tumor-suppressor gene CDKN2A. In contrast, targeted disruption of DNMT1 alleles in HCT116 human colon cancer cells produced clones that retained CpG island methylation and associated tumor-suppressor gene silencing, whereas HCT116 clones with inactivation of both DNMT1 and DNMT3B showed much lower levels of DNA methylation, suggesting that the two enzymes are highly cooperative. We used a combination of genetic (antisense and siRNA) and pharmacologic (5-aza-2'-deoxycytidine) inhibitors of DNA methyl transferases to study the contribution of the DNMT isotypes to cancer-cell methylation. Selective depletion of DNMT1 using either antisense or siRNA resulted in lower cellular maintenance methyltransferase activity, global and gene-specific demethylation and re-expression of tumor-suppressor genes in human cancer cells. Specific depletion of DNMT1 but not DNMT3A or DNMT3B markedly potentiated the ability of 5-aza-2'-deoxycytidine to reactivate silenced tumor-suppressor genes, indicating that inhibition of DNMT1 function is the principal means by which 5-aza-2'-deoxycytidine reactivates genes. These results indicate that DNMT1 is necessary and sufficient to maintain global methylation and aberrant CpG island methylation in human cancer cells.


Assuntos
Ilhas de CpG/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Sequência de Aminoácidos , Western Blotting , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , DNA (Citosina-5-)-Metiltransferases/deficiência , DNA (Citosina-5-)-Metiltransferases/genética , Genes p16 , Humanos , Dados de Sequência Molecular , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Células Tumorais Cultivadas
11.
Biochem Pharmacol ; 63(8): 1527-35, 2002 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-11996895

RESUMO

Phosphodiesterase 4 (PDE4) inhibitors elevate cyclic adenosine 5'-monophosphate (cAMP), and this elevation has been shown to inhibit inflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha). Using TNF-alpha as a biomarker, we have developed transcription-based assays to examine inhibition of PDE4 activity in human and guinea pig whole blood. In vitro inhibition by PDE4 inhibitors was measured using quantitative PCR (qPCR) analysis of TNF-alpha mRNA levels in whole blood stimulated with lipopolysaccharide (LPS). The kinetics of human TNF-alpha mRNA production were analyzed and shown to be highest 4 hr following LPS stimulation. The guinea pig displayed kinetics of TNF-alpha transcription similar to those of the human. Analysis of inhibition of human TNF-alpha protein production was performed by immunoassay and shown to correlate with inhibition of transcription for three of the four compounds tested. Roflumilast was found to be 9-fold more potent for TNF-alpha inhibition in the qPCR assay than in the protein assay. The potencies of L-826,141 and roflumilast were determined in human and guinea pig whole blood by qPCR, with IC(50) values of 270 and 20 nM, respectively, in humans and 100 and 10 nM, respectively, in guinea pigs. These results show that the potency of PDE4 inhibitors can be monitored in whole blood using a transcription-based assay, and that this type of assay can be adapted to various species provided the TNF-alpha nucleotide sequence is known. The in vitro whole blood IC(50) for TNF-alpha inhibition was compared to inhibition in the ovalbumin-challenged guinea pig model of bronchoconstriction. Obtaining plasma levels at the IC(50) determined in vitro for L-826,141 and roflumilast provides significant inhibition of bronchoconstriction. This suggests that TNF-alpha can be used as a whole blood biomarker in the guinea pig for PDE4 inhibition in this inflammatory model.


Assuntos
3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Broncoconstrição/efeitos dos fármacos , Ovalbumina/farmacologia , Inibidores de Fosfodiesterase/farmacologia , Piridinas/farmacologia , Fator de Necrose Tumoral alfa/metabolismo , 3',5'-AMP Cíclico Fosfodiesterases/antagonistas & inibidores , Animais , Broncoconstrição/fisiologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4 , Interações Medicamentosas , Feminino , Cobaias , Humanos , Lipopolissacarídeos/farmacologia , Masculino , Inibidores de Fosfodiesterase/química , Reação em Cadeia da Polimerase , Piridinas/química , RNA Mensageiro/sangue , RNA Mensageiro/efeitos dos fármacos , Fatores de Tempo , Fator de Necrose Tumoral alfa/genética
12.
J Biol Chem ; 277(31): 28176-81, 2002 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-12015329

RESUMO

Abnormal methylation and associated silencing of tumor suppressor genes is a common feature of many types of cancers. The observation of persistent methylation in human cancer cells lacking the maintenance methyltransferase DNMT1 suggests the involvement of other DNA methyltransferases in gene silencing in cancer. To test this hypothesis, we have evaluated methylation and gene expression in cancer cells specifically depleted of DNMT3A or DNMT3B, de novo methyltransferases that are expressed in adult tissues. Here we have shown that depletion of DNMT3B, but not DNMT3A, induced apoptosis of human cancer cells but not normal cells. DNMT3B depletion reactivated methylation-silenced gene expression but did not induce global or juxtacentromeric satellite demethylation as did specific depletion of DNMT1. Furthermore, the effect of DNMT3B depletion was rescued by exogenous expression of either of the splice variants DNMT3B2 or DNMT3B3 but not DNMT1. These results indicate that DNMT3B has significant site selectivity that is distinct from DNMT1, regulates aberrant gene silencing, and is essential for cancer cell survival.


Assuntos
Sobrevivência Celular/fisiologia , DNA (Citosina-5-)-Metiltransferases/metabolismo , Apoptose , Metilação de DNA , DNA de Neoplasias/metabolismo , Inativação Gênica , Humanos , Marcação In Situ das Extremidades Cortadas , Cinética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas , DNA Metiltransferase 3B
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