RESUMEN
Understanding the molecular dynamics of DNA replication in vivo has been a formidable challenge requiring the development of advanced technologies. Over the past 50 years or so, studies involving DNA autoradiography in bacterial cells have led to sophisticated DNA tract analyses in human cells to characterize replication dynamics at the single-molecule level. Our own lab has used DNA fiber analysis to characterize replication in helicase-deficient human cells. This work led us to propose a model in which the human DNA helicase RECQ1 acts as a governor of the single-stranded DNA binding protein RPA and regulates its bioavailability for DNA synthesis. We have also used the DNA fiber approach to investigate the interactive role of DDX11 helicase with a replication fork protection protein (Timeless) in human cells when they are under pharmacologically induced stress. In this methods chapter, we present a step-by-step protocol for the single-molecule DNA fiber assay. We describe experimental designs to study replication stress and staining patterns from pulse-chase labeling experiments to address the dynamics of replication forks in stressed cells.
Asunto(s)
Daño del ADN/genética , Replicación del ADN/genética , Imagen Individual de Molécula/métodos , Proteínas de Ciclo Celular/metabolismo , ARN Helicasas DEAD-box/metabolismo , Daño del ADN/efectos de los fármacos , ADN Helicasas/metabolismo , Replicación del ADN/efectos de los fármacos , ADN de Cadena Simple/metabolismo , Desoxiuridina/análogos & derivados , Desoxiuridina/toxicidad , Células HeLa , Humanos , Idoxuridina/análogos & derivados , Idoxuridina/toxicidad , Péptidos y Proteínas de Señalización Intracelular/metabolismo , RecQ Helicasas/metabolismo , Proteína de Replicación A/metabolismoRESUMEN
The regulated proliferation of cells is a critical factor in tumor progression, antineoplastic therapies, immune system regulation, and the cellular developmental of multicellular organisms. While measurement of cell cycle state by fluorescent flow cytometry is well established, mass cytometry allows the cell cycle to be measured along with large numbers of other antigens enabling characterization of the complex interactions between the cell cycle and wide variety of cellular processes. This method describes the use of mass cytometry for the analysis of cell cycle state for cells from three different sources: in vitro cultured cell lines, ex vivo human blood or bone marrow, and in vivo labeling of murine tissues. The method utilizes incorporation of 5-Iodo-2'-deoxyuridine (IdU), combined with measurement of phosphorylated retinoblastoma protein (pRb), Cyclin B1, and phosphorylated Histone H3 (pHH3). These measurements can be integrated into a gating strategy that enables clear separation of all five phases of the cell cycle.
Asunto(s)
Ciclo Celular , Ciclina B1/análisis , Citometría de Flujo/métodos , Histonas/análisis , Espectrometría de Masas/métodos , Proteína de Retinoblastoma/análisis , Coloración y Etiquetado/métodos , Animales , Células de la Médula Ósea/metabolismo , Humanos , Idoxuridina/análogos & derivados , Idoxuridina/metabolismo , Ratones , FosforilaciónRESUMEN
Halogenated nucleosides, such as 5-iodo-2'-deoxyuridine and 5-iodo-2'-deoxycytidine, are incorporated into the DNA of replicating cells to facilitate DNA single-strand breaks and intra- or interstrand crosslinks upon UV irradiation. In this work, it is shown that the naphthyl-based organoselenium compounds can mediate the dehalogenation of halogenated pyrimidine-based nucleosides, such as 5-X-2'-deoxyuridine and 5-X-2'-deoxycytidine (X=Br or I). The rate of deiodination was found to be significantly higher than that of the debromination for both nucleosides. Furthermore, the deiodination of iodo-cytidines was found to be faster than that of iodo-uridines. The initial rates of the deiodinations of 5-iodocytosine and 5-iodouracil indicated that the nature of the sugar moiety influences the kinetics of the deiodination. For both the nucleobases and nucleosides, the deiodination and debromination reactions follow a halogen-bond-mediated and addition/elimination pathway, respectively.
Asunto(s)
Nucleósidos/química , Compuestos de Organoselenio/química , Cristalografía por Rayos X , Halogenación , Idoxuridina/análogos & derivados , Idoxuridina/química , Espectroscopía de Resonancia Magnética , Conformación MolecularRESUMEN
The on-column functionalization of oligodeoxynucleotides via base-free Suzuki cross-coupling reactions is reported herein. These cross-coupling reactions were carried out with various boronic acids and either full-length modified oligonucleotides containing one or more 2'-deoxy-5-iodouridine (5IdU) monomer(s) or on oligonucleotide fragments immediately after incorporation of 5IdU. Five different functionalities were coupled to oligonucleotides containing one or three attachment points.
Asunto(s)
Oligodesoxirribonucleótidos/química , Ácidos Borónicos/química , Catálisis , Idoxuridina/análogos & derivados , Idoxuridina/química , Paladio/químicaRESUMEN
PURPOSE: Glioblastoma multiform (GBM) is the most prevalent and aggressive type of primary brain tumor. None of the current conventional treatment methods has improved treatment considerably. Therefore, in this study the effect of magnetic nanoparticles coated with poly (caprolactone)-poly (ethylene glycol) (PCL-PEG) as a 5-iodo 2'deoxyuridine (IUdR) carrier in the presence of hyperthermia and 6 MV (megavoltage) X-ray radiation, were investigated in a spheroid model of U87MG glioblastoma cell line using colony formation assay. MATERIALS AND METHODS: First, the human glioblastoma cell line U87MG was cultured as a spheroid using the liquid overlay technique. Spheroids on day 10 with 100 mm diameters were treated with 1 µM IUdR or nanoparticles as IUdR carriers for one volume doubling time (VDT) of spheroids (67 h) and hyperthermia at 43 °C for 1 h, and then irradiated with 2 Gy of 6 MV X-ray in different groups. Finally, the effect of treatment on colony-forming ability was obtained by colony formation and alkaline assay. RESULTS: Our results revealed that hyperthermia in combination with radiation could significantly reduce the colony number of glioblastoma spheroid cells treated with IUdR or nanoparticles as IUdR carriers. However, the extent of reduction in colony number following treatment with IUdR-loaded nanoparticles in combination with hyperthermia and then X-ray radiation was significantly more than free IUdR. CONCLUSION: According to this study, PCL-PEG-coated magnetic nanoparticles are effective delivery vehicles for IUdR into cells. Moreover, they can act as a radiosensitizer and thermosensitizer in the treatment of the glioblastoma cell line.
Asunto(s)
Portadores de Fármacos/química , Glioblastoma/patología , Hipertermia Inducida , Idoxuridina/análogos & derivados , Nanopartículas de Magnetita/química , Poliésteres/química , Polietilenglicoles/química , Transporte Biológico , Línea Celular Tumoral , Proliferación Celular/efectos de la radiación , Terapia Combinada , Portadores de Fármacos/metabolismo , Glioblastoma/radioterapia , Humanos , Idoxuridina/química , Tamaño de la Partícula , Esferoides Celulares/patología , Esferoides Celulares/efectos de la radiaciónRESUMEN
Pd-catalyzed C-C bond formation, an important vertebra in the spine of synthetic chemistry, is emerging as a valuable chemoselective transformation for post-synthetic functionalization of biomacromolecules. While methods are available for labeling protein and DNA, development of an analogous procedure to label RNA by cross-coupling reactions remains a major challenge. Herein, we describe a new Pd-mediated RNA oligonucleotide (ON) labeling method that involves post-transcriptional functionalization of iodouridine-labeled RNA transcripts by using Suzuki-Miyaura cross-coupling reaction. 5-Iodouridine triphosphate (IUTP) is efficiently incorporated into RNA ONs at one or more sites by T7 RNA polymerase. Further, using a catalytic system made of Pd(OAc)2 and 2-aminopyrimidine-4,6-diol (ADHP) or dimethylamino-substituted ADHP (DMADHP), we established a modular method to functionalize iodouridine-labeled RNA ONs in the presence of various boronic acid and ester substrates under very mild conditions (37°C and pH 8.5). This method is highly chemoselective, and offers direct access to RNA ONs labeled with commonly used fluorescent and affinity tags and new fluorogenic environment-sensitive nucleoside probes in a ligand-controlled stereoselective fashion. Taken together, this simple approach of generating functional RNA ON probes by Suzuki-Miyaura coupling will be a very important addition to the resources and tools available for analyzing RNA motifs.
Asunto(s)
Oligonucleótidos/química , Sondas ARN/química , ARN/química , Coloración y Etiquetado/métodos , Ácidos Borónicos/química , Catálisis , ARN Polimerasas Dirigidas por ADN/metabolismo , Idoxuridina/análogos & derivados , Idoxuridina/química , Estructura Molecular , Paladio/química , Pirimidinas/química , Proteínas Virales/metabolismoRESUMEN
Glioma is one of the most common malignant cancers of the central nervous system (CNS). Radiatherapy and chemotherapy may be used to slow the growth of tumors that cannot be removed with surgery. The current study developed a combination therapy tool using Nanographene oxide (NGO) functionalized with poly lactic-co-glycolic acid (PLGA) as a carrier of 5-iodo-2-deoxyuridine (IUdR) for glioma cancer treatment. U87MG cells were treated in different groups with IUdR, PLGA-coated Nanographene oxide (PLGA-NGO), IUdR-loaded PLGA-coated Nanographene oxide (IUdR-PLGA-NGO), 2Gy 6MV X-ray radiation, and near-infrared region (NIR) laser radiation. PLGA-NGO showed excellent biocompatibility, high storage capacity for IUdR and high photothermal conversion efficiency. It was effectively employed to create cell damage in the U87MG cell line in the presence of X-ray (6 MV) and NIR laser. Moreover, IUdR-PLGA-NGO+X-ray+NIR laser significantly reduced the plating efficiency of the cells in comparison with IUdR-PLGA-NGO+X-ray and IUdR-PLGA-NGO+NIR laser. Furthermore, Prussian blue staining showed that IUdR-PLGA-NGO-SPIONs were delivered into glioblastoma cells. The PLGA-NGO loaded with IUdR under NIR and X-ray radiation exhibited the highest cytotoxicity toward U87MG cells when compared with other treatment methods, indicating efficient radio-photothermal targeted therapy.
Asunto(s)
Portadores de Fármacos/química , Glioma/terapia , Idoxuridina/análogos & derivados , Terapia por Láser/métodos , Nanopartículas/química , Línea Celular Tumoral , Glioma/tratamiento farmacológico , Glioma/radioterapia , Grafito/química , Humanos , Idoxuridina/administración & dosificación , Idoxuridina/uso terapéutico , Tamaño de la Partícula , Poliésteres/química , Radiación IonizanteRESUMEN
The regulated progression of cells through the cell cycle during proliferation is a critical factor in tumor progression, anti-neoplastic therapy response, immune system regulation, and developmental biology. While flow cytometric measurement of cell cycle progression is well established, mass cytometry assays allow the cell cycle to be measured along with up to 39 other antigens enabling characterization of the complex interactions between the cell cycle and a wide variety of cellular processes. This method describes the use of mass cytometry for the analysis of cell cycle state for cells from three different sources: in vitro cultured cell lines, ex vivo human blood or bone marrow, and in vivo labeling and ex vivo analysis of murine tissues. The method utilizes incorporation of 5-Iodo-2'-deoxyuridine (IdU), combined with measurement of phosphorylated retinoblastoma protein (pRb), cyclin B1, and phosphorylated histone H3 (p-HH3). These measurements can be integrated into a gating strategy that allows for clear separation of all five phases of the cell cycle.
Asunto(s)
Ciclo Celular , Citofotometría/métodos , Citometría de Flujo/métodos , Coloración y Etiquetado/métodos , Animales , Biomarcadores/análisis , Ciclina B1/análisis , Histonas/análisis , Humanos , Idoxuridina/análogos & derivados , Idoxuridina/metabolismo , Ratones , Proteína de Retinoblastoma/análisisRESUMEN
The radiolysis of deoxygenated aqueous solution containing trimeric oligonucleotides labelled with iodinated pyrimidines and Tris-HCl as the hydroxyl radical scavenger leads to electron attachment to the halogenated bases that mainly results in single strand breaks. The iodinated trimers are 2-fold more sensitive to solvated electrons than the brominated oligonucleotides, which is explained by the barrier-free dissociation of the iodinated base anions. The present study fills the literature gap concerning the chemistry triggered by ionizing radiation in the iodinated pyrimidines incorporated into DNA.
Asunto(s)
Oligonucleótidos/química , Oligonucleótidos/efectos de la radiación , Cromatografía Líquida de Alta Presión , ADN de Cadena Simple , Electrones , Radical Hidroxilo , Idoxuridina/análogos & derivados , Idoxuridina/química , Yodo/química , Espectrometría de Masas/métodos , Pirimidinas/química , Radiación IonizanteRESUMEN
We have developed a simple system for the analysis of the affinity of anti-bromodeoxyuridine antibodies. The system is based on the anchored oligonucleotides containing 5-bromo-2'-deoxyuridine (BrdU) at three different positions. It allows a reliable estimation of the reactivity of particular clones of monoclonal anti-bromodeoxyuridine antibodies with BrdU in fixed and permeabilized cells. Using oligonucleotide probes and four different protocols for the detection of BrdU incorporated in cellular DNA, we identified two antibody clones that evinced sufficient reactivity to BrdU in all the tested protocols. One of these clones exhibited higher reactivity to 5-iodo-2'-deoxyuridine (IdU) than to BrdU. It allowed us to increase the sensitivity of the used protocols without a negative effect on the cell physiology as the cytotoxicity of IdU was comparable with BrdU and negligible when compared to 5-ethynyl-2'-deoxyuridine. The combination of IdU and the improved protocol for oxidative degradation of DNA provided a sensitive and reliable approach for the situations when the low degradation of DNA and high BrdU signal is a priority.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Bromodesoxiuridina/metabolismo , ADN/metabolismo , Mapeo Peptídico , Células Clonales , Células HCT116 , Células HeLa , Humanos , Idoxuridina/análogos & derivados , Idoxuridina/metabolismoRESUMEN
An efficient, reliable method for the chemical synthesis of (E)-5-[3-aminoallyl]-uridine-5'-O-triphosphate (AA-UTP), starting from 5-iodouridine, is described. This new strategy features the involvement of one-pot triphosphate formation and fluorous solid-phase extraction (F-SPE). The one-pot synthesis involves the mono phosphorylation of fluorous-tagged uridine, followed by the reaction with pyrophosphate to afford the fluorous-tagged AA-UTP. The F-SPE is achieved by installing a fluorous-tag onto the uridine prior to triphosphate formation, purification via F-SPE, and cleavage of the fluorous-tag. It is worth mentioning that this protocol produces AA-UTP in high yield and purity using one simple F-SPE; no conventional column chromatography is involved.
Asunto(s)
Idoxuridina/análogos & derivados , Extracción en Fase Sólida/métodos , Uridina Trifosfato/síntesis química , Cromatografía , Idoxuridina/síntesis química , Idoxuridina/química , Estructura Molecular , Técnicas de Síntesis en Fase Sólida , Uridina Trifosfato/análogos & derivados , Uridina Trifosfato/químicaRESUMEN
In the search for more selective anticancer drugs, we designed and synthesized seven conjugates varying the structure of the linker connecting the 5-iodo-2'-deoxyuridine (IUdR) to the ICF 01012 melanoma-carrier for potential intratumoural specific drug release. Chemical and in vitro metabolic stability evaluations showed that, except for the ester conjugate (1), the ketal (2b), acetal (2a), carbonate (4) and carbamate (3) conjugates were compatible with our approach. The acetal (2a) and its PEGylated derivative (2c) were of particular interest for further in vivo development owing to their respective pH-dependent stability and limited metabolic degradation in order to exploit the acidic subcellular environment of malignant melanocytes to trigger the release of therapeutics upon internalization in cells.
Asunto(s)
Antineoplásicos/síntesis química , Sistemas de Liberación de Medicamentos , Idoxuridina/análogos & derivados , Melanoma/tratamiento farmacológico , Acetales/síntesis química , Acetales/química , Antineoplásicos/química , Antineoplásicos/uso terapéutico , Células Cultivadas , Estabilidad de Medicamentos , Humanos , Idoxuridina/síntesis química , Idoxuridina/química , Estructura Molecular , Quinoxalinas/químicaRESUMEN
In this unit, an efficient method for the synthesis of alkyne-modified nucleosides in an aqueous solvent system is described. The method allows direct palladium-catalyzed alkynylation of readily available unprotected 8-bromo-2'-deoxyguanosine (8-BrdG), 8-bromo-2'-deoxyadenosene (8-BrdA), 8-bromoadenosine (8-BrA), and 5-iodo-2'-deoxyuridine (5-IdU) precursors. The optimal catalyst is derived from palladium acetate, tri-(2,4-dimethyl-5-sulfonatophenyl)phosphane (TXPTS), and CuI.
Asunto(s)
Alquinos/síntesis química , Bencenosulfonatos/química , Fosfinas/química , Nucleósidos de Purina/síntesis química , Nucleósidos de Pirimidina/síntesis química , Catálisis , Cobre/química , Desoxiguanosina/análogos & derivados , Desoxiguanosina/química , Idoxuridina/análogos & derivados , Idoxuridina/química , Yoduros/química , Modelos Moleculares , Paladio/químicaRESUMEN
Lentiviral vector containing the HSV1-tk and firefly luciferase (fLuc) gene was infected into C6 and C6-TL expressing HSV1-tk and fLuc gene was generated. C6-TL showed higher [(125)I]IVDU uptake than C6. The survival rate of C6-TL decreased more rapidly with increasing GCV dose and was well correlated with fLuc activity. The images of microPET clearly demonstrated higher uptake of [(18)F]FHBG into the C6-TL tumor. Inhibition of tumor growth was observed in C6-TL tumor-bearing mice treated with GCV through tumor size measurement and bioluminescence imaging. The therapeutic effect of HSV1-tk/GCV system can be monitored using bioluminescent imaging and tumor size measurement.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias Encefálicas/terapia , Ganciclovir/farmacología , Genes Transgénicos Suicidas , Terapia Genética/métodos , Glioblastoma/terapia , Herpesvirus Humano 1/enzimología , Mediciones Luminiscentes , Timidina Quinasa/biosíntesis , Animales , Neoplasias Encefálicas/enzimología , Neoplasias Encefálicas/genética , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Femenino , Ganciclovir/metabolismo , Genes Reporteros , Vectores Genéticos/genética , Glioblastoma/enzimología , Glioblastoma/genética , Herpesvirus Humano 1/genética , Idoxuridina/análogos & derivados , Idoxuridina/metabolismo , Cinética , Lentivirus/genética , Luciferasas de Luciérnaga/biosíntesis , Luciferasas de Luciérnaga/genética , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Tomografía de Emisión de Positrones , Ratas , Proteínas Recombinantes de Fusión/metabolismo , Timidina Quinasa/genética , Carga Tumoral/efectos de los fármacosRESUMEN
The aim of the present work was to study the role of DNA synthesis in the formation of different types of memory in neonatal chicks. The nucleotide analogs 5'-iodo-2'-deoxyuridine (IdU) and 5'-bromo-2'-deoxyuridine (BrdU) were used; these are incorporated into DNA, impairing its function, and have amnestic actions in defined models of learning in mice. We studied the effects of 5'-iodo-2'-deoxyuridine of the formation of long-term memory in chicks during training in different models: passive avoidance, imprinting, taste aversion, and spatial learning in a maze. In the taste aversion model, i.p. administration of IdU (10 mg/kg 5 min before or 50 min after training) had an amnestic effect on testing 1-2 days after training. IdU-induced amnesia developed more than 6 h after training, while administration of IdU 2 h after training had no amnestic effect. 5'-Bromo-2'-deoxyuridine also had a similar amnestic action in the taste aversion model. In the passive avoidance, imprinting, and spatial maze learning models, administration of IdU at the same dose before and after training did not induce amnesia. These data lead to the suggestion that DNA synthesis in the brain may play a critical role in the mechanisms of memory consolidation in chicks in types of learning such as taste aversion.
Asunto(s)
ADN/biosíntesis , Idoxuridina/análogos & derivados , Trastornos de la Memoria/fisiopatología , Memoria/fisiología , Amnesia Retrógrada/fisiopatología , Análisis de Varianza , Animales , Reacción de Prevención/fisiología , Encéfalo/fisiopatología , Bromodesoxiuridina/farmacología , Pollos , Idoxuridina/farmacología , Inmunohistoquímica , Impronta Psicológica/fisiología , Aprendizaje/fisiología , Pruebas Neuropsicológicas , Fotomicrografía , Percepción Espacial/fisiología , Percepción del Gusto/fisiología , Factores de TiempoRESUMEN
Novel transition metal catalysts based on oligonucleotides can be easily obtained by functionalization of 5-iodouridine with phosphine ligands, resulting in good asymmetric induction in palladium catalyzed allylic nucleophilic substitution.
Asunto(s)
ADN Catalítico/síntesis química , Idoxuridina/análogos & derivados , Oligonucleótidos/síntesis química , Paladio/química , Fosfinas/análisis , Catálisis , Cristalografía , ADN Catalítico/química , Idoxuridina/química , Ligandos , Oligonucleótidos/química , Elementos de Transición/químicaRESUMEN
INTRODUCTION: 3'-O-(3-Benzenesulfonylfuroxan-4-yl)-5-iodo-2'-deoxyuridine (1) is a cytotoxic nitric oxide (NO) donor-nucleoside dual action prodrug designed to exploit both NO and an antimetabolite nucleoside for cancer therapy. METHODS: 1 was radiolabeled by radioiodide exchange and purified by HPLC in 16% overall radiochemical yield. The specific activity of [(125)I]1 was 31.8 microCi/mug (680 MBq/microM). Protein binding, deiodination, cellular uptake and incorporation of 1 into cellular nucleic acids were measured by standard methods, and its in vivo biodistribution was determined in Balb/c mice bearing implanted EMT-6 tumors following intravenous injection. RESULTS: [(125)I]1 degraded rapidly during the in vitro tests, thus impeding unequivocal assessment but indicating that it was only weakly protein bound and that it was resistant to deiodination under test conditions. Uptake of [(125)I]1 by murine tumor cells (KBALB and KBALB-STK) in vitro was low (approximately 17 fmol/microg protein over 2 h) with only approximately 0.3% (0.04-0.06 fmol/microg protein) of total uptake present in the DNA fraction. In the murine tumor model, liver, kidney, intestine and tumor showed the greatest uptake, with liver, intestine and blood all containing >5 injected dose per gram of tissue (%ID/g) during the 15-min to 2-h postinjection period. Maximum tissue/blood level ratios were 3.6 (2 h) for tumor and 6.4 (24 h) for liver. Low uptake in thyroid and stomach was indicative of minimal in vivo deiodination. CONCLUSIONS: [(125)I]1 undergoes only minimal deoiodination under both in vitro and in vivo conditions. Under conditions of the in vitro NO release assay, 1 reacts to produce a single, major, unstable adduct that decomposes upon workup. Protein binding of [(125)I]1 could not be assessed because of similar chemical reaction with albumin. Incorporation of radioactivity into the cellular nucleic acid fraction was low, and in vivo distribution of [(125)I]1 was consistent with nonspecific reactivity towards tissue nucleophiles. The chemical reactivity of [(125)I]1 mitigates against its use as a NO donor and as a tracer for this class of compounds.
Asunto(s)
Idoxuridina/análogos & derivados , Donantes de Óxido Nítrico/síntesis química , Donantes de Óxido Nítrico/farmacocinética , Oxadiazoles/síntesis química , Oxadiazoles/farmacocinética , Sarcoma Experimental/metabolismo , Animales , Idoxuridina/síntesis química , Idoxuridina/farmacocinética , Radioisótopos de Yodo , Marcaje Isotópico , Masculino , Ratones , Ratones Endogámicos BALB C , Tomografía de Emisión de Positrones , Radioinmunodetección , Sarcoma Experimental/diagnóstico por imagen , Células Tumorales CultivadasRESUMEN
Elucidation of the three-dimensional (3D) structures of the two sequential active sites in spliceosomes is essential for understanding the mechanism of premessenger RNA splicing. The mechanism is predicted to be catalyzed by the small nuclear RNA (snRNA) components of spliceosomes. To obtain new tertiary constraints between the RNA components, we produced and mapped crosslinks between U6 snRNA and the proximal RNAs of active yeast spliceosomes ("yeast" in this report is Saccharomyces cerevisiae). Thus, specific sites in U6, when substituted with a photoreactive 4-thiouridine or 5-iodouridine, produced spliceosome-dependent crosslinks to U2 snRNA, or in one case, to the pre-mRNA substrate. One set of U2-U6 crosslinks formed before the Prp2p-dependent step of spliceosome assembly, whereas another set formed during or after this step but before the first chemical step of splicing. This latter set of crosslinks formed across U2-U6 helix I. Importantly, this set provides new tertiary constraints for developing 3D models of fully assembled yeast spliceosomes, which are poised for the first chemical step of splicing.
Asunto(s)
Idoxuridina/análogos & derivados , Conformación de Ácido Nucleico , Empalme del ARN/genética , ARN Nuclear Pequeño/genética , Empalmosomas/genética , Adenosina Trifosfato/metabolismo , ARN Helicasas DEAD-box , Idoxuridina/metabolismo , Empalme del ARN/fisiología , ARN Nuclear Pequeño/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
5-Iodo-2'-deoxyuridine (IUdR) is an effective radiosensitiser but its clinical development has been limited by toxicity. Prolonged intravenous infusions of IUdR are necessary for optimal tumour uptake but cause dose-limiting myelosuppression. The lack of selective tumour uptake can lead to radiosensitisation of adjacent normal tissues and enhanced local radiation toxicity. Liposomal IUdR delivery offers selective targeting of tumour tissues and avoidance of local and systemic toxicity. In these studies, we report the development of a pegylated liposome containing a lipophilic IUdR derivative (3', 5'-O-dipalmitoyl-5-iodo-2'-deoxyuridine) for use in a head and neck cancer xenograft model. Initial studies confirmed the ability of IUdR to sensitise two head and neck cancer cell lines to single fractions of radiotherapy (SFRT) and this effect was seen to correlate with the thymidine replacement index in KB cells. In vivo delivery of single doses of either unencapsulated IUdR or pegylated liposomal IUdR (PLIUdR) to nude mice bearing KB xenograft tumours did not enhance the effect of SFRT delivered 16 h later. When PLIUdR was delivered by a protracted administration schedule to a dose of 48 mg kg(-1) over 7 days, it enhanced the effect of both 4.5 Gy SFRT and fractionated radiotherapy. PLIUdR was at least as effective as unencapsulated IUdR delivered by multiple intravenous injections or continuous subcutaneous infusion. Immunohistochemistry with a specific anti-IUdR monoclonal antibody confirmed greater levels of tumour staining in tumours from animals treated with PLIUdR compared with those treated with unencapsulated IUdR.
Asunto(s)
Carcinoma de Células Escamosas/radioterapia , Neoplasias de Cabeza y Cuello/radioterapia , Idoxuridina/administración & dosificación , Polietilenglicoles/administración & dosificación , Animales , Carcinoma de Células Escamosas/patología , Relación Dosis-Respuesta en la Radiación , Portadores de Fármacos , Femenino , Neoplasias de Cabeza y Cuello/patología , Humanos , Idoxuridina/análogos & derivados , Inyecciones Intravenosas , Liposomas , Ratones , Ratones Desnudos , Tasa de Supervivencia , Timidina/metabolismo , Células Tumorales Cultivadas/trasplanteRESUMEN
Positron emission tomography (PET) imaging reporter genes, such as HSV1-tk and D(2) receptor genes, make it possible to visualise gene expression non-invasively and repetitively in vivo. However, these systems require the synthesis of complicated substrates and the availability of expensive PET equipment. Expression of the sodium/iodide symporter ( NIS) gene can be easily monitored with radioiodines and technetium-99m using a gamma camera. To evaluate the possibility of using NIS as an imaging reporter gene, we compared its characteristics with those of the conventional HSV1-tk gene. The CM cell line was made by transfecting the HSV1-tk gene into CT-26 (mouse colon carcinoma cell line). The CTN and CMN cell lines were then made by transfecting the NIS gene into CT-26 and CM. We measured the uptake of iodine-125 iodovinyldeoxyuridine ([(125)I]IVDU) and (125)I to evaluate the expression of the HSV1-tk and NIS genes, respectively. Each cell line was injected into four flank sites in Balb/c mice. The biodistribution study was performed after intravenously injecting [(125)I]IVDU and (131)I, and (131)I scintigraphy was performed for the evaluation of NIS expression. In vitro studies indicated that CTN and CMN had 40- to 79-fold and 150- to 256-fold higher uptake of (125)I than CT-26 and CM, respectively. Furthermore, CM and CMN showed 57- to 69-fold higher uptake of [(125)I]IVDU than CT-26 and CTN. NIS gene expression and (125)I accumulation were found to be directly correlated ( R(2)=0.923), as were HSV1-tk gene expression and [(125)I]IVDU accumulation ( R(2)=0.956). Calculated signal per unit NIS and HSV1-tk mRNA expression was 23,240+/-3,755 cpm and 34,039+/-5,346 cpm, respectively. In vivo study indicated that CTN and CMN had 2.3- and 5.8-fold higher uptake of (131)I than CT-26 and CM, and 1.8- and 3.5-fold higher uptake of [(125)I]IVDU than CT-26 and CTN. Scintigraphy using (131)I easily visualised CTN and CMN tumours. In conclusion, the NIS gene may be viewed as an imaging reporter gene with comparable performance to the HSV1-tk gene for monitoring target gene expression.