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1.
Mol Pharm ; 15(3): 759-767, 2018 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-29384380

RESUMO

The most promising F508del-CFTR corrector, VX-809, has been unsuccessful as an effective, stand-alone treatment for CF patients, but the rescue effect in combination with other drugs may confer an acceptable level of therapeutic benefit. Targeting cellular factors that modify trafficking may act to enhance the cell surface density of F508-CFTR with VX-809 correction. Our goal is to identify druggable kinases that enhance F508del-CFTR rescue and stabilization at the cell surface beyond that achievable with the VX-809 corrector alone. To achieve this goal, we implemented a new high-throughput screening paradigm that quickly and quantitatively measures surface density and total protein in the same cells. This allowed for rapid screening for increased surface targeting and proteostatic regulation. The assay utilizes fluorogen-activating-protein (FAP) technology with cell excluded and cell permeant fluorogenic dyes in a quick, wash-free fluorescent plate reader format on live cells to first measure F508del-CFTR expressed on the surface and then the total amount of F508del-CFTR protein present. To screen for kinase targets, we used Dharmacon's ON-TARGET plus SMARTpool siRNA Kinase library (715 target kinases) with and without 10 µM VX-809 treatment in triplicate at 37 °C. We identified several targets that had a significant interaction with VX-809 treatment in enhancing surface density with siRNA knockdown. Select small-molecule inhibitors of the kinase targets demonstrated augmented surface expression with VX-809 treatment.


Assuntos
Aminopiridinas/farmacologia , Benzodioxóis/farmacologia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/tratamento farmacológico , Ensaios de Triagem em Larga Escala/métodos , Fosfotransferases/genética , Inibidores de Proteínas Quinases/farmacologia , Aminopiridinas/uso terapêutico , Benzodioxóis/uso terapêutico , Membrana Celular/metabolismo , Fibrose Cística/genética , Fibrose Cística/terapia , Avaliação Pré-Clínica de Medicamentos , Sinergismo Farmacológico , Quimioterapia Combinada/métodos , Citometria de Fluxo , Corantes Fluorescentes/química , Técnicas de Silenciamento de Genes/métodos , Células HEK293 , Humanos , Mutação , Fosfotransferases/antagonistas & inibidores , Fosfotransferases/metabolismo , Inibidores de Proteínas Quinases/uso terapêutico , RNA Interferente Pequeno/metabolismo , Resultado do Tratamento
2.
Cytometry A ; 83(2): 220-6, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23303704

RESUMO

Monitoring the trafficking of multiple proteins simultaneously in live cells is of great interest because many receptor proteins are found to function together with others in the same cell. However, existing fluorescent labeling techniques have restricted the mechanistic study of functional receptor pairs. We have expanded a hybrid system combining fluorogen-activating protein (FAP) technology and high-throughput flow cytometry to a new type of biosensor that is robust, sensitive, and versatile. This provides the opportunity to study multiple trafficking proteins in the same cell. Human beta2 adrenergic receptor (ß2AR) fused with FAP AM2.2 and murine C-C chemokines receptor type 5 fused with FAP MG13 was chosen for our model system. The function of the receptor and the binding between MG13 and fluorogen MG-2p have been characterized by flow cytometry and confocal microscopy assays. The binding of fluorogen and the FAP pair is highly specific, while both FAP-tagged fusion proteins function similarly to their wild-type counterparts. The system has successfully served as a counter screen assay to eliminate false positive compounds identified in a screen against NIH Molecular Libraries Small Molecule Repository targeting regulators of the human ß2AR.


Assuntos
Técnicas Biossensoriais/métodos , Corantes Fluorescentes/química , Ensaios de Triagem em Larga Escala , Proteínas Recombinantes de Fusão/química , Agonistas de Receptores Adrenérgicos beta 2/farmacologia , Quimiocina CCL5/farmacologia , Relação Dose-Resposta a Droga , Citometria de Fluxo/métodos , Humanos , Concentração Inibidora 50 , Isoproterenol/farmacologia , Ligação Proteica , Receptores Adrenérgicos beta 2/biossíntese , Receptores CCR5/agonistas , Receptores CCR5/biossíntese , Proteínas Recombinantes de Fusão/biossíntese , Corantes de Rosanilina/química , Células U937
3.
Methods ; 57(3): 308-17, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22366230

RESUMO

The use of fluorescent proteins, particularly when genetically fused to proteins of biological interest, have greatly advanced many flow cytometry research applications. However, there remains a major limitation to this methodology in that only total cellular fluorescence is measured. Commonly used fluorescent proteins (e.g., EGFP and its variants) are fluorescent whether the fusion protein exists on the surface or in sub-cellular compartments. A flow cytometer cannot distinguish between these separate sources of fluorescence. This can be of great concern when using flow cytometry, plate readers or microscopy to quantify cell surface receptors or other surface proteins genetically fused to fluorescent proteins. Recently developed fluorogen activating proteins (FAPs) solve many of these issues by allowing the selective visualization of only those cell surface proteins that are exposed to the extracellular milieu. FAPs are GFP-sized single chain antibodies that specifically bind to and generate fluorescence from otherwise non-fluorescent dyes ('activate the fluorogen'). Like the fluorescent proteins, FAPs can be genetically fused to proteins of interest. When exogenously added fluorogens bind FAPs, fluorescence immediately increases by as much as 20,000-fold, rendering the FAP fusion proteins highly fluorescent. Moreover, since fluorogens can be made membrane impermeant, fluorescence can be limited to only those receptors expressed on the cell surface. Using cells expressing beta-2 adrenergic receptor (ß2AR) fused at its N-terminus to a FAP, flow cytometry based receptor internalization assays have been developed and characterized. The fluorogen/FAP system is ideally suited to the study of cell surface proteins by fluorescence and avoids drawbacks of using receptor/fluorescent protein fusions, such as internal accumulation. We also briefly comment on extending FAP-based technologies to the study of events occurring inside of the cell as well.


Assuntos
Receptores Adrenérgicos beta 2/metabolismo , Anticorpos de Cadeia Única/metabolismo , Antagonistas de Receptores Adrenérgicos beta 2/farmacologia , Agonistas Adrenérgicos beta/farmacologia , Animais , Bioensaio , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Citometria de Fluxo , Fluorescência , Corantes Fluorescentes , Proteínas de Fluorescência Verde , Humanos , Isoproterenol/farmacologia , Cinética , Camundongos , Propranolol/farmacologia , Ligação Proteica , Receptores Adrenérgicos beta 2/química , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/metabolismo , Anticorpos de Cadeia Única/química
4.
Mol Pharmacol ; 82(4): 645-57, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22767611

RESUMO

We developed a platform combining fluorogen-activating protein (FAP) technology with high-throughput flow cytometry to detect real-time protein trafficking to and from the plasma membrane in living cells. The hybrid platform facilitates drug discovery for trafficking receptors such as G protein-coupled receptors and was validated with the ß2-adrenergic receptor (ß2AR) system. When a chemical library containing ∼1200 off-patent drugs was screened against cells expressing FAP-tagged ß2ARs, all 33 known ß2AR-active ligands in the library were successfully identified, together with a number of compounds that might regulate receptor internalization in a nontraditional manner. Results indicated that the platform identified ligands of target proteins regardless of the associated signaling pathway; therefore, this approach presents opportunities to search for biased receptor modulators and is suitable for screening of multiplexed targets for improved efficiency. The results revealed that ligands may be biased with respect to the rate or duration of receptor internalization and that receptor internalization may be independent of activation of the mitogen-activated protein kinase pathway.


Assuntos
Ensaios de Triagem em Larga Escala/métodos , Receptores Adrenérgicos beta 2/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Agonistas de Receptores Adrenérgicos beta 2/farmacologia , Antagonistas de Receptores Adrenérgicos beta 2/farmacologia , Ligação Competitiva , Citometria de Fluxo/métodos , Proteínas de Fluorescência Verde/genética , Humanos , Ligantes , Transporte Proteico , Receptores Adrenérgicos beta 2/genética , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores Acoplados a Proteínas G/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Tempo , Transfecção , Células U937
5.
Cytometry A ; 77(8): 776-82, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20653017

RESUMO

This study explores the general utility of a new class of biosensor that allows one to selectively visualize molecules of a chosen membrane protein that are at the cell surface. These biosensors make use of recently described bipartite fluoromodules comprised of a fluorogen-activating protein (FAP) and a small molecule (fluorogen) whose fluorescence increases dramatically when noncovalently bound by the FAP (Szent-Gyorgyi et al., Nat Biotechnol 2010;00:000-000).


Assuntos
Técnicas Biossensoriais/métodos , Corantes Fluorescentes/metabolismo , Proteínas de Membrana/metabolismo , Agonistas de Receptores Adrenérgicos beta 2 , Animais , Membrana Celular/metabolismo , Sobrevivência Celular , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Endocitose , Corantes Fluorescentes/química , Transportador de Glucose Tipo 4/metabolismo , Humanos , Proteínas de Membrana/química , Camundongos , Microscopia de Fluorescência , Células NIH 3T3 , Estrutura Terciária de Proteína , Receptores Adrenérgicos beta 2/metabolismo
6.
Biomaterials ; 27(10): 2213-21, 2006 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-16325254

RESUMO

Immobilized patterns of unmodified fibroblast growth factor-2 (FGF-2), with varying surface concentrations, were inkjet printed onto physiologically relevant fibrin substrates. Printed patterns were characterized using iodinated FGF-2 to determine FGF-2 surface concentration and retention of FGF-2 binding in vitro. MG-63 cells were uniformly seeded onto patterned substrates. Cells were exposed to defined spatial FGF-2 surface concentrations of 1-22 pg/mm(2). Cell numbers were observed to increase in register with the printed FGF-2 patterns from an initial random uniform cell distribution across the patterned and non-patterned regions. Based on time-lapse image analysis, the primary organizational response of the cells was determined to be proliferation and not migration. Cell counts on and off the FGF-2 patterns over time demonstrated an increase in cell density up to a FGF-2 surface concentration of 14 pg/mm(2). Higher surface concentrations did not result in increased cell density. In addition, the cells on the FGF-2 patterns survived longer than the cells off patterns. Our inkjet printing approach permits the systematic study of cellular responses to defined spatial surface concentrations of immobilized growth factors.


Assuntos
Proliferação de Células/efeitos dos fármacos , Fibrina/metabolismo , Fator 2 de Crescimento de Fibroblastos/farmacologia , Impressão/métodos , Carbocianinas/metabolismo , Linhagem Celular , Relação Dose-Resposta a Droga , Corantes Fluorescentes/metabolismo , Humanos , Propriedades de Superfície
7.
J Biomol Screen ; 21(1): 74-87, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26442911

RESUMO

A new class of biosensors, fluorogen activating proteins (FAPs), has been successfully used to track receptor trafficking in live cells. Unlike the traditional fluorescent proteins (FPs), FAPs do not fluoresce unless bound to their specific small-molecule fluorogens, and thus FAP-based assays are highly sensitive. Application of the FAP-based assay for protein trafficking in high-throughput flow cytometry resulted in the discovery of a new class of compounds that interferes with the binding between fluorogens and FAP, thus blocking the fluorescence signal. These compounds are high-affinity, nonfluorescent analogs of fluorogens with little or no toxicity to the tested cells and no apparent interference with the normal function of FAP-tagged receptors. The most potent compound among these, N,4-dimethyl-N-(2-oxo-2-(4-(pyridin-2-yl)piperazin-1-yl)ethyl)benzenesulfonamide (ML342), has been investigated in detail. X-ray crystallographic analysis revealed that ML342 competes with the fluorogen, sulfonated thiazole orange coupled to diethylene glycol diamine (TO1-2p), for the same binding site on a FAP, AM2.2. Kinetic analysis shows that the FAP-fluorogen interaction is more complex than a homogeneous one-site binding process, with multiple conformational states of the fluorogen and/or the FAP, and possible dimerization of the FAP moiety involved in the process.


Assuntos
Ligação Proteica/efeitos dos fármacos , Proteínas/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Bioensaio/métodos , Técnicas Biossensoriais , Linhagem Celular Tumoral , Fluorescência , Corantes Fluorescentes/metabolismo , Humanos , Cinética , Transporte Proteico/efeitos dos fármacos , Células U937
8.
Biomaterials ; 26(33): 6762-70, 2005 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-15941581

RESUMO

The purpose of this study was to initiate the exploration of cell behavioral responses to inkjet printed spatial patterns of hormones biologically immobilized on biomimetic substrates. This approach was investigated using the example of preosteoblastic cell response in vitro to fibroblast growth factor-2 (FGF-2) printed on fibrin films. Concentration modulated patterns of FGF-2, including continuous concentration gradients, were created by overprinting dilute FGF-2 bioinks with a custom inkjet printer. The immobilized FGF-2 was biologically active and the printed patterns persisted up to 10 days under cell culture conditions. Cell numbers increased in register to printed patterns from an initial random uniform cell distribution across the patterned and non-patterned fibrin substrate. Patterned immobilized FGF-2, not cell attachment directed cell organization because the fibrin substrate was homogeneous. The capability to engineer arbitrary and persistent hormone patterns is relevant to basic studies across various fields including developmental biology and tissue regeneration. Furthermore, since this hormone inkjet printing methodology is extensible to create complex three-dimensional structures, this methodology has potential to create therapies for tissue engineering using spatial patterned delivery of exogenous hormones.


Assuntos
Fibrina/química , Fator 2 de Crescimento de Fibroblastos/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Células Cultivadas , Relação Dose-Resposta a Droga , Humanos , Processamento de Imagem Assistida por Computador , Osteoblastos/metabolismo , Especificidade por Substrato , Fatores de Tempo , Engenharia Tecidual/métodos
9.
J Biomol Screen ; 19(8): 1220-6, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24820110

RESUMO

G protein-coupled receptors (GPCRs) play stimulatory or modulatory roles in numerous physiological states and processes, including growth and development, vision, taste and olfaction, behavior and learning, emotion and mood, inflammation, and autonomic functions such as blood pressure, heart rate, and digestion. GPCRs constitute the largest protein superfamily in the human and are the largest target class for prescription drugs, yet most are poorly characterized, and of the more than 350 nonolfactory human GPCRs, over 100 are orphans for which no endogenous ligand has yet been convincingly identified. We here describe new live-cell assays that use recombinant GPCRs to quantify two general features of GPCR cell biology-receptor desensitization and resensitization. The assays employ a fluorogen-activating protein (FAP) reporter that reversibly complexes with either of two soluble organic molecules (fluorogens) whose fluorescence is strongly enhanced when complexed with the FAP. Both assays require no wash or cleanup steps and are readily performed in microwell plates, making them adaptable to high-throughput drug discovery applications.


Assuntos
Ensaios de Triagem em Larga Escala/métodos , Receptores Acoplados a Proteínas G/metabolismo , Linhagem Celular , Relação Dose-Resposta a Droga , Corantes Fluorescentes/metabolismo , Células HEK293 , Ensaios de Triagem em Larga Escala/instrumentação , Humanos , Ligantes , Microscopia de Fluorescência , Receptores Adrenérgicos alfa 2/genética , Receptores Adrenérgicos alfa 2/metabolismo , Receptores Adrenérgicos beta 1/genética , Receptores Adrenérgicos beta 1/metabolismo , Receptores Adrenérgicos beta 2/genética , Receptores Adrenérgicos beta 2/metabolismo , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores Acoplados a Proteínas G/genética
10.
J Biomol Screen ; 15(6): 703-9, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20488980

RESUMO

Ligand-dependent receptor internalization is a feature of numerous signaling systems. In this article, the authors describe a new kind of live-cell biosensor of receptor internalization that takes advantage of fluorogen-activating protein (FAP) technology. Recombinant genes that express the human beta2 adrenergic receptor (beta2AR) with FAP domains at their extracellular N-termini were transduced into mammalian cells. Exposure of the cells to membrane-impermeant fluorogens led to a strong fluorescent signal from the cell surface. Agonist-dependent translocation of the receptor from the surface to the cell interior was readily observed and quantified by fluorescence microscopy or flow cytometry in a homogeneous format without wash or separation steps. The approach described here is generalizable to other receptors and cell surface proteins and is adaptable to a variety of fluorescence-based high-throughput screening platforms.


Assuntos
Técnicas Biossensoriais/métodos , Endocitose , Corantes Fluorescentes/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Agonistas de Receptores Adrenérgicos beta 2 , Antagonistas de Receptores Adrenérgicos beta 2 , Animais , Bioensaio , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Endocitose/efeitos dos fármacos , Fluorescência , Humanos , Isoproterenol/farmacologia , Cinética , Camundongos , Células NIH 3T3 , Propranolol/farmacologia , Coloração e Rotulagem , Propriedades de Superfície/efeitos dos fármacos , Fatores de Tempo
11.
Comb Chem High Throughput Screen ; 12(6): 604-18, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19601758

RESUMO

Current methods for engineering immobilized, 'solid-phase' growth factor patterns have not addressed the need for presentation of the growth factors in a biologically-relevant context. We developed an inkjet printing methodology for creating solid-phase patterns of unmodified growth factors on native biological material substrates. We demonstrate this approach by printing gradients of fluorescently labeled bone morphogenetic protein-2 (BMP-2) and insulin-like growth factor-II (IGF-II) bio-inks on fibrin-coated surfaces. Concentration gradients were created by overprinting individual substrate locations using a dilute bio-ink to modulate the surface concentration of deposited growth factor. Persistence studies using fluorescently-labeled BMP-2 verified that the gradients retained their shape for up to 7 days. Desorption experiments performed with (125)I-BMP-2 and (125)I-IGF-II were used to quantify the surface concentration of growth factor retained on the substrate for up to 10 days in serum containing media after rinsing of the unbound growth factor. The inkjet method is programmable so the gradient shape can be easily modified as demonstrated by printed linear gradients with varying slopes and exponential gradients. In addition, the versatility of this method enabled combinatorial arrays of multiple growth factors to be created by printing overlapping patterns. The overlapping printing method was used to create a combinatorial square pattern array consisting of various surface concentrations of BMP-2 and fibroblast growth factor-2 (FGF-2). C2C12 myogenic precursor cells were seeded on the arrays and alkaline phosphatase staining was performed to determine the effect of FGF-2 and BMP-2 surface concentration on guiding C2C12 cells towards an osteogenic lineage. These results demonstrate the utility of inkjet printing for creating orthogonal growth factor gradients to investigate how combinations of immobilized growth factors influence cell fate.


Assuntos
Proteína Morfogenética Óssea 2/análise , Técnicas de Química Combinatória , Enzimas Imobilizadas/química , Fator 2 de Crescimento de Fibroblastos/análise , Impressão , Engenharia Tecidual , Animais , Linhagem Celular Tumoral , Substâncias de Crescimento/análise , Humanos , Camundongos , Impressão/métodos , Propriedades de Superfície
12.
Nat Biotechnol ; 26(2): 235-40, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18157118

RESUMO

Imaging of live cells has been revolutionized by genetically encoded fluorescent probes, most famously green and other fluorescent proteins, but also peptide tags that bind exogenous fluorophores. We report here the development of protein reporters that generate fluorescence from otherwise dark molecules (fluorogens). Eight unique fluorogen activating proteins (FAPs) have been isolated by screening a library of human single-chain antibodies (scFvs) using derivatives of thiazole orange and malachite green. When displayed on yeast or mammalian cell surfaces, these FAPs bind fluorogens with nanomolar affinity, increasing green or red fluorescence thousands-fold to brightness levels typical of fluorescent proteins. Spectral variation can be generated by combining different FAPs and fluorogen derivatives. Visualization of FAPs on the cell surface or within the secretory apparatus of mammalian cells can be achieved by choosing membrane permeant or impermeant fluorogens. The FAP technique is extensible to a wide variety of nonfluorescent dyes.


Assuntos
Anticorpos Monoclonais , Corantes Fluorescentes , Genes Reporter , Proteínas de Membrana/metabolismo , Microscopia de Fluorescência/métodos , Técnicas de Sonda Molecular , Fragmentos de Imunoglobulinas , Proteínas de Membrana/ultraestrutura
13.
J Am Chem Soc ; 129(7): 2025-34, 2007 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-17256855

RESUMO

Fluorescence detection and imaging are vital technologies in the life sciences and clinical diagnostics. The key to obtaining high-resolution images and sensitive detection is to use fluorescent molecules or particles that absorb and emit visible light with high efficiency. We have synthesized supramolecular complexes consisting of a branched DNA template and fluorogenic intercalating dyes. Because dyes can intercalate up to every other base pair, high densities of fluorophores are assembled yet the DNA template keeps them far enough away from each other to prevent self-quenching. The efficiency with which these noncovalent assemblies absorb light is more than 10-fold greater than that of the individual dye molecules. Förster resonance energy transfer from the intercalated dyes to covalently attached acceptor dyes is very efficient, allowing for wavelength shifting of the emission spectrum. Simple biotinylation of the DNA template allows for labeling of streptavidin-coated synthetic microspheres and mouse T-cells.


Assuntos
DNA/química , Corantes Fluorescentes/química , Substâncias Intercalantes/química , Nanopartículas/química , Animais , Membrana Celular/química , Cricetinae , Citometria de Fluxo , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/síntese química , Substâncias Intercalantes/síntese química , Camundongos , Camundongos Endogâmicos BALB C , Linfócitos T/química , Linfócitos T/citologia
14.
Microvasc Res ; 73(2): 75-83, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17070560

RESUMO

Quantum dots (QDs) are fluorescent semiconductor nanocrystals that possess a number of superior fluorescent properties compared to more established organic dyes and fluorescent proteins. As a result, QDs are being studied for use in a wide range of biological applications. We have examined QDs for one such application, visualization of blood vessels of the chick chorioallantoic membrane (CAM), a popular model for studying various aspects of blood vessel development including angiogenesis. Intravitally injected QDs were found to be biocompatible and were kept in circulation over the course of 4 days without any observed deleterious effects. QD vascular residence time was tunable through QD surface chemistry modification. We also found that use of QDs with higher emission wavelengths (>655 nm) virtually eliminated all chick-derived autofluorescence and improved depth-of-field imaging. QDs were compared to FITC-dextrans, a fluorescent dye commonly used for imaging CAM vessels. QDs were found to image vessels as well as or better than FITC-dextrans at 2-3 orders of magnitude lower concentration. We also demonstrated that QDs are fixable with low fluorescence loss and thus can be used in conjunction with histological processing for further sample analysis.


Assuntos
Alantoide/irrigação sanguínea , Córion/irrigação sanguínea , Pontos Quânticos , Animais , Embrião de Galinha , Dextranos , Fluoresceína-5-Isotiocianato/análogos & derivados , Corantes Fluorescentes , Microscopia Eletrônica , Microscopia de Fluorescência , Neovascularização Fisiológica , Fatores de Tempo
15.
Mol Med ; 8(2): 103-12, 2002 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-12080186

RESUMO

BACKGROUND: Two prominent biological features of the advanced stages of human melanoma are their high degree of vascularity and high-level expression of basic fibroblast growth factor (bFGF) and fibroblast growth factor receptor-1 (FGFR-1). Given these characteristics, human melanoma serves as an ideal model to address an important question regarding the efficacy of angiogenesis-based cancer therapy. To induce tumor growth arrest and regression, does it suffice to block expression of bFGF and/or FGFR-1 in only the melanoma cells, or is it essential to inhibit expression of bFGF and/or FGFR-1 in both the melanoma cells and the melanoma cell-interspersing vasculature? MATERIALS AND METHODS: Primary and metastatic human melanomas, grown as subcutaneous tumors in nude mice, were injected twice a week with vector constructs containing the human tyrosinase promoter and antisense- oriented human bFGF or FGFR-1 cDNA. On alternating days, the bFGF and FGFR-1 antisense-targeted tumors received injections of cyanine fluorochrome-conjugated antibodies to a human melanoma and mouse blood vessel marker. Noninvasive, dynamic fluorescence imaging was used to document the cellular events that took place inside the tumors as the result of blocking expression of bFGF or FGFR-1 in the melanoma cells. RESULTS: In vivo, ex vivo, and in vitro fluorescence imaging of the bFGF and FGFR-1 antisense-targeted tumors demonstrated that inhibiting bFGF and FGFR-1 signaling in only the melanoma cells suffices to inhibit tumor growth due to massive induction of melanoma cell apoptosis. CONCLUSIONS: The investigations presented in this study document that inhibiting expression of bFGF or FGFR-1 in only the melanoma cells is as effective in blocking tumor growth as simultaneously inhibiting bFGF or FGFR-1 synthesis in the melanoma cells and the melanoma cell-interspersing vasculature. Furthermore, blocking expression of bFGF or FGFR-1 in the melanoma cells did not lead to activation or increased production of another angiogenic molecule, suggesting the absence of a "salvage pathway" that can circumvent or rescue the blockage of bFGF/FGFR-1 in the melanoma cells.


Assuntos
Apoptose , Fator 2 de Crescimento de Fibroblastos/metabolismo , Melanoma/metabolismo , Melanoma/patologia , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Animais , Vírus do Sarcoma Aviário/genética , Divisão Celular , Fatores de Crescimento Endotelial/análise , Fator 2 de Crescimento de Fibroblastos/análise , Fator 2 de Crescimento de Fibroblastos/biossíntese , Fator 2 de Crescimento de Fibroblastos/genética , Imunofluorescência , Humanos , Injeções , Linfocinas/análise , Melanoma/genética , Camundongos , Camundongos Nus , Monofenol Mono-Oxigenase/genética , Oligonucleotídeos Antissenso/administração & dosagem , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/metabolismo , Regiões Promotoras Genéticas/genética , Receptores Proteína Tirosina Quinases/análise , Receptores Proteína Tirosina Quinases/biossíntese , Receptores Proteína Tirosina Quinases/genética , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos , Receptores de Fatores de Crescimento de Fibroblastos/análise , Receptores de Fatores de Crescimento de Fibroblastos/biossíntese , Receptores de Fatores de Crescimento de Fibroblastos/genética , Células Tumorais Cultivadas , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio Vascular
16.
J Am Chem Soc ; 125(23): 6878-9, 2003 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-12783535

RESUMO

Incorporation of a guanidine functional group into the PNA backbone facilitates cellular uptake of PNA into mammalian cells with efficiency comparable to that of the TAT transduction domain. The modified PNA recognizes and binds to the complementary DNA strand in accordance with Watson-Crick recognition rules. However, unlike polypyrimidine PNA which binds to DNA in 2:1 stoichiometry, the modified PNA binds to complementary DNA in a 1:1 ratio to form a highly stable duplex.


Assuntos
DNA/metabolismo , Guanidina/metabolismo , Ácidos Nucleicos Peptídicos/metabolismo , Dicroísmo Circular , DNA/química , Guanidina/química , Guanidina/farmacocinética , Humanos , Ácidos Nucleicos Peptídicos/química , Ácidos Nucleicos Peptídicos/farmacocinética , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta , Células Tumorais Cultivadas
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