RESUMO
Issues concerning non-specificity, degradation and hemolysis severely hamper the development of membranolytic amphiphilic peptides into safe and efficient anticancer agents. To increase the therapeutic potential, we have previously developed a strategy based on formulation of the peptides in biocompatible nanosized lipodisks. Studies using melittin as model peptide show that the proteolytic degradation and hemolytic effect of the peptide are substantially reduced upon loading in lipodisks. Here, we explored the possibilities to increase the specificity and boost the cytotoxicity of melittin to tumor cells by use of targeting lipodisk. We demonstrate that small (~20 nm) EGF-targeted lipodisks can be produced and loaded with substantial amounts of peptide (lipid/peptide molar ratio >7) by means of a simple and straightforward preparation protocol. In vitro cell studies confirm specific binding of the peptide-loaded disks to tumor cells and suggest that cellular internalization of the disks results in a significantly improved cell-killing effect.
Assuntos
Antineoplásicos/administração & dosagem , Portadores de Fármacos/química , Sistemas de Liberação de Medicamentos , Meliteno/administração & dosagem , Fosfatidilcolinas/química , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Humanos , Meliteno/farmacocinética , Meliteno/farmacologia , Neoplasias/tratamento farmacológico , Fosfatidiletanolaminas/química , Polietilenoglicóis/químicaRESUMO
Lipodisks are nanosized flat, circular, phospholipid bilayers that are edge-stabilized by polyethylene glycol-conjugated lipids (PEG-lipids). Over the last decade, lipodisks stabilized with PEG of molecular weight 2000 or 5000 have been shown to hold high potential as both biomimetic membranes and drug carriers. In this study we investigate the possibilities to optimize the properties of the lipodisks, and widen their applicability, by reducing the PEG molecular weight and/or the density of the PEG corona. Results obtained by cryo-transmission electron microscopy and dynamic light scattering show that stable, well-defined lipodisks can be produced from mixtures of distearoylphosphatidylcholine (DSPC) and distearoylphosphatidylethanolamine conjugated to PEG of molecular weight 1000 (DSPE-PEG1000). Preparations based on the use of DSPE-PEG750 tend, in contrast, to be polydisperse in size and structure. By comparing immobilization of lipodisks stabilized with DSPE-PEG1000, DSPE-PEG2000, and DSPE-PEG5000 to porous and smooth silica surfaces, we show that the amount of surface bound disks can be considerably improved by the use of PEG-lipids with reduced molecular weight. Further, a modified preparation protocol that enables production of lipodisks with very low PEG-lipid content is described. The reduced PEG density, which facilitates the incorporation of externally added ligand-linked PEG-lipids, is shown to be beneficial for the production of targeting lipodisks.
Assuntos
Portadores de Fármacos , Fator de Crescimento Epidérmico/química , Receptores ErbB/metabolismo , Bicamadas Lipídicas/química , Polietilenoglicóis/química , Linhagem Celular Tumoral , Fator de Crescimento Epidérmico/metabolismo , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Receptores ErbB/genética , Expressão Gênica , Humanos , Radioisótopos do Iodo/química , Terapia de Alvo Molecular , Peso Molecular , Fosfatidilcolinas/química , Fosfatidiletanolaminas/química , Porosidade , Ligação Proteica , Dióxido de Silício/química , Relação Estrutura-AtividadeRESUMO
Pax3 plays a role in regulating Hes1 and Neurog2 activity and thereby stem cell maintenance and neurogenesis. A mechanism for Pax3 regulation of these two opposing events, during caudal neural tube development, is examined in this study. Pax3 acetylation on C-terminal lysine residues K437 and K475 may be critical for proper regulation of Hes1 and Neurog2. Removal of these lysine residues increased Hes1 but decreased Neurog2 promoter activity. SIRT1 deacetylase may be a key component in regulating Pax3 acetylation. Chromatin immunoprecipitation assays showed that SIRT1 is associated with Hes1 and Neurog2 promoters during murine embryonic caudal neural tube development at E9.5, but not at E12.5. Overexpression of SIRT1 decreased Pax3 acetylation, Neurog2 and Brn3a positive staining. Conversely, siRNA-mediated silencing of SIRT1 increased these factors. These studies suggest that Pax3 acetylation results in decreased Hes1 and increased Neurog2 activity, thereby promoting sensory neuron differentiation.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas de Homeodomínio/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurogênese/genética , Fatores de Transcrição Box Pareados/metabolismo , Células Receptoras Sensoriais/metabolismo , Sirtuína 1/genética , Sirtuína 1/metabolismo , Acetilação , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina/métodos , Proteínas de Homeodomínio/genética , Humanos , Camundongos , Proteínas do Tecido Nervoso/genética , Tubo Neural/metabolismo , Fator de Transcrição PAX3 , Fatores de Transcrição Box Pareados/genética , Regiões Promotoras Genéticas/genética , Processamento de Proteína Pós-Traducional/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Fatores de Transcrição HES-1RESUMO
Affibody molecules are a class of small (ca. 7 kDa) robust scaffold proteins suitable for radionuclide molecular imaging of therapeutic targets in vivo. A hexahistidine tag at the N-terminus streamlines development of new imaging probes by enabling facile purification using immobilized metal ion affinity chromatography (IMAC), as well as convenient [99(m)Tc(CO)3](+)-labeling. However, previous studies in mice have demonstrated that Affibody molecules labeled by this method yield higher liver accumulation of radioactivity, compared to the same tracer lacking the hexahistidine tag and labeled by an alternative method. Two variants of the HER2-binding Affibody molecule Z(HER)2(:)342 were made in an attempt to create a tagged tracer that could be purified by immobilized metal affinity chromatography, yet would not result in anomalous hepatic radioactivity accumulation following labeling with [99(m)Tc(CO)3](+). In one construct, the hexahistidine tag was moved to the C-terminus. In the other construct, every second histidine residue in the hexahistidine tag was replaced by the more hydrophilic glutamate, resulting in a HEHEHE-tag. Both variants, denoted Z(HER)2(:)342-H6 and (HE)3-Z(HER)2(:)342, respectively, could be efficiently purified using IMAC and stably labeled with [99(m)Tc(CO)3](+) and were subsequently compared with the parental H6-Z(HER)2(:)342 having an N-terminal hexahistidine tag. All three variants were demonstrated to specifically bind to HER2-expressing cells in vitro. The hepatic accumulation of radioactivity in a murine model was 2-fold lower with [99(m)Tc(CO)3](+)-Z(HER2:342)-H6 compared to [99(m)Tc(CO)3](+)-H6-Z(HER)2(:)342, and more than 10-fold lower with [99(m)Tc(CO)3](+)-(HE)3-Z(HER)2(:)342. These differences translated into appreciably superior tumor-to-liver ratio for [99(m)Tc(CO)3](+)-(HE)3-Z(HER)2(:)342 compared to the alternative conjugates. This information might be useful for development of other scaffold-based molecular imaging probes.
Assuntos
Histidina/química , Fígado/metabolismo , Oligopeptídeos/química , Compostos de Organotecnécio/química , Compostos de Organotecnécio/metabolismo , Compostos Radiofarmacêuticos/química , Compostos Radiofarmacêuticos/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Animais , Linhagem Celular Tumoral , Cromatografia de Afinidade , Humanos , Marcação por Isótopo , Masculino , Camundongos , Camundongos Endogâmicos , Compostos de Organotecnécio/farmacocinética , Compostos Radiofarmacêuticos/farmacocinética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/farmacocinética , Distribuição TecidualRESUMO
INTRODUCTION: Molecular imaging of human epidermal growth factor receptor type 2 (HER2)-expression in malignant tumors provides potentially important information for patient management. Affibody molecules have shown to be suitable tracers for imaging applications using single photon emission computed tomography or positron emission tomography. Results from an earlier evaluation of the application of site-specific (99m)Tc-labeling of the Affibody molecule, Z(HER2:2395)-C, were favorable. METHODS: As a preparation for clinical application of this tracer, we have developed and evaluated a robust single-vial freeze-dried kit, allowing labeling of the Affibody molecule, Z(HER2:2395)-C, with (99m)Tc. RESULTS: The composition of the kit [containing glucoheptonate, EDTA and tin(II)-chloride], as well as the protein amount and the pertechnetate volume were optimized for a high labeling yield (>90%) and minimal presence of reduced hydrolyzed technetium colloids (<1%). The specificity to HER2 receptors, the binding competence and the stability in phosphate-buffered saline and murine serum were verified in vitro. The shelf-life was also evaluated in vitro, showing no reduction in labeling yield or binding capacity to HER2-expressing cells after over 400 days of storage of the single-vial freeze-dried kit. CONCLUSIONS: Z(HER2:2395)-C labeled with (99m)Tc using the lyophilized kit was stable and resulted in a favorable biodistribution in an in vivo evaluation in normal Naval Medical Research Institute mice.
Assuntos
Anticorpos/química , Anticorpos/imunologia , Cisteína/metabolismo , Marcação por Isótopo/métodos , Compostos de Organotecnécio/química , Engenharia de Proteínas , Receptor ErbB-2/imunologia , Animais , Anticorpos/genética , Anticorpos/metabolismo , Linhagem Celular Tumoral , Cisteína/química , Cisteína/genética , Ácido Edético/química , Feminino , Liofilização , Humanos , Camundongos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/farmacocinética , Reprodutibilidade dos Testes , Pertecnetato Tc 99m de Sódio/química , Açúcares Ácidos/química , Fatores de Tempo , Compostos de Estanho/químicaRESUMO
UNLABELLED: Overexpression of the human epidermal growth factor receptor type 2 (HER2) in breast carcinomas predicts response to trastuzumab therapy. Affibody molecules based on a nonimmunoglobulin scaffold have demonstrated a high potential for in vivo molecular imaging of HER2-expressing tumors. The reengineering of the molecular scaffold has led to a second generation of optimized Affibody molecules that have a surface distinctly different from the parental protein domain from staphylococcal protein A. Compared with the parental molecule, the new tracer showed a further increased melting point, stability, and overall hydrophilicity and was more amenable to chemical peptide synthesis. The goal of this study was to assess the potential effects of this extensive reengineering on HER2 targeting, using ABY-025, a DOTA-conjugated variant of the novel tracer. METHODS: (111)In-ABY-025 was compared with previously evaluated parent HER2-binding Affibody tracers in vitro and in vivo. The in vivo behavior was further evaluated in mice bearing SKOV-3 xenografts, rats, and cynomolgus macaques (Macaca fascicularis). RESULTS: (111)In-ABY-025 bound specifically to HER2 in vitro and in vivo. Direct comparison with the previous generation of HER2-binding tracers showed that ABY-025 retained excellent targeting properties. Rapid blood clearance was shown in mice, rats, and macaques. A highly specific tumor uptake of 16.7 +/- 2.5 percentage injected activity per gram of tissue was seen at 4 h after injection. The tumor-to-blood ratio was 6.3 at 0.5 h and 88 at 4 h and increased up to 3 d after injection. gamma-camera imaging of tumors was already possible at 0.5 h after injection. Furthermore, the repeated intravenous administration of ABY-025 did not induce antibody formation in rats. CONCLUSION: The biodistribution of (111)In-ABY-025 was in remarkably good agreement with the parent tracers, despite profound reengineering of the nonbinding surface. The molecule displayed rapid blood clearance in all species investigated and excellent targeting capacity in tumor-bearing mice, leading to high tumor-to-organ-ratios and high-contrast imaging shortly after injection.
Assuntos
Neoplasias da Mama/diagnóstico por imagem , Neoplasias da Mama/metabolismo , Fragmentos de Peptídeos , Compostos Radiofarmacêuticos , Receptor ErbB-2/metabolismo , Proteína Estafilocócica A , Sequência de Aminoácidos , Aminoácidos/química , Animais , Ensaio de Imunoadsorção Enzimática , Feminino , Câmaras gama , Humanos , Imunoquímica , Radioisótopos de Índio , Marcação por Isótopo , Macaca fascicularis , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Transplante de Neoplasias , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacocinética , Cintilografia , Compostos Radiofarmacêuticos/química , Compostos Radiofarmacêuticos/farmacocinética , Ratos , Ratos Sprague-Dawley , Receptor ErbB-2/genética , Proteína Estafilocócica A/química , Distribuição TecidualRESUMO
The current way to increase efficacy of cancer therapy is the use of molecular recognition of aberrantly expressed gene products for selective treatment. However, only a fraction of the patients have tumors with a particular molecular target. Radionuclide imaging of molecular targets might help to stratify patient for cancer treatment. Affibody molecules are scaffold proteins, which can be selected for high affinity recognition of proteinaceous molecular targets. The capacity to re-fold under physiological conditions allows labeling of Affibody molecules in a broad range of pH and temperatures with preserved binding properties. Peptide synthesis or introduction of a unique cysteine enables site-specific labeling of Affibody molecules, resulting in uniform conjugates with well-defined pharmacological characteristics. The small size (7 kDa) of Affibody molecules provides rapid extravasation, rapid tumor penetration, and rapid clearance of unbound tracer from healthy organs and tissues. In combination with sub-nanomolar affinity, this results in high contrast in vivo imaging a few hours after injection. Excellent targeting has been demonstrated in pre-clinical studies with HER2-targeting Affibody molecules labeled with (99m)Tc and (111)In for single photon computed tomography (SPECT), and (18)F, (64)Cu, (124)I and (68)Ga for positron emission tomography (PET). Pilot clinical data confirm the high potential of Affibody molecules.
Assuntos
Imagem Molecular/métodos , Neoplasias/diagnóstico por imagem , Tomografia por Emissão de Pósitrons/métodos , Radioisótopos , Compostos Radiofarmacêuticos , Proteínas Recombinantes de Fusão , Humanos , Aumento da Imagem/métodos , Marcação por Isótopo/métodosRESUMO
BACKGROUND: The Hedgehog (Hh)-signaling pathway plays a crucial role in the development and maintenance of multiple vertebrate and invertebrate organ systems. Gli transcription factors are regulated by Hh-signaling and act as downstream effectors of the pathway to activate Hh-target genes. Understanding the requirements for Hh-signaling in organisms can be gained by assessing Gli activity in a spatial and temporal fashion. METHODOLOGY/PRINCIPAL FINDINGS: We have generated a Gli-dependent (Gli-d) transgenic line, Tg(Gli-d:mCherry), that allows for rapid and simple detection of Hh-responding cell populations in both live and fixed zebrafish. This transgenic line expresses a mCherry reporter under the control of a Gli responsive promoter, which can be followed by using fluorescent microscopy and in situ hybridization. Expression of the mCherry transgene reporter during embryogenesis and early larval development faithfully replicated known expression domains of Hh-signaling in zebrafish, and abrogating Hh-signaling in transgenic fish resulted in the suppression of reporter expression. Moreover, ectopic shh expression in Tg(Glid:mCherry) fish led to increased transgene production. Using this transgenic line we investigated the nature of Hh-pathway response during early craniofacial development and determined that the neural crest skeletal precursors do not directly respond to Hh-signaling prior to 48 hours post fertilization, suggesting that earlier requirements for pathway activation in this population of facial skeleton precursors are indirect. CONCLUSION/SIGNIFICANCE: We have determined that early Hh-signaling requirements in craniofacial development are indirect. We further demonstrate the Tg(Gli-d:mCherry) fish are a highly useful tool for studying Hh-signaling dependent processes during embryogenesis and larval stages.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Proteínas Oncogênicas/metabolismo , Transativadores/metabolismo , Animais , Animais Geneticamente Modificados , Condrócitos/citologia , Condrogênese , Cruzamentos Genéticos , Epitélio/metabolismo , Genes Reporter , Técnicas Genéticas , Proteínas Hedgehog/metabolismo , Modelos Biológicos , Transdução de Sinais , Crânio/metabolismo , Peixe-Zebra , Proteína GLI1 em Dedos de ZincoRESUMO
PURPOSE: Imaging using positron emission tomography (PET) in the field of nuclear medicine is becoming increasingly important. The aim of this study was to develop a method for labeling of affibody molecules with radiocobalt for PET applications. PROCEDURES: The human epidermal growth factor receptors type 2 (HER2) binding affibody molecule DOTA-Z(2395)-C was radiolabeled with (57)Co (used as a surrogate of (55)Co). The binding specificity and cellular processing of the labeled compound was studied in vitro followed by in vivo characterization in normal and tumor-bearing mice. Furthermore, a comparative biodistribution study was performed with a (111)In-labeled counterpart. RESULTS: DOTA-Z(2395)-C was successfully labeled with radiocobalt with nearly quantitative yield. The compound displayed good retention on cells over time and high tumor accumulation of radioactivity in animal studies. Imaging studies showed clear visualization of HER2-positive tumors. Furthermore, the radiocobalt label provided better tumor-to-organ ratios than (111)In. CONCLUSIONS: Radiocobalt is a promising label for affibody molecules for future PET applications.
Assuntos
Neoplasias/diagnóstico por imagem , Receptor ErbB-2/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Animais , Linhagem Celular Tumoral , Radioisótopos de Cobalto , Câmaras gama , Humanos , Camundongos , Camundongos Nus , Cintilografia , Distribuição Tecidual , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: The purpose of this study was to design and evaluate a novel platform for labelling of Affibody molecules, enabling both recombinant and synthetic production and site-specific labelling with (99m)Tc or trivalent radiometals. METHODS: The HER2-specific Affibody molecule PEP05352 was made by peptide synthesis. The chelator sequence SECG (serine-glutamic acid-cysteine-glycine) was anchored on the C-terminal to allow (99m)Tc labelling. The cysteine can alternatively serve as a conjugation site of the chelator DOTA for indium labelling. The resulting (99m)Tc- and (111)In-labelled Affibody molecules were evaluated both in vitro and in vivo. RESULTS: Both conjugates retained their capacity to bind to HER2 receptors in vitro and in vivo. The tumour to blood ratio in LS174T xenografts was 30 at 4 h post-injection for both conjugates. Biodistribution data showed that the (99m)Tc-labelled Affibody molecule had a fourfold lower kidney accumulation compared with the (111)In-labelled Affibody molecule while the accumulation in other organs was similar. Gamma camera imaging of the conjugates could clearly visualise the tumours 4 h after injection. CONCLUSION: Incorporation of the C-terminal SECG sequence in Affibody molecules provides a general multifunctional platform for site-specific labelling with different nuclides (technetium, indium, gallium, cobalt or yttrium) and for a flexible production (chemical synthesis or recombinant).
Assuntos
Desenho de Fármacos , Receptor ErbB-2/metabolismo , Proteínas Recombinantes de Fusão/síntese química , Proteínas Recombinantes de Fusão/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Feminino , Humanos , Marcação por Isótopo , Cinética , Camundongos , Imagem Molecular , Dados de Sequência Molecular , Neoplasias/diagnóstico por imagem , Neoplasias/metabolismo , Traçadores Radioativos , Cintilografia , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/farmacocinética , Especificidade por Substrato , Distribuição TecidualRESUMO
UNLABELLED: The detection of human epidermal growth factor receptor type 2 (HER2) expression in malignant tumors provides important information influencing patient management. Radionuclide in vivo imaging of HER2 may permit the detection of HER2 in both primary tumors and metastases by a single noninvasive procedure. Small (7 kDa) high-affinity anti-HER2 Affibody molecules may be suitable tracers for SPECT visualization of HER2-expressing tumors. The use of generator-produced (99m)Tc as a label would facilitate the prompt translation of anti-HER2 Affibody molecules into use in clinics. METHODS: A C-terminal cysteine was introduced into the Affibody molecule Z(HER2:342) to enable site-specific labeling with (99m)Tc. Two recombinant variants, His(6)-Z(HER2:342)-Cys (dissociation constant [K(D)], 29 pM) and Z(HER2:2395)-Cys, lacking a His tag (K(D), 27 pM), were labeled with (99m)Tc in yields exceeding 90%. The binding specificity and the cellular processing of Affibody molecules were studied in vitro. Biodistribution and gamma-camera imaging studies were performed in mice bearing HER2-expressing xenografts. RESULTS: (99m)Tc-His(6)-Z(HER2:342)-Cys was capable of targeting HER2-expressing SKOV-3 xenografts in SCID mice, but the liver radioactivity uptake was high. A series of comparative biodistribution experiments indicated that the presence of the His tag caused elevated accumulation in the liver. (99m)Tc-Z(HER2:2395)-Cys, not containing a His tag, showed low uptake in the liver and high and specific uptake in HER2-expressing xenografts. Four hours after injection, the radioactivity uptake values (percentage of injected activity per gram of tissue [%IA/g]) were 6.9 +/- 2.5 (mean +/- SD) %IA/g in LS174T xenografts (moderate level of HER2 expression) and 15 +/- 3 %IA/g in SKOV-3 xenografts (high level of HER2 expression). The corresponding tumor-to-blood ratios were 88 +/- 24 and 121 +/- 24, respectively. Both LS174T and SKOV-3 xenografts were clearly visualized with a clinical gamma-camera 1 h after injection of (99m)Tc-Z(HER2:2395)-Cys. CONCLUSION: The Affibody molecule (99m)Tc-Z(HER2:2395)-Cys is a promising tracer for SPECT visualization of HER2-expressing tumors.
Assuntos
Adenocarcinoma/diagnóstico por imagem , Adenocarcinoma/metabolismo , Cisteína , Técnicas de Sonda Molecular , Compostos de Organotecnécio/farmacocinética , Receptor ErbB-2/metabolismo , Proteínas Recombinantes de Fusão/farmacocinética , Tomografia Computadorizada de Emissão de Fóton Único/métodos , Animais , Cisteína/farmacocinética , Sistemas de Liberação de Medicamentos/métodos , Feminino , Taxa de Depuração Metabólica , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Camundongos SCID , Especificidade de Órgãos , Compostos Radiofarmacêuticos/farmacocinética , Proteínas Recombinantes/farmacocinética , Distribuição TecidualRESUMO
Affibody molecules are a new class of small targeting proteins based on a common three-helix bundle structure. Affibody molecules binding a desired target may be selected using phage-display technology. An Affibody molecule Z HER2:342 binding with subnanomolar affinity to the tumor antigen HER2 has recently been developed for radionuclide imaging in vivo. Introduction of a single cysteine into the cysteine-free Affibody scaffold provides a unique thiol group for site-specific labeling of recombinant Affibody molecules. The recently developed maleimido-CHX-A'' DTPA was site-specifically conjugated at the C-terminal cysteine of Z HER2:2395-C, a variant of Z HER2:342, providing a homogeneous conjugate with a dissociation constant of 56 pM. The yield of labeling with (111)In was >99% after 10 min at room temperature. In vitro cell tests demonstrated specific binding of (111)In-CHX-A'' DTPA-Z 2395-C to HER2-expressing cell-line SKOV-3 and good cellular retention of radioactivity. In normal mice, the conjugate demonstrated rapid clearance from all nonspecific organs except kidney. In mice bearing SKOV-3 xenografts, the tumor uptake of (111)In-CHX-A'' DTPA-Z 2395-C was 17.3 +/- 4.8% IA/g and the tumor-to-blood ratio 86 +/- 46 (4 h postinjection). HER2-expressing xenografts were clearly visualized 1 h postinjection. In conclusion, coupling of maleimido-CHX-A'' DTPA to cysteine-containing Affibody molecules provides a well-defined uniform conjugate, which can be rapidly labeled at room temperature and provides high-contrast imaging of molecular targets in vivo.
Assuntos
Isotiocianatos/química , Isotiocianatos/metabolismo , Maleimidas/química , Ácido Pentético/análogos & derivados , Proteínas Recombinantes de Fusão/metabolismo , Coloração e Rotulagem/métodos , Animais , Anticorpos/imunologia , Sítios de Ligação , Linhagem Celular Tumoral , Quelantes/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Compostos Heterocíclicos com 1 Anel/metabolismo , Humanos , Radioisótopos de Índio , Camundongos , Ácido Pentético/química , Ácido Pentético/metabolismo , Receptor ErbB-2/imunologia , Receptor ErbB-2/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/farmacocinética , Especificidade por Substrato , Distribuição TecidualRESUMO
Affibody molecules are a new class of small (7 kDa) scaffold affinity proteins, which demonstrate promising properties as agents for in vivo radionuclide targeting. The Affibody scaffold is cysteine-free and therefore independent of disulfide bonds. Thus, a single thiol group can be engineered into the protein by introduction of one cysteine. Coupling of thiol-reactive bifunctional chelators can enable site-specific labeling of recombinantly produced Affibody molecules. In this study, the use of 1,4,7,10-tetraazacyclododecane-1,4,7-tris-acetic acid-10-maleimidoethylacetamide (MMA-DOTA) for 111 In-labeling of anti-HER2 Affibody molecules His 6-Z HER2:342-Cys and Z HER2:2395-Cys has been evaluated. The introduction of a cysteine residue did not affect the affinity of the proteins, which was 29 pM for His 6-Z HER2:342-Cys and 27 pM for Z HER2:2395-Cys, comparable with 22 pM for the parental Z HER2:342. MMA-DOTA was conjugated to DTT-reduced Affibody molecules with a coupling efficiency of 93% using a 1:1 molar ratio of chelator to protein. The conjugates were labeled with 111 In to a specific radioactivity of up to 7 GBq/mmol, with preserved binding for the target HER2. In vivo, the non-His-tagged variant 111 In-[MMA-DOTA-Cys61]-Z HER2:2395-Cys demonstrated appreciably lower liver uptake than its His-tag-containing counterpart. In mice bearing HER2-expressing LS174T xenografts, 111 In-[MMA-DOTA-Cys61]-Z HER2:2395-Cys showed specific and rapid tumor localization, and rapid clearance from blood and nonspecific compartments, leading to a tumor-to-blood-ratio of 18 +/- 8 already 1 h p.i. Four hours p.i., the tumor-to-blood ratio was 138 +/- 8. Xenografts were clearly visualized already 1 h p.i.
Assuntos
Compostos Heterocíclicos com 1 Anel/química , Maleimidas/química , Peptídeos/química , Proteínas Recombinantes de Fusão/química , Animais , Sítios de Ligação , Quelantes/química , Cisteína/metabolismo , Histidina/metabolismo , Radioisótopos de Índio , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias/diagnóstico por imagem , Cintilografia , Receptor ErbB-2/metabolismo , Sensibilidade e Especificidade , Transplante HeterólogoRESUMO
The winged helix transcription factor FoxG1 (Bf-1, qin) plays multiple roles in the development of the telencephalon, with different parts of the protein affecting either proliferation or differentiation. We examined the consequences of over-expression, via retroviral expression, of FoxG1 on the growth of different regions of the chicken brain. Excess expression of FoxG1 caused a thickening of the neuroepithelium, and ultimately large outgrowths of the telencephalon and mesencephalon. In contrast, the myelencephalon appeared unaffected, exhibiting normal apoptosis and growth characteristics. A DNA binding defective form of FoxG1 did not exhibit these abnormalities, suggesting that these effects are due to FoxG1's function as a transcriptional repressor. To examine the means by which excess FoxG1 caused overgrowth of the brain, we examined alterations in cell proliferation and death. No increase in proliferation was noted in any portion of the neural tube, rather a significant decrease in neuroepithelial apoptosis was seen. These results demonstrate a previously unrecognized role for winged helix factors in the regulation of neural cell apoptosis.
Assuntos
Apoptose/fisiologia , Encéfalo/embriologia , Proteínas de Ligação a DNA/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Defeitos do Tubo Neural/genética , Animais , Encéfalo/virologia , Divisão Celular/genética , Embrião de Galinha , Desenvolvimento Embrionário e Fetal , Regulação da Expressão Gênica no Desenvolvimento , Hiperplasia/genética , Hibridização In Situ , Defeitos do Tubo Neural/virologia , RetroviridaeRESUMO
Alcohol is a teratogen that induces a variety of abnormalities including brain and facial defects [Jones, K. & Smith, D. (1973) Lancet 2, 999-1001], with the exact nature of the deficit depending on the time and magnitude of the dose of ethanol to which developing fetuses are exposed. In addition to abnormal facial structures, ethanol-treated embryos exhibit a highly characteristic pattern of cell death. Dying cells are observed in the premigratory and migratory neural crest cells that normally populate most facial structures. The observation that blocking Sonic hedgehog (Shh) signaling results in similar craniofacial abnormalities prompted us to examine whether there was a link between this aspect of fetal alcohol syndrome and loss of Shh. We demonstrate that administration of ethanol to chick embryos results in a dramatic loss of Shh, as well as a loss of transcripts involved in Shh signaling pathways. In contrast, other signaling molecules examined do not demonstrate such dramatic changes. Furthermore, we demonstrate that both the ethanol-induced cranial neural crest cell death and the associated craniofacial growth defect can be rescued by application of Shh. These data suggest that craniofacial anomalies resulting from fetal alcohol exposure are caused at least partially by loss of Shh and subsequent neural crest cell death.