RESUMO
Neuropilin 1 (NRP1), a non-tyrosine kinase receptor for vascular endothelial growth factor and class 3 Semaphorins, is highly expressed in many human tumour cell lines, but its function is poorly understood. Here, we describe the expression of a new chondroitin sulphate-modified NRP1 (NRP1-CS) in human tumour cell lines. Expression of a non-modifiable NRP1 mutant (S612A) in U87MG human glioma cells results in enhanced invasion in three dimensions (3D), whereas wild-type NRP1 has no effect. Furthermore, the S612A NRP1 cells show a significant increase in p130Cas tyrosine phosphorylation compared with control and wild-type NRP1 cells. Silencing of p130Cas in S612A NRP1 cells resulted in a loss of increased invasive phenotype. Interestingly, p130Cas silencing does not inhibit basal 3D invasion, but leads to a mesenchymal to amoeboid transition. Biopsies from both low- and high-grade human gliomas show strong expression of NRP1, and little expression of NRP1-CS. Our data establish distinct roles for NRP1 and NRP1-CS in modulating a new NRP1-p130Cas signalling pathway contributing to glioblastoma cell invasion in 3D.
Assuntos
Sulfatos de Condroitina/fisiologia , Proteína Substrato Associada a Crk/fisiologia , Glioblastoma/metabolismo , Glioblastoma/patologia , Neuropilina-1/genética , Transdução de Sinais/fisiologia , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Glioblastoma/genética , Humanos , Camundongos , Dados de Sequência Molecular , Invasividade Neoplásica , Neuropilina-1/biossíntese , Interferência de RNA , Ratos , SuínosRESUMO
OBJECTIVES: We sought to magnetically tag endothelial progenitor cells (EPCs) with a clinical agent and target them to a site of arterial injury using a magnetic device positioned outside the body. BACKGROUND: Circulating EPCs are involved in physiological processes such as vascular re-endothelialization and post-ischemic neovascularization. However, the success of cell therapies depends on the ability to deliver the cells to the site of injury. METHODS: Human EPCs were labeled with iron oxide superparamagnetic nanoparticles. Cell viability and differentiation were tested using flow cytometry. Following finite element modeling computer simulations and flow testing in vitro, angioplasty was performed on rat common carotid arteries to denude the endothelium and EPCs were administered with and without the presence of an external magnetic device for 12 min. RESULTS: Computer simulations indicated successful external magnetic cell targeting from a vessel with flow rate similar to a rat common carotid artery; correspondingly there was a 6-fold increase in cell capture in an in vitro flow system. Targeting enhanced cell retention at the site of injury by 5-fold at 24 h after implantation in vivo. CONCLUSIONS: Using an externally applied magnetic device, we have been able to enhance EPC localization at a site of common carotid artery injury. This technology could be more widely adapted to localize cells in other organs and may provide a useful tool for the systemic injection of cell therapies.
Assuntos
Lesões das Artérias Carótidas/cirurgia , Movimento Celular , Células Endoteliais/transplante , Óxido Ferroso-Férrico , Magnetismo , Coloração e Rotulagem/métodos , Transplante de Células-Tronco/métodos , Células-Tronco , Antígeno AC133 , Animais , Antígenos CD/análise , Apoptose , Lesões das Artérias Carótidas/patologia , Diferenciação Celular , Sobrevivência Celular , Células Cultivadas , Simulação por Computador , Dextranos , Modelos Animais de Doenças , Células Endoteliais/imunologia , Estudos de Viabilidade , Análise de Elementos Finitos , Citometria de Fluxo , Glicoproteínas/análise , Humanos , Nanopartículas de Magnetita , Masculino , Microscopia Confocal , Modelos Cardiovasculares , Nanopartículas , Peptídeos/análise , Projetos Piloto , Ratos , Ratos Sprague-Dawley , Células-Tronco/imunologia , Fatores de TempoRESUMO
A novel, stable, biotin aldehyde derivative is reported in which the biotin moiety is N1,N3-protected by the allyloxycarbonyl group. The derivative is stable to sodium cyanoborohydride mediated reductive alkylation and is cleaved under mild Pd [0] catalysis. This novel biotin aldehyde should have wide application in avidin- and streptavidin-based detection systems and bioassays. The derivative is utilized in the synthesis of a biotinylated doxorubicin analogue that retains topoisomerase activity.
Assuntos
Marcadores de Afinidade/química , Aldeídos/química , Biotinilação , Doxorrubicina/química , Alquilação , DNA Topoisomerases Tipo II/efeitos dos fármacos , Doxorrubicina/análogos & derivados , Doxorrubicina/farmacologiaRESUMO
Adenovirus is a widely used vector in gene transfer experiments because it produces high transduction efficiency in vitro and in vivo by means of the coxsackie-adenovirus receptor (CAR) and major histocompatibility complex (MHC) class I alpha-2 domain. Adenoviral gene transfer efficiency has been reported to correlate with cellular CAR expression. We report here a simple method to increase adenoviral gene transfer efficiency in cells that do not express high levels of CAR: preincubation of adenovirus for 30-40 minutes at +37 degrees C significantly increased the transduction efficiency in vitro in CHO and BALB/3T3 cells, in which CAR is expressed at very low levels. Increased transduction efficiency of preincubated adenovirus was also detected in vivo in rat brain tissue. In addition, we found that adenoviruses were rapidly inactivated in human serum in a complement-independent manner, whereas fetal bovine serum (FBS) had hardly any effects on the viral infectivity. We conclude that preincubation of adenoviral vectors at +37 degrees C may substantially increase gene transfer efficiency in applications in which target cells do not express high levels of CAR.
Assuntos
Adenoviridae/genética , Técnicas de Transferência de Genes , Temperatura Alta , Células 3T3 , Animais , Células CHO , Cricetinae , Proteínas de Fluorescência Verde , Proteínas Luminescentes/genética , Camundongos , Camundongos Endogâmicos BALB C , Transdução GenéticaRESUMO
We have developed an ex vivo gene transfer technique to rabbit arterial wall using autologous smooth muscle cells (SMCs). SMCs were harvested from rabbit ear artery, transduced in vitro with vesicular stomatitis virus G-glycoprotein pseudotyped retrovirus or feline immunodeficiency virus (FIV) and returned to the adventitial surface of the carotid artery using a periadventitial silicone collar or collagen sheet placed around the artery. Beta-galactosidase (lacZ) and human apolipoprotein E3 (apoE3) cDNAs were used as transgenes. After retrovirus-mediated gene transfer of lacZ the selected cells implanted with high efficiency and expressed lacZ marker gene at a very high level 7 and 14 days after the operation. The level of lacZ expression decreased thereafter but was still detectable 12 weeks after the gene transfer, and was exclusively localized to the site of cell implantation inside the collar. Utilizing FIV vector expressing apoE3, low levels of apoE were measured from serum collected from a low-density lipoprotein receptor deficient Watanabe heritable hyperlipidemic rabbits 1 month after the gene transfer. The physiological effect of apoE expression was detected as transiently elevated serum cholesterol levels. The results indicate that the model can be used for high efficiency local gene transfer in arteries, e.g. during vascular surgery. The model is also valuable for studying expression, stability and safety of new gene transfer vectors and their expression products in vivo.
Assuntos
Vetores Genéticos , Vírus da Imunodeficiência Felina , Miócitos de Músculo Liso/fisiologia , Transdução Genética , Animais , Apolipoproteína E3 , Apolipoproteínas E/genética , Artérias Carótidas/citologia , Artérias Carótidas/fisiologia , Gatos , Terapia Genética/métodos , Humanos , Glicoproteínas de Membrana/genética , Miócitos de Músculo Liso/transplante , Coelhos , Transdução Genética/métodos , Transplante Autólogo , Doenças Vasculares/terapia , Procedimentos Cirúrgicos Vasculares , Proteínas do Envelope Viral/genética , beta-Galactosidase/genéticaRESUMO
We have constructed a novel fusion protein "Scavidin" consisting of the macrophage scavenger receptor class A and avidin. The Scavidin fusion protein is transported to plasma membranes where the avidin portion of the fusion protein binds biotin with high affinity and forms the basis for the targeted delivery of biotinylated molecules. Subcellular fractionation analysis, immunostaining, and electron microscopy demonstrated endosomal localization of the fusion protein. According to pulse-labeling and cross-linking studies Scavidin is found as monomers (55 kDa), dimers, and multimers, of which the 220-kDa form was the most abundant. The biotin binding capacity and active endocytosis of the biotinylated ligands were demonstrated in rat malignant glioma. Local Scavidin gene transfer to target tissues could have general utility as a universal tool to deliver biotinylated molecules at systemic low concentrations for therapeutic and imaging purposes, whereby high local concentration is achieved.
Assuntos
Avidina/química , Biotinilação , Membrana Celular/metabolismo , Receptores Imunológicos/química , Proteínas Recombinantes de Fusão/química , Animais , Avidina/genética , Avidina/metabolismo , Northern Blotting , Western Blotting , Clonagem Molecular , Reagentes de Ligações Cruzadas/farmacologia , Dimerização , Endocitose , Técnicas de Transferência de Genes , Vetores Genéticos , Glioma/metabolismo , Imuno-Histoquímica , Ligantes , Microscopia de Fluorescência , Microscopia Imunoeletrônica , Modelos Genéticos , Testes de Precipitina , Ligação Proteica , Estrutura Terciária de Proteína , Ratos , Receptores Imunológicos/genética , Receptores Depuradores , Proteínas Recombinantes de Fusão/genética , Retroviridae/genética , Receptores Depuradores Classe A , Transdução Genética , Células Tumorais CultivadasRESUMO
Flexible alteration of virus surface properties would be beneficial for enhanced and targeted gene delivery. A useful approach could be based on a high-affinity receptor-ligand pair, such as avidin and biotin. In this study, we have constructed an avidin-displaying baculovirus, Baavi. Avidin display was expected to enhance cell transduction due to the high positive charge of avidin in physiological pH and to provide a binding site for covering the virus with desired biotinylated ligands. Successful incorporation of avidin on the virus envelope was detected by immunoblotting and electron microscopy. Multiple biotin-binding sites per virus were detected with fluorescence-correlation spectroscopy and tight biotin binding was observed using an optical biosensor, IAsys. Baavi showed a 5-fold increase in transduction efficiency in rat malignant glioma cells (BT4C) and a 26-fold increase in rabbit aortic smooth muscle (RAASMC) cells compared to wild-type baculovirus. Enhanced transduction was also observed with biotinylated target cells. Biotinylated epidermal growth factor (EGF) enabled specific targeting of the virus with high efficiency to EGF receptor-expressing (SKOV-3) cells. An additional advantage of the avidin display was demonstrated with biotinylated paramagnetic particles, which enabled magnetic targeting. Altogether, we show that avidin display is a rapid and versatile method to improve viral properties for gene delivery.
Assuntos
Avidina/metabolismo , Baculoviridae/genética , Baculoviridae/fisiologia , Técnicas de Transferência de Genes , Animais , Avidina/genética , Técnicas Biossensoriais , Biotina/metabolismo , Biotinilação , Linhagem Celular , Linhagem Celular Tumoral , Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/metabolismo , Vetores Genéticos/genética , Vetores Genéticos/fisiologia , Ligação Proteica , Coelhos , Ratos , Espectrometria de Fluorescência , Transdução Genética , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/metabolismoRESUMO
Baculoviruses are enveloped insect viruses that can carry large quantities of foreign DNA in their genome. Baculoviruses have proved to be very promising gene therapy vectors but little is known about their transduction mechanisms in mammalian cells. We show in this study that Autographa californica multiple nuclear polyhedrosis virus capsid is compatible with the incorporation of desired proteins in large quantities. Fusions can be made to the N-terminus or C-terminus of the major capsid protein vp39 without compromising the viral titer or functionality. As an example of the baculovirus capsid display we show a tracking of the baculovirus transduction in mammalian cells by an enhanced green fluorescent protein (EGFP)-displaying virus. Our confocal and electron microscopy results suggest that the transduction block in mammalian cells is not in the endosomal escape, as previously proposed, but rather in the cytoplasmic transport or nuclear entry of the virus capsid. Our results also suggest that the EGFP-tagged virus can be used for visualization of the virus biodistribution in vivo. Furthermore, capsid-modified baculoviruses hold great promise for the nuclear and subcellular targeting of transgenes and as a novel peptide display system for a variety of eukaryotic applications.