RESUMO
Galectin-3 (Gal-3) is a multifunctional glycan-binding protein that participates in many pathophysiological events and has been described as a biomarker and potential therapeutic target for severe disorders, such as cancer. Several probes for Gal-3 or its ligands have been developed, however both the pathophysiological mechanisms and potential biomedical applications of Gal-3 remain not fully assessed. Molecular imaging using bioluminescent probes provides great sensitivity for in vivo and in vitro analysis for both cellular and whole multicellular organism tracking and target detection. Here, we engineered a chimeric molecule consisting of Renilla luciferase fused with mouse Gal-3 (RLuc-mGal-3). RLuc-mGal-3 preparation was highly homogenous, soluble, active, and has molecular mass of 65,870.95â¯Da. This molecule was able to bind to MKN45â¯cell surface, property which was inhibited by the reduction of Gal-3 ligands on the cell surface by the overexpression of ST6GalNAc-I. In order to obtain an efficient and stable delivery system, RLuc-mGal-3 was adsorbed to poly-lactic acid nanoparticles, which increased binding to MKN45â¯cells in vitro. Furthermore, bioluminescence imaging showed that RLuc-mGal-3 was able to indicate the presence of implanted tumor in mice, event drastically inhibited by the presence of lactose. This novel bioluminescent chimeric molecule offers a safe and highly sensitive alternative to fluorescent and radiolabeled probes with potential application in biomedical research for a better understanding of the distribution and fate of Gal-3 and its ligands in vitro and in vivo.
Assuntos
Galectina 3/metabolismo , Luciferases de Renilla/metabolismo , Substâncias Luminescentes/metabolismo , Neoplasias/diagnóstico por imagem , Polissacarídeos/metabolismo , Animais , Linhagem Celular Tumoral , Galectina 3/análise , Galectina 3/genética , Humanos , Luciferases de Renilla/análise , Luciferases de Renilla/genética , Substâncias Luminescentes/análise , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias/metabolismo , Imagem Óptica , Polissacarídeos/análise , Ligação Proteica , Engenharia de Proteínas , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
The outstanding potential of Extracellular Vesicles (EVs) in medicine, deserves a detailed study of the molecular aspects regulating their incorporation into target cells. However, because EV size lies below the limit of resolution of optical techniques, quantification together with discrimination between EV binding to the target cell and uptake is usually not completely achieved with current techniques. Human tetraspanins CD9 and CD63 were fused to a dual EGFP-Renilla-split tag. Subcellular localization and incorporation of these fusion proteins into EVs was assessed by western-blot and fluorescence microscopy. EV binding and uptake was measured using either a classical Renilla substrate or a cytopermeable one. Incubation of target cells expressing DSP2 with EVs containing the complementary DSP1 portion could not recover fluorescence or luciferase activity. However, using EVs carrying the fully reconstituted Dual-EGFP-Renilla protein and the cytopermeable Renilla luciferase substrate, we could distinguish EV binding from uptake. We provide proof of concept of the system by analysing the effect of different chemical inhibitors, demonstrating that this method is highly sensitive and quantitative, allowing a dynamic follow-up in a high-throughput scheme to unravel the molecular mechanisms of EV uptake in different biological systems.
Assuntos
Vesículas Extracelulares/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Tetraspanina 29/metabolismo , Tetraspanina 30/metabolismo , Transporte Biológico , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Genes Reporter , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Humanos , Imidazóis/metabolismo , Luciferases de Renilla/análise , Luciferases de Renilla/genética , Medições Luminescentes , Nanopartículas , Pirazinas/metabolismo , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/metabolismo , Sensibilidade e Especificidade , Frações Subcelulares/química , Tetraspanina 29/genética , Tetraspanina 30/genéticaRESUMO
The rapid development of CRISPR-Cas technologies brought a personalized and targeted treatment of genetic disorders into closer reach. To render CRISPR-based therapies precise and safe, strategies to confine the activity of Cas(9) to selected cells and tissues are highly desired. Here, we developed a cell type-specific Cas-ON switch based on miRNA-regulated expression of anti-CRISPR (Acr) proteins. We inserted target sites for miR-122 or miR-1, which are abundant specifically in liver and cardiac muscle cells, respectively, into the 3'UTR of Acr transgenes. Co-expressing these with Cas9 and sgRNAs resulted in Acr knockdown and released Cas9 activity solely in hepatocytes or cardiomyocytes, while Cas9 was efficiently inhibited in off-target cells. We demonstrate control of genome editing and gene activation using a miR-dependent AcrIIA4 in combination with different Streptococcus pyogenes (Spy)Cas9 variants (full-length Cas9, split-Cas9, dCas9-VP64). Finally, to showcase its modularity, we adapted our Cas-ON system to the smaller and more target-specific Neisseria meningitidis (Nme)Cas9 orthologue and its cognate inhibitors AcrIIC1 and AcrIIC3. Our Cas-ON switch should facilitate cell-specific activity of any CRISPR-Cas orthologue, for which a potent anti-CRISPR protein is known.
Assuntos
Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas , Edição de Genes/métodos , Regulação da Expressão Gênica , Transgenes , Regiões 3' não Traduzidas/genética , Sítios de Ligação , Proteína 9 Associada à CRISPR/antagonistas & inibidores , Proteína 9 Associada à CRISPR/biossíntese , Dependovirus/genética , Ativação Enzimática , Indução Enzimática , Genes Reporter , Células HEK293 , Células HeLa , Hepatócitos/metabolismo , Humanos , Luciferases de Renilla/análise , Luciferases de Renilla/genética , MicroRNAs , Miócitos Cardíacos/metabolismo , Especificidade de Órgãos , Isoformas de Proteínas/antagonistas & inibidoresRESUMO
To develop a photoactivatable bioluminescence imaging technique, a set of high and efficient photoactivatable substrates for Renilla luciferase has been well designed and synthesized. Surprisingly, all of them could release the free luciferin that presented robust bioluminescent signals ex vivo and in living animals after UV irradiation at 365 nm.
Assuntos
Imidazóis/química , Luciferases de Renilla/análise , Substâncias Luminescentes/química , Imagem Óptica , Pirazinas/química , Animais , Imidazóis/síntese química , Luciferases de Renilla/metabolismo , Substâncias Luminescentes/síntese química , Medições Luminescentes , Camundongos , Camundongos Nus , Estrutura Molecular , Neoplasias Experimentais/diagnóstico por imagem , Processos Fotoquímicos , Pirazinas/síntese química , Especificidade por Substrato , Raios UltravioletaRESUMO
Macroautophagy (autophagy) is a dynamic intracellular degradation pathway. Monitoring the flux through the autophagy pathway is experimentally challenging but obviously a prerequisite for the proper investigation of the process. Here, we present an indirect autophagy flux assay based on monitoring the degradation of an autophagosome-associated fusion protein Rluc-LC3 by luminescence detection. The method is suitable for screening purposes with a high number of parallel samples and can be used for measurements in cell lysates as well as in living cells. The Rluc-LC3 assay has proven useful for the identification of genes, miRNAs, and small molecules that regulate autophagy flux in mammalian cells.
Assuntos
Autofagia , Luciferases de Renilla/análise , Animais , Ensaios de Triagem em Larga Escala/métodos , Humanos , Luciferases de Renilla/metabolismo , Células MCF-7 , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/metabolismo , Transdução de SinaisRESUMO
Aberrant activation of the Src kinase is implicated in the development of a variety of human malignancies. However, it is almost impossible to monitor Src activity in an in vivo setting with current biochemical techniques. To facilitate the noninvasive investigation of the activity of Src kinase both in vitro and in vivo, we developed a genetically engineered, activatable bioluminescent reporter using split-luciferase complementation. The bioluminescence of this reporter can be used as a surrogate for Src activity in real time. This hybrid luciferase reporter was constructed by sandwiching a Src-dependent conformationally responsive unit (SH2 domain-Srcpep) between the split luciferase fragments. The complementation bioluminescence of this reporter was dependent on the Src activity status. In our study, Src kinase activity in cultured cells and tumor xenografts was monitored quantitatively and dynamically in response to clinical small-molecular kinase inhibitors, dasatinib and saracatinib. This system was also applied for high-throughput screening of Src inhibitors against a kinase inhibitor library in living cells. These results provide unique insights into drug development and pharmacokinetics/phoarmocodynamics of therapeutic drugs targeting Src signaling pathway enabling the optimization of drug administration schedules for maximum benefit. Using both Firefly and Renilla luciferase imaging, we have successfully monitored Src tyrosine kinase activity and Akt serine/threonine kinase activity concurrently in one tumor xenograft. This dual luciferase reporter imaging system will be helpful in exploring the complex signaling networks in vivo. The strategies reported here can also be extended to study and image other important kinases and the cross-talks among them.
Assuntos
Genes Reporter , Luciferases de Renilla/análise , Medições Luminescentes , Neoplasias/diagnóstico por imagem , Quinases da Família src/análise , Animais , Linhagem Celular Tumoral , Xenoenxertos , Luciferases de Renilla/genética , Camundongos , Análise de Sequência de DNARESUMO
BACKGROUND: Recently, Japanese Red Cross blood centers have changed the confirmatory test method from an indirect immunofluorescence (IF) technique to Western blotting (WB) for antibodies against human T-cell leukemia virus Type 1 (HTLV-1). In this study, these HTLV-1 tests were assessed using another sensitive method, that is, a luciferase immunoprecipitation system (LIPS), to identify a better confirmatory test for HTLV-1 infection. STUDY DESIGN AND METHODS: Plasma samples from 54 qualified donors and 114 HTLV-1 screening-positive donors were tested by LIPS for antibodies against HTLV-1 Gag, Tax, Env, and HBZ recombinant proteins. The donors were categorized into six groups, namely, (Group I) qualified donors, screening positive; (Group II) IF positive; (Group III) IF negative; (Group IV) WB positive; (Group V) WB negative; and (Group VI) screening positive in the previous blood donation, but WB-indeterminate during this study period. RESULTS: In Groups II and IV, all plasma samples tested positive by LIPS for antibodies against Gag and Env proteins. In Group V, all samples tested negative by LIPS, whereas some Group III samples reacted with single or double antigens in LIPS. In Group VI, the LIPS test identified a donor with suspected HTLV-1 infection. The first case of a blood donor with plasma that reacted with HBZ was identified by LIPS. CONCLUSION: Reevaluation of the current HTLV-1 screening method using the LIPS test showed that both confirmatory tests had similar sensitivity and specificity only when WB indeterminate results were eliminated. LIPS is a promising method for detecting and characterizing HTLV-1 antibodies.
Assuntos
Doadores de Sangue , Segurança do Sangue/métodos , Seleção do Doador/métodos , Anticorpos Anti-HTLV-I/sangue , Infecções por HTLV-I/diagnóstico , Vírus Linfotrópico T Tipo 1 Humano/isolamento & purificação , Viremia/diagnóstico , Fatores de Transcrição de Zíper de Leucina Básica/imunologia , Western Blotting , Seleção do Doador/legislação & jurisprudência , Ensaio de Imunoadsorção Enzimática , Reações Falso-Negativas , Reações Falso-Positivas , Genes Reporter , Vetores Genéticos , Células HEK293 , Antígenos HTLV-I/genética , Antígenos HTLV-I/imunologia , Infecções por HTLV-I/sangue , Infecções por HTLV-I/epidemiologia , Infecções por HTLV-I/prevenção & controle , Infecções por HTLV-I/virologia , Vírus Linfotrópico T Tipo 1 Humano/imunologia , Humanos , Imunoprecipitação , Japão/epidemiologia , Luciferases de Renilla/análise , Luciferases de Renilla/genética , Medições Luminescentes , Valor Preditivo dos Testes , Proteínas dos Retroviridae/imunologia , Sensibilidade e Especificidade , Viremia/virologiaRESUMO
BACKGROUND: New compounds for the treatment of human African trypanosomiasis (HAT) are urgently required. Trypanosoma brucei (T.b.) gambiense is the leading cause of HAT, yet T.b. gambiense is often not the prime target organism in drug discovery. This may be attributed to the difficulties in handling this subspecies and the lack of an efficient viability assay to monitor drug efficacy. METHODS: In this study, a T.b. gambiense strain, recently isolated in the D.R. Congo, was made bioluminescent by transfection with Renilla luciferase (RLuc) without altering its in vitro and in vivo growth characteristics. A luminescent multiplex viability assay (LMVA), based on measurement of the Renilla luciferase activity and the ATP content of the cells within the same experiment, was investigated as an alternative to the standard fluorimetric resazurin viability assay for drug sensitivity testing of T.b. gambiense. RESULTS: In a 96-well format, the RLuc transfected strain showed a detection limit of 2 × 10(4) cells ml(-1) for the Renilla luciferase measurement and 5 × 10(3) cells ml(-1) for the ATP measurement. Both assays of the LMVA showed linearity up to 10(6) cells ml(-1) and correlated well with the cell density during exponential growth of the long slender bloodstream forms. The LMVA was compared to the fluorimetric resazurin viability assay for drug sensitivity testing of pentamidine, eflornithine, nifurtimox and melarsoprol with both the wild type and the RLuc transfected population. For each drug, the IC50 value of the RLuc population was similar to that of the wild type when determined with either the fluorimetric resazurin method or the LMVA. For eflornithine, nifurtimox and melarsoprol we found no difference between the IC50 values in both viability assays. In contrast, the IC50 value of pentamidine was higher when determined with the fluorimetric resazurin method than in both assays of the LMVA. CONCLUSIONS: LMVA has some advantages for viability measurement of T.b. gambiense: it requires less incubation time for viability detection than the fluorimetric resazurin assay and in LMVA, two sensitive and independent viability assays are performed in the same experiment.
Assuntos
Antiprotozoários/farmacologia , Parasitologia/métodos , Trypanosoma brucei gambiense/efeitos dos fármacos , Trypanosoma brucei gambiense/fisiologia , Trifosfato de Adenosina/análise , Sobrevivência Celular , Concentração Inibidora 50 , Luciferases de Renilla/análise , Luciferases de Renilla/genética , Medições Luminescentes/métodos , Testes de Sensibilidade Parasitária/métodos , Proteínas Recombinantes/análise , Proteínas Recombinantes/genética , Coloração e Rotulagem/métodos , Trypanosoma brucei gambiense/químicaRESUMO
Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate since the advent of in vivo bioluminescent imaging technologies. Indeed, the ability to locate and quantify tumor growth longitudinally in a single cohort of animals to completion of the study as opposed to sacrificing individual groups of animals at specific assay times has revolutionized how researchers investigate breast cancer metastasis. Unfortunately, current methodologies preclude the real-time assessment of critical changes that transpire in cell signaling systems as breast cancer cells (i) evolve within primary tumors, (ii) disseminate throughout the body, and (iii) reinitiate proliferative programs at sites of a metastatic lesion. However, recent advancements in bioluminescent imaging now make it possible to simultaneously quantify specific spatiotemporal changes in gene expression as a function of tumor development and metastatic progression via the use of dual substrate luminescence reactions. To do so, researchers take advantage for two light-producing luciferase enzymes isolated from the firefly (Photinus pyralis) and sea pansy (Renilla reniformis), both of which react to mutually exclusive substrates that previously facilitated their wide-spread use in in vitro cell-based reporter gene assays. Here we demonstrate the in vivo utility of these two enzymes such that one luminescence reaction specifically marks the size and location of a developing tumor, while the second luminescent reaction serves as a means to visualize the activation status of specific signaling systems during distinct stages of tumor and metastasis development. Thus, the objectives of this study are two-fold. First, we will describe the steps necessary to construct dual bioluminescent reporter cell lines, as well as those needed to facilitate their use in visualizing the spatiotemporal regulation of gene expression during specific steps of the metastatic cascade. Using the 4T1 model of breast cancer metastasis, we show that the in vivo activity of a synthetic Smad Binding Element (SBE) promoter was decreased dramatically in pulmonary metastasis as compared to that measured in the primary tumor. Recently, breast cancer metastasis was shown to be regulated by changes within the primary tumor microenvironment and reactive stroma, including those occurring in fibroblasts and infiltrating immune cells. Thus, our second objective will be to demonstrate the utility of dual bioluminescent techniques in monitoring the growth and localization of two unique cell populations harbored within a single animal during breast cancer growth and metastasis.
Assuntos
Luciferases de Vaga-Lume/análise , Luciferases de Renilla/análise , Medições Luminescentes/métodos , Neoplasias Mamárias Experimentais/patologia , Animais , Feminino , Luciferases de Vaga-Lume/biossíntese , Luciferases de Vaga-Lume/genética , Luciferases de Renilla/biossíntese , Luciferases de Renilla/genética , Neoplasias Mamárias Experimentais/enzimologia , Neoplasias Mamárias Experimentais/genética , Camundongos , Metástase Neoplásica , TransfecçãoRESUMO
Cytosine methylation is a vital biology event. However, it is also the source of genomic instability due to deamination of 5'-methylcytosine by spontaneous hydrolysis, which produces thymine and results in G:T mismatches. Thymine DNA glycosylase and methyl-CpG-binding protein 4 are major DNA glycosylases involved in the mismatch repair progress, and their activities have been measured in many related researches. In this study, we developed a convenient spectrometric assay system for specific and quantitative measurement of intracellular DNA glycosylase activity. A G:T mismatch was introduced into the upstream region of firefly luciferase-coding sequence in the pGL3-control plasmid. Only if the G:T mismatches were repaired to G:C, will luciferase be expressed in transfected cells. By measuring luciferase activity, which is simple and convenient, the intracellular DNA glycosylase activity can be determined.
Assuntos
Endodesoxirribonucleases/análise , Espectrometria de Fluorescência/métodos , Timina DNA Glicosilase/análise , Animais , Pareamento Incorreto de Bases , Linhagem Celular Tumoral , Reparo do DNA , Genes Reporter , Vetores Genéticos , Luciferases de Vaga-Lume/metabolismo , Luciferases de Renilla/análise , Camundongos , Plasmídeos , Timina/metabolismoRESUMO
Sequence-specific binding by the human p53 master regulator is critical to its tumor suppressor activity in response to environmental stresses. p53 binds as a tetramer to two decameric half-sites separated by 0-13 nucleotides (nt), originally defined by the consensus RRRCWWGYYY (n = 0-13) RRRCWWGYYY. To better understand the role of sequence, organization, and level of p53 on transactivation at target response elements (REs) by wild type (WT) and mutant p53, we deconstructed the functional p53 canonical consensus sequence using budding yeast and human cell systems. Contrary to early reports on binding in vitro, small increases in distance between decamer half-sites greatly reduces p53 transactivation, as demonstrated for the natural TIGER RE. This was confirmed with human cell extracts using a newly developed, semi-in vitro microsphere binding assay. These results contrast with the synergistic increase in transactivation from a pair of weak, full-site REs in the MDM2 promoter that are separated by an evolutionary conserved 17 bp spacer. Surprisingly, there can be substantial transactivation at noncanonical (1/2)-(a single decamer) and (3/4)-sites, some of which were originally classified as biologically relevant canonical consensus sequences including PIDD and Apaf-1. p53 family members p63 and p73 yielded similar results. Efficient transactivation from noncanonical elements requires tetrameric p53, and the presence of the carboxy terminal, non-specific DNA binding domain enhanced transactivation from noncanonical sequences. Our findings demonstrate that RE sequence, organization, and level of p53 can strongly impact p53-mediated transactivation, thereby changing the view of what constitutes a functional p53 target. Importantly, inclusion of (1/2)- and (3/4)-site REs greatly expands the p53 master regulatory network.
Assuntos
DNA/metabolismo , Mutação Puntual , Saccharomyces cerevisiae/genética , Ativação Transcricional , Proteína Supressora de Tumor p53/genética , Sequência de Bases , Sítios de Ligação/genética , Bioensaio , Linhagem Celular Tumoral , Sequência Consenso , DNA/genética , DNA Intergênico/genética , Dimerização , Diploide , Genes Reporter , Humanos , Luciferases de Renilla/análise , Luciferases de Renilla/metabolismo , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Estrutura Terciária de Proteína/genética , Proteínas Proto-Oncogênicas c-mdm2/genética , Elementos de Resposta/genética , Transfecção , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor p53/fisiologiaRESUMO
Oxidative stress induced by glutathione depletion in the mouse HT22 neuroblastoma cell line and embryonic rat immature cortical neurons causes a delayed, sustained activation of extracellular signal-regulated kinase (ERK) 1/2, which is required for cell death. This sustained activation of ERK1/2 is mediated primarily by a selective inhibition of distinct ERK1/2-directed phosphatases either by enhanced degradation (i.e., for mitogen-activated protein kinase phosphatase-1) or as shown here by reductions in enzymatic activity (i.e., for protein phosphatase type 2A). The inhibition of ERK1/2 phosphatases in HT22 cells and immature neurons subjected to glutathione depletion results from oxidative stress because phosphatase activity is restored in cells treated with the antioxidant butylated hydroxyanisole. This leads to reduced ERK1/2 activation and neuroprotection. Furthermore, an increase in free intracellular zinc that accompanies glutathione-induced oxidative stress in HT22 cells and immature neurons contributes to selective inhibition of ERK1/2 phosphatase activity and cell death. Finally, ERK1/2 also functions to maintain elevated levels of zinc. Thus, the elevation of intracellular zinc within neurons subjected to oxidative stress can trigger a robust positive feedback loop operating through activated ERK1/2 that rapidly sets into motion a zinc-dependent pathway of cell death.
Assuntos
Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosfatases da Proteína Quinase Ativada por Mitógeno/antagonistas & inibidores , Neurônios/efeitos dos fármacos , Zinco/farmacologia , Animais , Técnicas de Cultura de Células , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/embriologia , Embrião de Mamíferos/citologia , Luciferases de Renilla/análise , Luciferases de Renilla/metabolismo , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/análise , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/análise , Proteína Quinase 3 Ativada por Mitógeno/genética , Modelos Biológicos , Neuroblastoma/enzimologia , Neuroblastoma/patologia , Neurônios/citologia , Oxirredução/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , TransfecçãoRESUMO
The product of the Snail1 gene is a transcriptional repressor required for triggering the epithelial-to-mesenchymal transition. Furthermore, ectopic expression of Snail1 in epithelial cells promotes resistance to apoptosis. In this study, we demonstrate that this resistance to gamma radiation-induced apoptosis caused by Snail1 is associated with the inhibition of PTEN phosphatase. In MDCK cells, mRNA levels of the p53 target gene PTEN are induced after gamma radiation; the transfection of Snail1 prevents this up-regulation. Decreased mRNA levels of PTEN were also detected in RWP-1 cells after the ectopic expression of this transcriptional factor. Snail1 represses and associates to the PTEN promoter as detected both by the electrophoretic mobility shift assay and chromatin immunoprecipitation experiments performed with either endogenous or ectopic Snail1. The binding of Snail1 to the PTEN promoter increases after gamma radiation, correlating with the stabilization of Snail1 protein, and prevents the association of p53 to the PTEN promoter. These results stress the critical role of Snail1 in the control of apoptosis and demonstrate the regulation of PTEN phosphatase by this transcriptional repressor.
Assuntos
Apoptose/efeitos da radiação , Raios gama , Regulação da Expressão Gênica , PTEN Fosfo-Hidrolase/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Dano ao DNA , DNA Complementar , Cães , Fase G2 , Genes Reporter , Humanos , Luciferases de Vaga-Lume/análise , Luciferases de Vaga-Lume/metabolismo , Luciferases de Renilla/análise , Luciferases de Renilla/metabolismo , Substâncias Luminescentes/metabolismo , Neoplasias Pancreáticas/patologia , Regiões Promotoras Genéticas , Inibidores da Síntese de Proteínas/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Puromicina/farmacologia , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/farmacologia , Seleção Genética , Fatores de Transcrição da Família Snail , Fatores de Tempo , Fatores de Transcrição/genética , Fatores de Transcrição/farmacologia , TransfecçãoRESUMO
Heat shock protein 90 alpha (Hsp90 alpha)/p23 and Hsp90 beta/p23 interactions are crucial for proper folding of proteins involved in cancer and neurodegenerative diseases. Small molecule Hsp90 inhibitors block Hsp90 alpha/p23 and Hsp90 beta/p23 interactions in part by preventing ATP binding to Hsp90. The importance of isoform-selective Hsp90 alpha/p23 and Hsp90 beta/p23 interactions in determining the sensitivity to Hsp90 was examined using 293T human kidney cancer cells stably expressing split Renilla luciferase (RL) reporters. Interactions between Hsp90 alpha/p23 and Hsp90 beta/p23 in the split RL reporters led to complementation of RL activity, which was determined by bioluminescence imaging of intact cells in cell culture and living mice using a cooled charge-coupled device camera. The three geldanamycin-based and seven purine-scaffold Hsp90 inhibitors led to different levels of inhibition of complemented RL activities (10-70%). However, there was no isoform selectivity to both classes of Hsp90 inhibitors in cell culture conditions. The most potent Hsp90 inhibitor, PU-H71, however, led to a 60% and 30% decrease in RL activity (14 hr) in 293T xenografts expressing Hsp90 alpha/p23 and Hsp90 beta/p23 split reporters respectively, relative to carrier control-treated mice. Molecular imaging of isoform-specific Hsp90 alpha/p23 and Hsp90 beta/p23 interactions and efficacy of different classes of Hsp90 inhibitors in living subjects have been achieved with a novel genetically encoded reporter gene strategy that should help in accelerating development of potent and isoform-selective Hsp90 inhibitors.
Assuntos
Antineoplásicos/farmacologia , Benzodioxóis/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Chaperonas Moleculares/antagonistas & inibidores , Fosfoproteínas/antagonistas & inibidores , Purinas/farmacologia , Animais , Antineoplásicos/química , Benzoquinonas/química , Linhagem Celular Tumoral , Genes Reporter/genética , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Lactamas Macrocíclicas/química , Luciferases de Renilla/análise , Luciferases de Renilla/genética , Camundongos , Chaperonas Moleculares/metabolismo , Mutação , Fosfoproteínas/metabolismo , Prostaglandina-E Sintases , Mapeamento de Interação de Proteínas/métodos , Purinas/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Eukaryotic initiation factor 2B (eIF2B) plays a key role in controlling the initiation of mRNA translation. eIF2B is heteropentamer whose catalytic (epsilon) subunit promotes GDP/GTP exchange on eIF2. We show here that depriving human cells of amino acids rapidly results in the inhibition of eIF2B, independently of changes in eIF2 phosphorylation. Although amino acid deprivation also inhibits signaling through the mammalian target of rapamycin complex 1 (mTORC1), the inhibition of eIF2B activity by amino acid starvation is independent of mTORC1. Instead, amino acids repress the phosphorylation of a novel site in eIF2Bepsilon. We identify this site as Ser525, located adjacent to the known phosphoregulatory region in eIF2Bepsilon. Mutation of Ser525 to Ala abolishes the regulation of eIF2B and protein synthesis by amino acids. This indicates that phosphorylation of this site is crucial for the control of eIF2B and protein synthesis by amino acids. These findings identify a new way in which amino acids regulate a key step in translation initiation and indicate that this involves a novel amino acid-sensitive signaling mechanism.
Assuntos
Aminoácidos/metabolismo , Fator de Iniciação 2B em Eucariotos/metabolismo , Biossíntese de Proteínas , Alanina/metabolismo , Substituição de Aminoácidos , Animais , Carcinoma Hepatocelular/patologia , Domínio Catalítico , Linhagem Celular , Linhagem Celular Tumoral , Células Cultivadas , Cicloeximida/farmacologia , Embrião de Mamíferos , Fator de Iniciação 2B em Eucariotos/análise , Fator de Iniciação 2B em Eucariotos/química , Fator de Iniciação 2B em Eucariotos/isolamento & purificação , Fibroblastos/metabolismo , Genes Reporter , Histidina/metabolismo , Humanos , Células Jurkat , Rim/citologia , Neoplasias Hepáticas/patologia , Luciferases de Renilla/análise , Luciferases de Renilla/metabolismo , Camundongos , Modelos Biológicos , Mapeamento de Peptídeos , Fosforilação , Estrutura Terciária de Proteína , Inibidores da Síntese de Proteínas/farmacologia , Serina/metabolismo , Fatores de Tempo , TransfecçãoRESUMO
Long double-stranded RNAs (dsRNAs) may undergo extensive modification (hyperediting) by adenosine deaminases that act on RNA (ADARs), where up to 50% of adenosine (A) residues are changed to inosine (I). Traditionally, consequences of A-to-I editing were thought to be limited to modified RNA itself. We show here, however, that hyperedited dsRNA (I-dsRNA) is able to downregulate gene expression in trans. Furthermore, we show that both endogenous expression and reporter gene expression were substantially reduced in the presence of I-dsRNA. This was due to a reduction in reporter mRNA levels and also translation inhibition. Importantly, we show that I-dsRNA interferes with translation initiation. We also show that I-dsRNA specifically binds a stress-granule-like complex. Stress granules (SGs) are important for translational silencing during stress. Finally, we propose a model whereby editing by ADARs results in downregulation of gene expression via SG formation.
Assuntos
Regulação para Baixo , Modelos Genéticos , RNA de Cadeia Dupla/fisiologia , Genes Reporter , Globinas/análise , Proteínas de Fluorescência Verde/análise , Células HeLa , Humanos , Inosina/química , Luciferases de Vaga-Lume/análise , Luciferases de Renilla/análise , Biossíntese de Proteínas , RNA de Cadeia Dupla/química , RNA de Cadeia Dupla/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismoRESUMO
BACKGROUND: Epidermal growth factor receptor (EGFR) stimulation enhances intestinal adaptation after massive small bowel resection (SBR), measured by taller villi, deeper crypts, and augmented enterocyte proliferation. Min mice with constitutively active beta-catenin signaling demonstrate enhanced villus growth after SBR, suggesting a role for the Wnt pathway during adaptation. Because there is crosstalk between EGFR signaling and the Wnt pathway, we hypothesized that beta-catenin is modulated by EGFR-induced enterocyte proliferation. METHODS: Rat intestinal epithelial cells were stimulated with EGF and cytoplasmic to nuclear trafficking of beta-catenin was measured. Beta-catenin-directed transcription was also tested via transfection with a TOP/FOP luciferase reporter. Downstream transcriptional target expression was measured in murine intestine after SBR. RESULTS: Epidermal growth factor-treated rat intestinal epithelial cells exhibited increased proliferation compared to serum-deficient cells in the face of no detectable accumulation of nuclear beta-catenin. The luciferase assay results showed minimal transcription activity in response to EGF. In vivo experiments revealed no significant difference in expression of beta-catenin targeted genes in crypt enterocytes after SBR. CONCLUSIONS: The mechanism for EGFR-induced proliferation of enterocytes does not appear to involve a transcriptional role for beta-catenin. The effects of EGFR signaling on beta-catenin-mediated cell adhesion remain to be investigated.
Assuntos
Enterócitos/citologia , Receptores ErbB/fisiologia , Síndrome do Intestino Curto/fisiopatologia , Transdução de Sinais/fisiologia , Proteínas Wnt/fisiologia , beta Catenina/fisiologia , Transporte Ativo do Núcleo Celular/fisiologia , Adaptação Fisiológica , Animais , Divisão Celular , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/metabolismo , Ciclina D , Ciclinas/biossíntese , Ciclinas/genética , Citoplasma/metabolismo , Enterócitos/efeitos dos fármacos , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/efeitos dos fármacos , Perfilação da Expressão Gênica , Genes Reporter , Genes myc , Intestino Delgado/cirurgia , Luciferases de Renilla/análise , Luciferases de Renilla/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Proto-Oncogênicas c-myc/biossíntese , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Síndrome do Intestino Curto/genética , Síndrome do Intestino Curto/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transcrição Gênica , TransfecçãoRESUMO
An infectious hepatitis C virus (HCV) cDNA clone (JFH1) was generated recently. However, quantitative analysis of HCV infection and observation of infected cells have proved to be difficult because the yield of HCV in cell cultures is fairly low. We generated infectious HCV clones containing the convenient reporters green fluorescent protein (GFP) and Renilla luciferase in the NS5a-coding sequence. The new viruses responded to antiviral agents in a dose-dependent manner. Responses of individual cells containing HCV to alpha interferon (IFN-alpha) were monitored using GFP-tagged HCV and time-lapse confocal microscopy. Marked variations in the response to IFN-alpha were observed among HCV-containing cells.
Assuntos
Antivirais/farmacologia , Hepacivirus/efeitos dos fármacos , Interferon-alfa/farmacologia , Linhagem Celular Tumoral , Genes Reporter , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Hepacivirus/genética , Hepacivirus/imunologia , Humanos , Luciferases de Renilla/análise , Luciferases de Renilla/genética , Internalização do Vírus/efeitos dos fármacosRESUMO
The zinc finger transcription factor, WT1, regulates many growth control genes, repressing or activating transcription depending on the gene and cell type. Based on earlier analyses of the effect of WT1 on androgen responsive genes, we hypothesized that there may be an interaction between the androgen signaling pathway and WT1, such that the commonly used Renilla luciferase control vectors were activated in LNCaP prostate cancer cells. Using cotransfection assays we tested the effects of WT1 and/or the androgen analog, R1881, on two Renilla luciferase vectors, pRL-SV40 and the promoter-less pRL-null. To determine whether the zinc finger DNA binding domain was required, the zinc finger mutant DDS-WT1 (R394W) was tested; but it had no significant effect on the Renilla luciferase vectors. To determine whether the androgen signaling pathway was required, WT1 was co-transfected with Renilla vectors in cells with varied hormone responsiveness. The WT1 effect on pRL-null varied from no significant effect in 293 and PC3 cells to very strong enhancement in LNCaP cells treated with 5 nM R1881. Overall, these results suggest that hormone enhanced WT1 mediated activation of Renilla luciferase and that these interactions require an intact WT1 zinc finger DNA binding domain.
Assuntos
Luciferases de Renilla/análise , Congêneres da Testosterona/farmacologia , Ativação Transcricional , Proteínas WT1/metabolismo , Linhagem Celular Tumoral , Genes Reporter , Humanos , Luciferases de Renilla/genética , Metribolona/farmacologia , Ativação Transcricional/efeitos dos fármacosRESUMO
The oncogenic transcription factor forkhead box M1 (FoxM1) is overexpressed in a number of different carcinomas, whereas its expression is turned off in terminally differentiated cells. For this reason, FoxM1 is an attractive target for therapeutic intervention in cancer treatment. As a first step toward realizing this goal, in this study, using a high-throughput, cell-based assay system, we screened for and isolated the antibiotic thiazole compound Siomycin A as an inhibitor of FoxM1. Interestingly, we observed that Siomycin A was able to down-regulate the transcriptional activity as well as the protein and mRNA abundance of FoxM1. Consequently, we found that the downstream target genes of FoxM1, such as Cdc25B, Survivin, and CENPB, were repressed. Also, we observed that consistent with earlier reports of FoxM1 inhibition, Siomycin A was able to reduce anchorage-independent growth of cells in soft agar. Furthermore, we found that Siomycin A was able to induce apoptosis selectively in transformed but not normal cells of the same origin. Taken together, our data suggest that FoxM1 inhibitor Siomycin A could represent a useful starting point for the development of anticancer therapeutics.