RESUMEN
Codon usage bias is a universal feature of all genomes. Although codon usage has been shown to regulate mRNA and protein levels by influencing mRNA decay and transcription in eukaryotes, little or no genome-wide correlations between codon usage and mRNA levels are detected in mammalian cells, raising doubt on the significance of codon usage effect on gene expression. Here we show that gene-specific regulation reduces the genome-wide codon usage and mRNA correlations: Constitutively expressed genes exhibit much higher genome-wide correlations than differentially expressed genes from fungi to human cells. Using Drosophila S2 cells as a model system, we showed that the effect of codon usage on mRNA expression level is promoter-dependent. Regions downstream of the core promoters of differentially expressed genes can repress the codon usage effects on mRNA expression. An element in the Hsp70 promoter was identified to be necessary and sufficient for this inhibitory effect. The promoter-dependent codon usage effects on mRNA levels are regulated at the transcriptional level through modulation of histone modifications, nucleosome densities and premature termination. Together, our results demonstrate that promoters play a major role in determining whether codon usage influences gene expression and further establish the transcription-dependent codon usage effects on gene expression.
Asunto(s)
Uso de Codones , Drosophila melanogaster/genética , Regulación de la Expresión Génica/genética , Regiones Promotoras Genéticas/genética , Acetilación , Animales , Composición de Base , Línea Celular , Cromatina/genética , Cromatina/ultraestructura , Codón sin Sentido , Proteínas de Drosophila/fisiología , Drosophila melanogaster/citología , Genes Reporteros , Código de Histonas , Humanos , Luciferasas de Renilla/biosíntesis , Luciferasas de Renilla/genética , Ratones , Neurospora crassa/genética , Nucleosomas/metabolismo , Procesamiento Proteico-Postraduccional , Estabilidad del ARN , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Saccharomyces cerevisiae/genética , Especificidad de la EspecieRESUMEN
HTLV-1 and HTLV-2 viruses express Tax transactivator proteins required for viral genome transcription and capable of transforming cells in vivo and in vitro. Although Tax oncogenic potential needs to be further elucidated, it is well established that Tax proteins activate, among others, transcription factors of the NF-ĸB family, which are involved in immune and inflammatory responses, cell growth, apoptosis, stress responses and oncogenesis. Here, we describe a reporter gene assay applied for quantitative analysis of Tax-dependent NF-ĸB activation. The procedure is based on co-transfection of two individual vectors containing the cDNA for firefly and Renilla luciferase enzymes and vectors expressing Tax proteins. The luciferase expression is driven by cis-NF-ĸB promoter regulatory elements responsive to Tax transactivating factor. This assay is particularly useful to investigate Tax influence on NF-ĸB activation mediated by viral or host factors.
Asunto(s)
Productos del Gen tax , Virus Linfotrópico T Tipo 1 Humano , Virus Linfotrópico T Tipo 2 Humano , Luciferasas de Renilla , FN-kappa B , Activación Transcripcional , Productos del Gen tax/genética , Productos del Gen tax/metabolismo , Células HEK293 , Virus Linfotrópico T Tipo 1 Humano/genética , Virus Linfotrópico T Tipo 1 Humano/metabolismo , Virus Linfotrópico T Tipo 2 Humano/genética , Virus Linfotrópico T Tipo 2 Humano/metabolismo , Humanos , Luciferasas de Renilla/biosíntesis , Luciferasas de Renilla/genética , FN-kappa B/genética , FN-kappa B/metabolismo , Elementos de RespuestaRESUMEN
Renilla luciferase reporter is a widely used internal control in dual luciferase reporter assay system, where its transcription is driven by a constitutively active promoter. However, the authenticity of the Renilla luciferase response in some experimental settings has recently been questioned. Testicular receptor 4 (TR4, also known as NR2C2) belongs to the subfamily 2 of nuclear receptors. TR4 binds to a direct repeat regulatory element in the promoter of a variety of target genes and plays a key role in tumorigenesis, lipoprotein regulation, and central nervous system development. In our experimental system using murine pituitary corticotroph tumor AtT20 cells to investigate TR4 actions on POMC transcription, we found that overexpression of TR4 resulted in reduced Renilla luciferase expression whereas knockdown TR4 increased Renilla luciferase expression. The TR4 inhibitory effect was mediated by the TR4 DNA-binding domain and behaved similarly to the GR and its agonist, Dexamethasone. We further demonstrated that the chimeric intron, commonly present in various Renilla plasmid backbones such as pRL-Null, pRL-SV40, and pRL-TK, was responsible for TR4's inhibitory effect. The results suggest that an intron-free Renilla luciferase reporter may provide a satisfactory internal control for TR4 at certain dose range. Our findings advocate caution on the use of Renilla luciferase as an internal control in TR4-directed studies to avoid misleading data interpretation.
Asunto(s)
Dexametasona/farmacología , Genes Reporteros , Luciferasas de Renilla/biosíntesis , Proteínas de Neoplasias/metabolismo , Miembro 2 del Grupo C de la Subfamilia 2 de Receptores Nucleares/metabolismo , Neoplasias Hipofisarias/metabolismo , Animales , Línea Celular Tumoral , Reacciones Falso Positivas , Luciferasas de Renilla/genética , Ratones , Proteínas de Neoplasias/agonistas , Proteínas de Neoplasias/genética , Miembro 2 del Grupo C de la Subfamilia 2 de Receptores Nucleares/agonistas , Miembro 2 del Grupo C de la Subfamilia 2 de Receptores Nucleares/genética , Neoplasias Hipofisarias/genética , Neoplasias Hipofisarias/patología , Proopiomelanocortina/biosíntesis , Proopiomelanocortina/genéticaRESUMEN
MicroRNAs are small noncoding RNAs that regulate gene expression by repressing translation of target cellular transcripts. Increasing evidences indicate that miRNAs have different expression profiles and play crucial roles in numerous cellular processes. Delivery and expression of transgenes for cancer therapy must be specific for tumors to avoid killing of healthy tissues. Many investigators have shown that transgene expression can be suppressed in normal cells using vectors that are responsive to microRNA regulation. To overcome this problem, miR-145 that exhibits downregulation in many types of cancer cells was chosen for posttranscriptional regulatory systems mediated by microRNAs. In this study, a psiCHECK-145T vector carrying four tandem copies of target sequences of miR-145 into 3'-UTR of the Renilla luciferase gene was constructed. Renilla luciferase activity from the psiCHECK-145T vector was 57% lower in MCF10A cells with high miR-145 expression as compared to a control condition. Additionally, overexpression of miR-145 in MCF-7 cells with low expression level of miR-145 showed more than 76% reduction in the Renilla luciferase activity from the psiCHECK-145T vector. Inclusion of miR-145 target sequences into the 3'-UTR of the Renilla luciferase gene is a feasible strategy for restricting transgene expression in a breast cancer cell line while sparing a breast normal cell line.
Asunto(s)
Neoplasias de la Mama/genética , Terapia Genética , Luciferasas de Renilla/genética , MicroARNs/biosíntesis , Neoplasias de la Mama/patología , Femenino , Regulación Neoplásica de la Expresión Génica , Vectores Genéticos , Humanos , Luciferasas de Renilla/biosíntesis , Células MCF-7 , MicroARNs/genética , TransgenesRESUMEN
HMGA2 is an important chromatin factor that interacts with DNA via three AT-hook domains, thereby regulating chromatin architecture and transcription during embryonic and fetal development. The protein is absent from differentiated somatic cells, but aberrantly re-expressed in most aggressive human neoplasias where it is causally linked to cell transformation and metastasis. DNA-binding also enables HMGA2 to protect cancer cells from DNA-damaging agents. HMGA2 therefore is considered to be a prime drug target for many aggressive malignancies. Here, we have developed a broadly applicable cell-based reporter system which can identify HMGA2 antagonists targeting functionally important protein domains, as validated with the known AT-hook competitor netropsin. In addition, high-throughput screening can uncover functional links between HMGA2 and cellular factors important for cell transformation. This is demonstrated with the discovery that HMGA2 potentiates the clinically important topoisomerase I inhibitor irinotecan/SN-38 in trapping the enzyme in covalent DNA-complexes, thereby attenuating transcription.
Asunto(s)
Camptotecina/análogos & derivados , ADN-Topoisomerasas de Tipo I/fisiología , Proteína HMGA2/fisiología , Inhibidores de Topoisomerasa I/farmacología , Secuencia de Aminoácidos , Camptotecina/farmacología , Diferenciación Celular/efectos de los fármacos , Estabilidad de Enzimas , Genes Reporteros , Células HEK293 , Células HeLa , Ensayos Analíticos de Alto Rendimiento , Humanos , Irinotecán , Luciferasas de Renilla/biosíntesis , Luciferasas de Renilla/genética , Netropsina/farmacología , Regiones Promotoras Genéticas , Transcripción Genética/efectos de los fármacos , Activación TranscripcionalRESUMEN
Computational techniques have been used to design a novel class of RNA architecture with expected improved resistance to nuclease degradation, while showing interference RNA activity. The in silico designed structure consists of a 24-29 bp duplex RNA region linked on both ends by N-alkyl-N dimeric nucleotides (BCn dimers; n = number of carbon atoms of the alkyl chain). A series of N-alkyl-N capped dumbbell-shaped structures were efficiently synthesized by double ligation of BCn-loop hairpins. The resulting BCn-loop dumbbells displayed experimentally higher biostability than their 3'-N-alkyl-N linear version, and were active against a range of mRNA targets. We studied first the effect of the alkyl chain and stem lengths on RNAi activity in a screen involving two series of dumbbell analogues targeting Renilla and Firefly luciferase genes. The best dumbbell design (containing BC6 loops and 29 bp) was successfully used to silence GRB7 expression in HER2+ breast cancer cells for longer periods of time than natural siRNAs and known biostable dumbbells. This BC6-loop dumbbell-shaped structure displayed greater anti-proliferative activity than natural siRNAs.
Asunto(s)
Técnicas de Silenciamiento del Gen/métodos , ARN/genética , Alquilación , Secuencia de Bases , Proteína Adaptadora GRB7/biosíntesis , Proteína Adaptadora GRB7/genética , Expresión Génica , Células HeLa , Humanos , Luciferasas de Luciérnaga/biosíntesis , Luciferasas de Luciérnaga/genética , Luciferasas de Renilla/biosíntesis , Luciferasas de Renilla/genética , Células MCF-7 , Nanoestructuras , ARN/síntesis química , Interferencia de ARN , Estabilidad del ARNRESUMEN
WNT5A is a secreted ligand involved in Wnt pathway signaling and has a role in cell movement and differentiation. Altered WNT5A expression is associated with various cancers, although in most studies the focus has been on only one of the known WNT5A isoforms. In this study, we analyzed expression from two of the major WNT5A promoters, termed promoter A and promoter B, in normal human osteoblasts, SaOS-2 and U2OS osteosarcoma cell lines, and osteosarcoma tumor tissue. We found that both promoters A and B are active in normal osteoblasts with nearly 11-fold more promoter B than A transcripts. Promoter B but not promoter A transcripts are decreased or nearly undetectable in the SaOS-2 and U2OS cell lines and osteosarcoma tumor tissues. Transient transfection of promoter A and promoter B reporter constructs confirmed that SaOS-2 cells have the necessary factors to transcribe both promoters. Bisulfite sequencing analysis revealed that three CpG enriched regions upstream of the promoter B exon 1ßare highly methylated in both SaOS-2 and U2OS cells. The CpG island sub-region R6 located in promoter B exon 1ß was approximately 51% methylated in SaOS-2 and 25% methylated in U2OS. Region 3 was approximately 28% methylated in normal osteoblasts, whereas the others were unmethylated. Promoter B was re-activated by treatment of SaOS-2 cells with 1 µM 5-azacytidine, which was associated with only a small insignificant change in methylation of sub-region R6. ChIP analysis of U2OS and SaOS-2 cells indicated that the promoter B region is less enriched in the active histone mark H3K4me3, in comparison to promoter A and that there is increased enrichment of the repressive mark H3K27me3 in association with the promoter B genomic region in the cell line SaOS-2. These findings show that epigenetic inactivation of the WNT5A promoter B involves both DNA methylation and histone modifications and suggest that differential expression of the WNT5A alternative promoters A and B is a characteristic of osteosarcomas.
Asunto(s)
Neoplasias Óseas/patología , Metilación de ADN , Regulación Neoplásica de la Expresión Génica/genética , Código de Histonas , Proteínas de Neoplasias/genética , Osteosarcoma/patología , Regiones Promotoras Genéticas/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Wnt/genética , Azacitidina/farmacología , Neoplasias Óseas/genética , Línea Celular Tumoral , Inmunoprecipitación de Cromatina , Islas de CpG/genética , Metilación de ADN/efectos de los fármacos , ADN de Neoplasias/genética , ADN de Neoplasias/metabolismo , Exones/genética , Genes Reporteros , Humanos , Luciferasas de Renilla/biosíntesis , Luciferasas de Renilla/genética , Metilación , Proteínas de Neoplasias/biosíntesis , Análisis de Secuencia por Matrices de Oligonucleótidos , Osteoblastos/metabolismo , Osteoblastos/patología , Osteosarcoma/genética , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Recombinantes de Fusión/biosíntesis , Transfección , Proteínas Wnt/biosíntesis , Vía de Señalización Wnt/genética , Proteína Wnt-5aRESUMEN
Surface-mediated gene transfer systems using biocompatible calcium phosphate (CaP)-based composite layers have attracted attention as a tool for controlling cell behaviors. In the present study we aimed to demonstrate the potential of CaP-based composite layers to mediate area-specific dual gene transfer and to stimulate cells on an area-by-area basis in the same well. For this purpose we prepared two pairs of DNA-fibronectin-apatite composite (DF-Ap) layers using a pair of reporter genes and pair of differentiation factor genes. The results of the area-specific dual gene transfer successfully demonstrated that the cells cultured on a pair of DF-Ap layers that were adjacently placed in the same well showed specific gene expression patterns depending on the gene that was immobilized in the underlying layer. Moreover, preliminary real-time PCR results indicated that multipotential C3H10T1/2 cells may have a potential to change into different types of cells depending on the differentiation factor gene that was immobilized in the underlying layer, even in the same well. Because DF-Ap layers have a potential to mediate area-specific cell stimulation on their surfaces, they could be useful in tissue engineering applications.
Asunto(s)
Apatitas/química , Transfección/métodos , Animales , Células CHO , Diferenciación Celular , Cricetinae , Cricetulus , ADN/química , Fibronectinas/química , Genes Reporteros , Luciferasas de Luciérnaga/biosíntesis , Luciferasas de Luciérnaga/genética , Luciferasas de Renilla/biosíntesis , Luciferasas de Renilla/genética , Ratones , Propiedades de SuperficieRESUMEN
PURPOSE: Deregulation of miRNA has been implicated in the pathogenesis of multiple myeloma. We identified miR-137 and miR-197, mapped to the chromosome 1p (12)-(21) deletion region, and examined their antimyeloma activity as tumor suppressors. EXPERIMENTAL DESIGN: The expression of miR-137/197 was examined in multiple myeloma and normal plasma cells by qRT-PCR. Functional effect of miR-137/197 was analyzed by cell viability, apoptosis, clonogenic, and migration assays. Antimyeloma activity of miR-137/197 was further evaluated in vivo by lentiviral-based or lipid-based delivery in a mouse xenograft model of multiple myeloma. RESULTS: miR-137/197 expression was significantly lower in multiple myeloma cell lines and multiple myeloma patient samples compared with normal plasma cells. Transfection of miR-137/197 resulted in reduction of MCL-1 protein expression, as well as alteration of apoptosis-related genes, and induction of apoptosis, inhibition of viability, colony formation, and migration in multiple myeloma cells. MCL-1 was further validated as a direct target of miR-137/197. Conversely, overexpression of MCL-1 partially reverted the effect of miR-137/197. Importantly, in vivo lentiviral-mediated or intratumor delivery of miR-137/197 induced regression of tumors in murine xenograft models of multiple myeloma. CONCLUSIONS: Our study reveals a novel role of miR-137/197 as tumor suppressors in mediating apoptosis in multiple myeloma cells by targeting MCL-1. Our findings provide a proof-of-principle that lentivirus-based or formulated synthetic miR-137/197 exerts therapeutic activity in preclinical models, and support a framework for development of miR-137/197-based treatment strategies in patients with multiple myeloma.
Asunto(s)
Apoptosis , MicroARNs/genética , Mieloma Múltiple/metabolismo , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/genética , Regiones no Traducidas 3' , Animales , Secuencia de Bases , Sitios de Unión , Carcinogénesis/genética , Carcinogénesis/metabolismo , Línea Celular Tumoral , Movimiento Celular , Supervivencia Celular , Expresión Génica , Regulación Neoplásica de la Expresión Génica , Genes Reporteros , Terapia Genética , Células HEK293 , Humanos , Luciferasas de Luciérnaga/biosíntesis , Luciferasas de Luciérnaga/genética , Luciferasas de Renilla/biosíntesis , Luciferasas de Renilla/genética , Ratones SCID , MicroARNs/metabolismo , Mieloma Múltiple/genética , Mieloma Múltiple/terapia , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Trasplante de Neoplasias , Interferencia de ARNRESUMEN
Heat shock gene promoters represent a highly conserved and universal system for the rapid induction of transcription after various stressful stimuli. We chose pairs of mammalian and insect species that significantly differ in their thermoresistance and constitutive levels of Hsp70 to compare hsp promoter strength under normal conditions and after heat shock (HS). The first pair includes the HSPA1 gene promoter of camel (Camelus dromedarius) and humans. It was demonstrated that the camel HSPA1A and HSPA1L promoters function normally in vitro in human cell cultures and exceed the strength of orthologous human promoters under basal conditions. We used the same in vitro assay for Drosophila melanogaster Schneider-2 (S2) cells to compare the activity of the hsp70 and hsp83 promoters of the second species pair represented by Diptera, i.e., Stratiomys singularior and D. melanogaster, which dramatically differ in thermoresistance and the pattern of Hsp70 accumulation. Promoter strength was also monitored in vivo in D. melanogaster strains transformed with constructs containing the S. singularior hsp70 ORF driven either by its own promoter or an orthologous promoter from the D. melanogaster hsp70Aa gene. Analysis revealed low S. singularior hsp70 promoter activity in vitro and in vivo under basal conditions and after HS in comparison with the endogenous promoter in D. melanogaster cells, which correlates with the absence of canonical GAGA elements in the promoters of the former species. Indeed, the insertion of GAGA elements into the S. singularior hsp70 regulatory region resulted in a dramatic increase in promoter activity in vitro but only modestly enhanced the promoter strength in the larvae of the transformed strains. In contrast with hsp70 promoters, hsp83 promoters from both of the studied Diptera species demonstrated high conservation and universality.
Asunto(s)
Proteínas de Choque Térmico/genética , Regiones Promotoras Genéticas , Animales , Secuencia de Bases , Camelus/genética , Línea Celular , Drosophila melanogaster/genética , Genes Reporteros , Humanos , Luciferasas de Renilla/biosíntesis , Luciferasas de Renilla/genética , Datos de Secuencia Molecular , Especificidad de la Especie , TATA Box , Activación TranscripcionalRESUMEN
The human airway serves as the entry point of human respiratory viruses, including human coronaviruses. In this chapter we outline the methods by which we establish fully differentiated airway epithelium and its use for human coronavirus propagation. Additionally, we outline methods for immunofluorescence staining of these cultures for virus detection, characterization of cell tropism, and how to perform antiviral assays and quantify viral replication.
Asunto(s)
Coronavirus/fisiología , Células Epiteliales/virología , Bronquios/citología , Técnicas de Cultivo de Célula , Células Cultivadas , Genes Reporteros , Humanos , Luciferasas de Renilla/biosíntesis , Luciferasas de Renilla/genética , Tráquea/citología , Tropismo Viral , Cultivo de Virus , Replicación ViralRESUMEN
Many of the cancer cell lines derived from solid tumors are difficult to transfect using commonly established transfection approaches. This hurdle for some DNA transfection systems has hindered cancer biology studies. Moreover, there are limited tools for studying pathway activities. Therefore, highly efficient improved gene transfer and versatile genetic tools are required. In this study, we established and developed a comprehensive set of new lentiviral tools to study gene functions and pathway activities. Using the optimized conditions, cancer cell lines achieved >90% transduction efficiency. Novel lentiviral doxycycline-regulated pTet-IRES-EGFP (pTIE) systems for transgene expression and TRE reporters used for pathway activity determination were developed and tested. The pTIE Tet-Off system showed in vitro doxycycline-sensitive responses with low or undetectable leakage of protein expression and in vivo tumor suppression as illustrated using candidate tumor suppressors, Fibulin-2 and THY1. In contrast, the Tet-On system showed dose-dependent responses. The pTRE-EGFP (pTE) and pTRE-FLuc-EF1α-RLuc (pT-FER) reporters with the NFκB p65 subunit consensus sequence showed GFP and firefly luciferase responses, which were directly correlated with TNFα stimulation, respectively. Taken together, these newly developed lentiviral systems provide versatile in vitro and in vivo platforms to strengthen our capabilities for cancer biology studies.
Asunto(s)
Terapia Genética , Lentivirus/genética , Neoplasias/terapia , Activación Transcripcional , Animales , Células COS , Carcinogénesis/patología , Línea Celular Tumoral , Chlorocebus aethiops , Femenino , Expresión Génica , Genes Reporteros , Vectores Genéticos , Células HEK293 , Humanos , Luciferasas de Renilla/biosíntesis , Luciferasas de Renilla/genética , Ratones Endogámicos BALB C , Ratones Desnudos , Trasplante de Neoplasias , Neoplasias/patología , Plásmidos/genética , Regiones Promotoras Genéticas , Transducción Genética , TransgenesRESUMEN
Site specificity is pivotal in obtaining homogeneously labeled proteins without batch-to-batch variations. More importantly, precisely controlled modification at specific sites avoids potential pitfalls that could otherwise interfere with protein folding, structure, and function. Inspired by the chemical synthesis of D-luciferin, we have developed an efficient strategy (second-order rate constant k 2 = 9.2 M(-1) s(-1)) for labeling of proteins containing 1,2-aminothiol via reaction with 2-cyanobenzothiazole (CBT). In addition, the CBT condensation enjoys the convenience of protein engineering, as production of N-terminal cysteine-containing proteins has been well developed for native chemical ligation. This protocol describes the preparation of Renilla luciferase (rLuc) with 1,2-aminothiol at either its N- or C-terminus, and site-specific labeling of rLuc with fluorescein or (18)F via CBT condensation.
Asunto(s)
Benzotiazoles/química , Colorantes Fluorescentes/química , Luciferasas de Renilla/química , Nitrilos/química , Proteínas Recombinantes de Fusión/química , Cisteína/química , Escherichia coli , Fluoresceína-5-Isotiocianato/química , Luciferasas de Renilla/biosíntesis , Proteínas Recombinantes de Fusión/biosíntesis , Coloración y EtiquetadoRESUMEN
Cell free protein synthesis systems (CFPS) have been widely used to express proteins and to explore the pathways of gene expression. In the present manuscript, we describe the design of a novel adaptable hybrid in vitro translation system which is assembled with ribosomes isolated from many different origins. We first show that this hybrid system exhibits all important features such as efficiency, sensitivity, reproducibility and the ability to translate specialized mRNAs in less than 1 h. In addition, the unique design of this cell free assay makes it highly adaptable to utilize ribosomes isolated from many different organs, tissues or cell types.
Asunto(s)
Biosíntesis de Proteínas , Ribosomas/metabolismo , Regiones no Traducidas 5' , Animales , Línea Celular , Sistema Libre de Células , Células Cultivadas , Cricetinae , Células HeLa , Humanos , Técnicas In Vitro , Células Jurkat , Luciferasas de Renilla/biosíntesis , Luciferasas de Renilla/genética , Ratones , Poliovirus/genética , Biosíntesis de Proteínas/genética , ARN Mensajero/genética , Conejos , Reticulocitos/metabolismo , Globinas beta/biosíntesis , Globinas beta/genéticaRESUMEN
Polyethyleneimine (PEI) is a cost-effective and non-viral vector for gene transfer, but the factors determining gene transfer efficiency and cytotoxicity of PEI in different mammalian cell lines remain largely unknown. In the present study, three different cell lines were chosen for investigation. Using pEGFP DNA and PEI, 21.5, 29.2, and 92.1 % of GFP-positive cells were obtained in BMSC, Hela, and 293T, respectively. In luciferase reporter assay, similar results were obtained (for luciferase activity, BMSC < Hela < 293T cells). By MTT test and cell apoptotic marker analysis, we demonstrated that high gene transfer efficiency is accompanied with high cytotoxicity of PEI. Moreover, we found that high expression level of caveolin-1 was accompanied with high gene transfer efficiency and cytotoxicity of PEI in 293T cells. More convincingly, caveolin-1 silencing in 293T could reduce both gene transfer efficiency and cytotoxicity of PEI. In contrast, caveolin-1 overexpression in BMSCs increases both gene transfer efficiency and cytotoxicity of PEI. Taken together, our study suggests that caveolin-1 may at least in part determine gene transfer efficiency and cytotoxicity of PEI in mammalian cell lines, providing caveolin-1 as a potential target for improving gene transfer efficiency when applying positively charged polyplexes to cell transfection.
Asunto(s)
Caveolina 1/fisiología , Polietileneimina/toxicidad , Animales , Genes Reporteros , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/genética , Células HEK293 , Células HeLa , Humanos , Luciferasas de Renilla/biosíntesis , Luciferasas de Renilla/genética , Ratones , TransfecciónRESUMEN
Nuclear receptors (NRs) are an important group of ligand-dependent transcriptional factors. Presently, no natural or synthetic ligand has been identified for a large group of orphan NRs. Small molecules to target these orphan NRs will provide unique resources for uncovering regulatory systems that impact human health and to modulate these pathways with drugs. The orphan NR tailless (TLX, NR2E1), a transcriptional repressor, is a major player in neurogenesis and Neural Stem Cell (NSC) derived brain tumors. No chemical probes that modulate TLX activity are available, and it is not clear whether TLX is druggable. To assess TLX ligand binding capacity, we created homology models of the TLX ligand binding domain (LBD). Results suggest that TLX belongs to an emerging class of NRs that lack LBD helices α1 and α2 and that it has potential to form a large open ligand binding pocket (LBP). Using a medium throughput screening strategy, we investigated direct binding of 20,000 compounds to purified human TLX protein and verified interactions with a secondary (orthogonal) assay. We then assessed effects of verified binders on TLX activity using luciferase assays. As a result, we report identification of three compounds (ccrp1, ccrp2 and ccrp3) that bind to recombinant TLX protein with affinities in the high nanomolar to low micromolar range and enhance TLX transcriptional repressive activity. We conclude that TLX is druggable and propose that our lead compounds could serve as scaffolds to derive more potent ligands. While our ligands potentiate TLX repressive activity, the question of whether it is possible to develop ligands to de-repress TLX activity remains open.
Asunto(s)
Didrogesterona/farmacología , Piperazinas/farmacología , Pirazoles/farmacología , Receptores Citoplasmáticos y Nucleares/fisiología , Activación Transcripcional/efectos de los fármacos , Secuencia de Aminoácidos , Sitios de Unión , Factor de Transcripción COUP II/antagonistas & inhibidores , Factor de Transcripción COUP II/fisiología , Receptor beta de Estrógeno/antagonistas & inhibidores , Receptor beta de Estrógeno/fisiología , Genes Reporteros , Células HeLa , Humanos , Concentración 50 Inhibidora , Ligandos , Luciferasas de Renilla/biosíntesis , Luciferasas de Renilla/genética , Modelos Moleculares , Datos de Secuencia Molecular , Receptores Nucleares Huérfanos , Unión Proteica , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores , Receptores Citoplasmáticos y Nucleares/química , Receptor alfa X Retinoide/antagonistas & inhibidores , Receptor alfa X Retinoide/fisiología , Transcripción Genética/efectos de los fármacosRESUMEN
Peroxisome proliferator-activated receptor α (PPARα) is an important regulator of hepatic lipid metabolism which functions through ligand binding. Despite high amino acid sequence identity (>90%), marked differences in PPARα ligand binding, activation and gene regulation have been noted across species. Similar to previous observations with synthetic agonists, we have recently reported differences in ligand affinities and extent of activation between human PPARα (hPPARα) and mouse PPARα (mPPARα) in response to long chain fatty acids (LCFA). The present study was aimed to determine if structural alterations could account for these differences. The binding of PPARα to LCFA was examined through in silico molecular modeling and docking simulations. Modeling suggested that variances at amino acid position 272 are likely to be responsible for differences in saturated LCFA binding to hPPARα and mPPARα. To confirm these results experimentally, LCFA binding, circular dichroism, and transactivation studies were performed using a F272I mutant form of mPPARα. Experimental data correlated with in silico docking simulations, further confirming the importance of amino acid 272 in LCFA binding. Although the driving force for evolution of species differences at this position are yet unidentified, this study enhances our understanding of ligand-induced regulation by PPARα and demonstrates the efficacy of molecular modeling and docking simulations.
Asunto(s)
Ácidos Grasos/química , PPAR alfa/química , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Sitios de Unión , Células COS , Chlorocebus aethiops , Ácidos Grasos/fisiología , Genes Reporteros , Humanos , Luciferasas de Renilla/biosíntesis , Luciferasas de Renilla/genética , Ratones , Simulación del Acoplamiento Molecular , Datos de Secuencia Molecular , PPAR alfa/fisiología , Fenilalanina/química , Fenilalanina/genética , Unión Proteica , Estructura Secundaria de Proteína , Receptor alfa X Retinoide/fisiología , Homología de Secuencia de Aminoácido , Termodinámica , Activación TranscripcionalRESUMEN
Egg activation at fertilization in mammals is initiated by prolonged Ca(2+) oscillations that trigger the completion of meiosis and formation of pronuclei. A fall in mitogen-activated protein kinase (MAPK) activity is essential for pronuclear formation, but the precise timing and mechanism of decline are unknown. Here, we have measured the dynamics of MAPK pathway inactivation during fertilization of mouse eggs using novel chemiluminescent MAPK activity reporters. This reveals that the MAPK activity decrease begins during the Ca(2+) oscillations, but MAPK does not completely inactivate until after pronuclear formation. The MAPKs present in eggs are Mos, MAP2K1 and MAP2K2 (MEK1 and MEK2, respectively) and MAPK3 and MAPK1 (ERK1 and ERK2, respectively). Notably, the MAPK activity decline at fertilization is not explained by upstream destruction of Mos, because a decrease in the signal from a Mos-luciferase reporter is not associated with egg activation. Furthermore, Mos overexpression does not affect the timing of MAPK inactivation or pronuclear formation. However, the late decrease in MAPK could be rapidly reversed by the protein phosphatase inhibitor, okadaic acid. These data suggest that the completion of meiosis in mouse zygotes is driven by an increased phosphatase activity and not by a decline in Mos levels or MEK activity.
Asunto(s)
Proteínas Quinasas Activadas por Mitógenos/metabolismo , Óvulo/enzimología , Animales , Señalización del Calcio , Inhibidores Enzimáticos/farmacología , Femenino , Fertilización , Genes Reporteros , Luciferasas de Renilla/biosíntesis , Luciferasas de Renilla/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ácido Ocadaico/farmacología , Proteínas Oncogénicas v-mos/genética , Proteínas Oncogénicas v-mos/metabolismo , Monoéster Fosfórico Hidrolasas/antagonistas & inhibidores , Monoéster Fosfórico Hidrolasas/metabolismo , Fosforilación , Procesamiento Proteico-Postraduccional , Espermatozoides/fisiologíaRESUMEN
Eukaryotic cell-free in vitro translation systems have been in use since the 1970s. These systems can faithfully synthesize polypeptides when programmed with mRNA, enabling the production of polypeptides for analysis as well as permitting analyses of the cis- and trans-acting factors that regulate translation. Here we describe the preparation and use of cell-free translation systems from the yeast Saccharomyces cerevisiae.
Asunto(s)
Extractos Celulares/aislamiento & purificación , Biosíntesis de Proteínas , Saccharomyces cerevisiae , Tampones (Química) , Fraccionamiento Celular , Genes Reporteros , Luciferasas de Renilla/biosíntesis , Luciferasas de Renilla/genética , Inhibidores de Proteasas/química , Saccharomyces cerevisiae/citología , SolucionesRESUMEN
RNAi is a powerful tool for the regulation of gene expression. It is widely and successfully employed in functional studies and is now emerging as a promising therapeutic approach. Several RNAi-based clinical trials suggest encouraging results in the treatment of a variety of diseases, including cancer. Here we present miR-Synth, a computational resource for the design of synthetic microRNAs able to target multiple genes in multiple sites. The proposed strategy constitutes a valid alternative to the use of siRNA, allowing the employment of a fewer number of molecules for the inhibition of multiple targets. This may represent a great advantage in designing therapies for diseases caused by crucial cellular pathways altered by multiple dysregulated genes. The system has been successfully validated on two of the most prominent genes associated to lung cancer, c-MET and Epidermal Growth Factor Receptor (EGFR). (See http://microrna.osumc.edu/mir-synth).