RESUMEN

The cardiac natriuretic peptides (NPs) control pivotal physiological actions such as fluid and electrolyte balance, cardiovascular homeostasis, and adipose tissue metabolism by activating their receptor enzymes [natriuretic peptide receptor-A (NPRA) and natriuretic peptide receptor-B (NPRB)]. These receptors are homodimers that generate intracellular cyclic guanosine monophosphate (cGMP). The natriuretic peptide receptor-C (NPRC), nicknamed the clearance receptor, lacks a guanylyl cyclase domain; instead, it can bind the NPs to internalize and degrade them. The conventional paradigm is that by competing for and internalizing NPs, NPRC blunts the ability of NPs to signal through NPRA and NPRB. Here we show another previously unknown mechanism by which NPRC can interfere with the cGMP signaling function of the NP receptors. By forming a heterodimer with monomeric NPRA or NPRB, NPRC can prevent the formation of a functional guanylyl cyclase domain and thereby suppress cGMP production in a cell-autonomous manner.

Asunto(s)

Guanilato Ciclasa , Receptores del Factor Natriurético Atrial , Guanilato Ciclasa/metabolismo , Receptores del Factor Natriurético Atrial/metabolismo , Receptores de Péptidos/metabolismo , Péptidos Natriuréticos , Transducción de Señal , Factor Natriurético Atrial/metabolismo , GMP Cíclico/metabolismo

RESUMEN

Cyclophosphamide (CP) has been proven to be an embryo-fetal toxic. However, the mechanism responsible for the toxicity of the teratogenic agent has not been fully explored. This study aimed to examine the teratogenicity of CP when administered in the sensitive period of pregnant rats. The effect of CP on the lipid and metabolic profiles of amniotic fluid was evaluated using a UHPLC-Q-Exactive Orbitrap MS-based method. Metabolome analysis was performed using the MS-DIAL software with LipidBlast and NIST. Initially, we identified 636 and 154 lipid compounds in the positive and negative ion modes and 118 metabolites for differential analysis. Mainly 4 types of oxidized lipids in the amniotic fluid were found to accumulate most significantly after CP treatment, including very-long-chain unsaturated fatty acids (VLCUFAs), polyunsaturated fatty acid (PUFA)-containing triglycerides (TGs), oxidized phosphatidylcholine (PC), and sphingomyelin (SM). Tryptophan and some long-chain saturated fatty acids were lowered pronouncedly after CP treatment. These findings suggest that CP may exert teratogenic toxicity on pregnant rats through maternal and fetal oxidative stress. The UHPLC-Q-Exactive Orbitrap MS-based lipidomics approach is worthy of wider application for evaluating the potential toxicity of other agents (toxicants) during embryonic development.

RESUMEN

In addition to controlling blood pressure, cardiac natriuretic peptides (NPs) can stimulate lipolysis in adipocytes and promote the "browning" of white adipose tissue. NPs may also increase the oxidative capacity of skeletal muscle. To unravel the contribution of NP-stimulated metabolism in adipose tissue compared to that in muscle in vivo, we generated mice with tissue-specific deletion of the NP clearance receptor, NPRC, in adipose tissue (NprcAKO ) or in skeletal muscle (NprcMKO ). We showed that, similar to Nprc null mice, NprcAKO mice, but not NprcMKO mice, were resistant to obesity induced by a high-fat diet. NprcAKO mice exhibited increased energy expenditure, improved insulin sensitivity, and increased glucose uptake into brown fat. These mice were also protected from diet-induced hepatic steatosis and visceral fat inflammation. These findings support the conclusion that NPRC in adipose tissue is a critical regulator of energy metabolism and suggest that inhibiting this receptor may be an important avenue to explore for combating metabolic disease.

Asunto(s)

Tejido Adiposo/metabolismo , Grasas de la Dieta/efectos adversos , Resistencia a la Insulina , Obesidad , Receptores del Factor Natriurético Atrial , Transducción de Señal , Tejido Adiposo/patología , Animales , Grasas de la Dieta/farmacología , Ratones , Ratones Noqueados , Obesidad/inducido químicamente , Obesidad/genética , Obesidad/metabolismo , Receptores del Factor Natriurético Atrial/genética , Receptores del Factor Natriurético Atrial/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética

RESUMEN

A classic metabolic concept posits that insulin promotes energy storage and adipose expansion, while catecholamines stimulate release of adipose energy stores by hydrolysis of triglycerides through ß-adrenergic receptor (ßARs) and protein kinase A (PKA) signaling. Here, we have shown that a key hub in the insulin signaling pathway, activation of p70 ribosomal S6 kinase (S6K1) through mTORC1, is also triggered by PKA activation in both mouse and human adipocytes. Mice with mTORC1 impairment, either through adipocyte-specific deletion of Raptor or pharmacologic rapamycin treatment, were refractory to the well-known ßAR-dependent increase of uncoupling protein UCP1 expression and expansion of beige/brite adipocytes (so-called browning) in white adipose tissue (WAT). Mechanistically, PKA directly phosphorylated mTOR and RAPTOR on unique serine residues, an effect that was independent of insulin/AKT signaling. Abrogation of the PKA site within RAPTOR disrupted ßAR/mTORC1 activation of S6K1 without affecting mTORC1 activation by insulin. Conversely, a phosphomimetic RAPTOR augmented S6K1 activity. Together, these studies reveal a signaling pathway from ßARs and PKA through mTORC1 that is required for adipose browning by catecholamines and provides potential therapeutic strategies to enhance energy expenditure and combat metabolic disease.

Asunto(s)

Tejido Adiposo Pardo/metabolismo , Complejos Multiproteicos/metabolismo , Transducción de Señal/fisiología , Serina-Treonina Quinasas TOR/metabolismo , Células 3T3-L1 , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Tejido Adiposo Pardo/citología , Tejido Adiposo Blanco/citología , Tejido Adiposo Blanco/metabolismo , Animales , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Células HEK293 , Humanos , Insulina/genética , Insulina/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Ratones Noqueados , Complejos Multiproteicos/genética , Receptores Adrenérgicos beta/genética , Proteína Reguladora Asociada a mTOR , Proteínas Quinasas S6 Ribosómicas 70-kDa/genética , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Proteínas Quinasas S6 Ribosómicas 90-kDa/genética , Proteínas Quinasas S6 Ribosómicas 90-kDa/metabolismo , Serina-Treonina Quinasas TOR/genética , Proteína Desacopladora 1/biosíntesis , Proteína Desacopladora 1/genética

RESUMEN

Despite the great potential of small interfering RNA (siRNA) as a therapeutic agent, progress in this area has been hampered by a lack of efficient biocompatible transfection agents. Recently, cationic shell-crosslinked knedel-like nanoparticles (cSCKs) were found to possess lower cytotoxicity and better transfection ability for phosphorothioate ODNs and plasmid DNA than the commonly used cationic lipid-based agent Lipofectamine. To determine the usefulness of cSCKs for siRNA transfection, a small library of cSCKs with varying percentage of primary and tertiary amines was assessed for its ability to bind to siRNA, inhibit siRNA degradation in human serum, and to transfect HeLa and mouse macrophage cell lines. The silencing efficiency in HeLa cells was greatest with the cSCK with 100% primary amines (pa100) as determined by their viability following transfection with cytotoxic and non-cytotoxic siRNAs. cSCK-pa100 showed greater silencing efficiency than Lipofectamine 2000 in the HeLa cells, as well in 293T and human bronchial epithelial (HEK) cells, but was comparable in human bronchial epithelial (BEAS-2B) cells and human mammary epithelial (MCF10a) cells. cSCK-pa100 also showed greater silencing of iNOS expression than Lipofectamine 2000 in a mouse macrophage cell line, and provided greater protection from serum degradation, demonstrating its potential usefulness as an siRNA transfection agent. The siRNA silencing of iNOS at lower concentrations of siRNA could be enhanced by complexation with the fusogenic GALA peptide, which was shown to enhance endosomal escape following uptake.

Asunto(s)

Cationes/química , Sistemas de Liberación de Medicamentos , Nanopartículas/química , ARN Interferente Pequeño , Cationes/administración & dosificación , Regulación de la Expresión Génica , Células HeLa , Humanos , Nanopartículas/administración & dosificación , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/metabolismo , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/química , ARN Interferente Pequeño/genética , Transfección

RESUMEN

4-Hydroxyalkenal species are a class of peroxidative products of polyunsaturated fatty acids, which serve as "toxic second messengers" in cellular systems. Investigation of their cellular role is hindered due to the lack of sensitive, reliable, robust method for identification and quantification of these metastable metabolites. Herein, we explored the facile Michael adduct of carnosine with 4-hydroxyalkenal species and developed a sensitive, facile, shotgun lipidomics-based method for quantification of these compounds directly from organic solvent lipid extracts of biological samples. In the study, we extensively examined the factors that may affect the accurate quantification of 4-hydroxyalkenal species and found that this method possessed high reproducibility (<8%) and nearly 3 orders of linear dynamic range with a limit of quantification at lower than 0.56 fmol/µL. Mass levels of 4-hydroxyalkenal species in various biological samples, including mouse heart, kidney, liver, and skeletal muscle, were determined by this developing method. In addition, the effects of sample collection methods and sample storage time on 4-hydroxyalkenal mass levels were also determined. We believe that development of this novel methodology should provide a powerful tool for us to better understand the role of 4-hydroxyalkenal species in biological processes.

Asunto(s)

Aldehídos/análisis , Lípidos/química , Espectrometría de Masa por Ionización de Electrospray , Animales , Carnosina/química , Masculino , Ratones , Ratones Endogámicos C57BL

RESUMEN

A procedure for rapidly generating a library of antisense-accessible sites on native mRNAs (mRNA antisense-accessible sites library [MASL]) is described that involves reverse transcription of whole cell mRNA extracts with a random oligodeoxynucleotide primer followed by mRNA-specific polymerase chain reaction (PCR). Antisense phosphorothioate oligodeoxynucleotides (ODNs), peptide nucleic acids (PNAs), and small interfering RNAs (siRNAs) can then be identified by screening against the antisense-accessible sites. The utility of this methodology is demonstrated for the identification of more effective inhibitors of inducible nitric oxide synthase (iNOS) induction than have previously been reported. This method may also be useful for constraining folding calculations of native mRNAs and for designing mRNA imaging probes.

Asunto(s)

Técnicas Genéticas , Oligonucleótidos Antisentido/genética , Ácidos Nucleicos de Péptidos/genética , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Animales , Secuencia de Bases , Línea Celular , Datos de Secuencia Molecular , Óxido Nítrico Sintasa de Tipo II

RESUMEN

Cationic nanoparticles are a promising class of transfection agents for oligonucleotide and gene delivery, but vary greatly in their effectiveness and cytotoxicity. Recently, we developed a new class of cationic transfection agents based on cationic shell-crosslinked knedel-like nanoparticles (cSCKs) that efficiently transfect mammalian cells with both oligonucleotides and plasmid DNA. In an effort to further improve transfection efficiency without increasing cytotoxicity, we examined the effects of the composition of primary amine (pa), tertiary amine (ta) and carboxylic acid (ca) groups in the shell of these nanoparticles. A series of discrete complexes of the cSCKs with plasmid DNA (pDNA) or phosphorothioate 2'-OMe oliogonucleotides (ps-MeON) were prepared over a broad range of amine to phosphate ratios (N/P ratio) of 4:1-40:1. The sizes of the complexes and the ability of the nanoparticles to completely bind ODNs were found to depend on the cSCK amine to DNA phosphate (N/P) ratio and the cSCK buffering capacity. The cSCKs were then evaluated for their ability to transfect cells with plasmid DNA by monitoring fluorescence from an encoded EGFP, and for delivery of ps-MeON by monitoring luminescence from luciferase resulting from ps-MeON-mediated splicing correction. Whereas the cationic cSCK-pa(25)-ta(75) was found to be best for transfecting plasmid DNA into HeLa cells at an N/P ratio of 20:1, cSCK-pa(50)-ta(50), at an N/P ratio 10:1 was best for ps-MeON delivery. We also found that increasing the proportion of tertiary relative to primary amine reduced the cytotoxicity. These results demonstrate that a dramatic improvement in gene and oligonucleotide delivery efficiency with decreased cytotoxicity in HeLa cells can be achieved by incorporation of tertiary amines into the shells of cSCKs.

Asunto(s)

Cationes/química , Reactivos de Enlaces Cruzados/farmacología , Nanopartículas/química , Transfección , Tampones (Química) , Muerte Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , ADN/metabolismo , ADN/ultraestructura , Ensayo de Cambio de Movilidad Electroforética , Citometría de Flujo , Células HeLa , Humanos , Luciferasas/metabolismo , Microscopía Confocal , Nanopartículas/ultraestructura , Oligonucleótidos/química , Oligonucleótidos/farmacología , Plásmidos/genética , Relación Estructura-Actividad

RESUMEN

Peptide nucleic acids (PNAs) have a number of attractive features that have made them an ideal choice for antisense and antigene-based tools, probes, and drugs, but their poor membrane permeability has limited their application as therapeutic or diagnostic agents. Herein, we report a general method for the synthesis of phospholipid-PNAs (LP-PNAs) and compare the effect of noncleavable lipids and bioreductively cleavable lipids (L and LSS) and phospholipid (LP) on the splice-correcting bioactivity of a PNA bearing the cell penetrating Arg9 group (PNA-R9). While the three constructs show similar and increasing bioactivity at 1-3 microM, the activity of LP-PNA-R9 continues to increase from 4-6 microM, while the activity of L-PNA-R9 remains constant and that of LSS-PNA-R9 decreases rapidly in parallel with their relative cytotoxicity. The activity of both LP-PNA-R9 and L-PNA-R9 dramatically increased in the presence of chloroquine, as expected for an endocytotic entry mechanism. The constructs were also found to have CMC values of 1.0 and 4.5 microM, respectively, in 150 mM NaCl, pH 7 water, suggesting that micelle formation may play a hitherto unrecognized role in modulating toxicity and/or facilitating endocytosis.

Asunto(s)

Portadores de Fármacos/síntesis química , Sistemas de Liberación de Medicamentos , Ácidos Nucleicos de Péptidos/química , Fosfolípidos/química , Animales , Muerte Celular , Línea Celular , Permeabilidad de la Membrana Celular , Cloroquina/farmacología , Relación Dosis-Respuesta a Droga , Portadores de Fármacos/farmacocinética , Humanos , Micelas , Ácidos Nucleicos de Péptidos/farmacocinética , Fosfolípidos/farmacocinética , Relación Estructura-Actividad

RESUMEN

This mini-review highlights developments that have been made over the past year to advance the construction of well-defined nanoscale objects to serve as devices for cell transfection. Design of the nanoscale objects originated from biomimicry concepts, using histones as the model, to afford cationic shell crosslinked knedel-like (cSCK) nanoparticles. Packaging and delivery of plasmid DNA, oligonucleotides, and peptide nucleic acids were studied by dynamic light scattering, transmission electron microscopy, gel electrophoresis, biological activity assays, RT-PCR measurements, flow cytometry, and confocal fluorescence microscopy. With the demonstration of more efficient cell transfection in vitro than that achieved using commercially-available transfection agents, together with the other features offered by the robust nanostructural framework, work continues toward the application of these cSCKs for in vivo molecular recognition of genetic material, for imaging and therapy targeted specifically to pulmonary injury and disease.

Asunto(s)

ADN , Nanopartículas , Ácidos Nucleicos de Péptidos , Transfección/métodos , ADN/genética , ADN/uso terapéutico , Humanos , Nanopartículas/química , Nanopartículas/uso terapéutico , Ácidos Nucleicos de Péptidos/genética

RESUMEN

The unusual structural forms of telomere DNA, which protect the ends of chromosomes during replication, may render it vulnerable to unprecedented photodamage, possibly involving nonadjacent bases that are made proximate by folding. The G-quadruplex for the human telomere sequence consisting of a repeating d(TTAGGG) is one unusual form. Tel22, d[AGGG(TTAGGG)(3)], forms a basket structure in the presence of Na(+) and may form multiple equilibrating structures in the presence of K(+) with hybrid-type structures predominating. UVB irradiation of d[AGGG(TTAGGG)(3)] in the presence of Na(+) results in a cis,syn thymine dimer between two adjacent Ts in a TTA loop and a mixture of nonadjacent anti thymine dimers between various loops. Irradiation in the presence of K(+), however, produces, in addition to these same products, a large amount of specific anti thymine dimers formed between either T in loop 1 and the central T in loop 3. These latter species were not observed in the presence of Na(+). Interloop-specific anti thymine dimers are incompatible with hybrid-type structures, but could arise from a chair or basket-type structure or from triplex intermediates involved in interconverting these structures. If these unique nonadjacent anti thymine dimer photoproducts also form in vivo, they would constitute a previously unrecognized type of DNA photodamage that may interfere with telomere replication and present a unique challenge to DNA repair. Furthermore, these unusual anti photoproducts may be used to establish the presence of G-quadruplex or quadruplex-like structures in vivo.

Asunto(s)

G-Cuádruplex , Dímeros de Pirimidina/química , Telómero , Cromatografía Líquida de Alta Presión , Electroforesis en Gel de Poliacrilamida , Proteínas Fúngicas/farmacología , Humanos , Fotoquímica , Endonucleasas Específicas del ADN y ARN con un Solo Filamento/farmacología , Espectrometría de Masa por Ionización de Electrospray

RESUMEN

Polymer vesicles with diameters of ca. 100-600 nm and bearing benzaldehyde functionalities within the vesicular walls were constructed through self-assembly of an amphiphilic block copolymer PEO(45)-b-PVBA(26) in water. The reactivity of the benzaldehyde functionalities was verified by cross-linking the polymersomes and also by a one-pot cross-linking and functionalization approach to further render the vesicles fluorescent, each via reductive amination. In vitro studies found these labeled nanostructures to undergo cell association.

Asunto(s)

Benzaldehídos/química , Nanoestructuras/química , Polímeros/química , Animales , Células CHO , Cricetinae , Cricetulus , Reactivos de Enlaces Cruzados/química , Células HeLa , Humanos , Agua/química

RESUMEN

Peptide nucleic acids have a number of features that make them an ideal platform for the development of in vitro biological probes and tools. Unfortunately, their inability to pass through membranes has limited their in vivo application as diagnostic and therapeutic agents. Herein, we describe the development of cationic shell-cross-linked knedel-like (cSCK) nanoparticles as highly efficient vehicles for the delivery of PNAs into cells, either through electrostatic complexation with a PNA * ODN hybrid, or through a bioreductively cleavable disulfide linkage to a PNA. These delivery systems are better than the standard Lipofectamine/ODN-mediated method and much better than the Arg(g)-mediated method for PNA delivery in HeLa cells, showing lower toxicity and higher bioactivity. The cSCKs were also found to facilitate both endocytosis and endosomal release of the PNAs, while themselves remaining trapped in the endosomes.

Asunto(s)

Cationes/química , Sistemas de Liberación de Medicamentos , Nanopartículas , Oligonucleótidos/administración & dosificación , Ácidos Nucleicos de Péptidos/administración & dosificación , Supervivencia Celular/efectos de los fármacos , Endocitosis , Endosomas , Células HeLa , Humanos , Lípidos , Luciferasas/genética , Luciferasas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transfección

RESUMEN

In this work, a robust synthetic nanostructure was designed for the effective packaging of DNA and it was shown to be an efficient agent for cell transfection. An amphiphilic block copolymer, poly(acrylamidoethylamine)(128)-b-polystyrene(40) (PAEA(128)-b-PS(40)), was synthesized, micellized in water and shell-crosslinked using a diacid-derivatized crosslinker, to give cationic shell-crosslinked nanoparticles (cSCKs) with a mean hydrodynamic diameter of 14 +/- 2 nm. A series of discrete complexes of the cSCKs with plasmid DNA (pDNA) was able to be formed over a broad range of polymer amine:pDNA phosphate ratios (N/P ratio), 2:1-20:1. The sizes of the complexes and their ability to fully bind the pDNA were dependent upon the N/P ratio, as characterized by dynamic light scattering (DLS), transmission electron microscopy (TEM) and gel retardation assay. A luciferase activity assay and EGFP expression were used to evaluate intracellular delivery of a splice-correcting phosphorothioate and genetic material, respectively, by the cSCKs, which indicated that an N/P ratio of 6:1 gave the highest transfection. It was shown by both luciferase activity assay (48 h) and EGFP transfection data that high transfection efficiencies were achieved for HeLa cells transfected by cSCK/CCUCUUACCUCAGUUACA and cSCK/pEGFP-N1 plasmid, respectively. The cSCK/pEGFP-N1 plasmid transfection efficiency of 27% far exceeded the performance of Polyfect (PAMAM dendrimers), which achieved only 12% transfection efficiency, under the same conditions. Cytotoxicities for the cSCKs were evaluated for HeLa and CHO cells.

Asunto(s)

Nanopartículas/química , Transfección/métodos , Animales , Células CHO , Supervivencia Celular/efectos de los fármacos , Cricetinae , Cricetulus , ADN/química , ADN/metabolismo , Citometría de Flujo , Técnicas de Transferencia de Gen , Células HeLa , Humanos , Microscopía Confocal , Microscopía Fluorescente , Nanopartículas/efectos adversos , Oligonucleótidos/química , Oligonucleótidos/metabolismo , Polímeros/química

RESUMEN

Many mutations occur as a result of DNA synthesis past the site of DNA damage by DNA damage bypass polymerases. The frequency and types of mutations not only depend on the nature of the damage, but also on the sequence context, as revealed from analysis of mutation spectra of DNA exposed to mutagens. Herein we report a new method for the rapid determination of the effect of sequence context on mutagenesis called SAMS for serial analysis of mutation spectra. This technique makes use of the methodology that underlies serial analysis of gene expression (SAGE) to analyze mutations that result from DNA synthesis past a DNA lesion site-specifically embedded in a library of DNA sequences. To illustrate our technique we determined the effect of sequence context on mutations generated by DNA synthesis past a tetrahydrofuran abasic site model by the DNA damage bypass polymerase yeast polymerase eta.

Asunto(s)

Daño del ADN , Análisis Mutacional de ADN/métodos , Cartilla de ADN , ADN Polimerasa Dirigida por ADN , Modelos Genéticos , Reacción en Cadena de la Polimerasa , Eliminación de Secuencia , Moldes Genéticos

RESUMEN

In order to probe the nanoparticle shape/size effect on cellular uptake, a spherical and two cylindrical nanoparticles, whose lengths were distinctively varied, were constructed by the selective cross-linking of amphiphilic block copolymer micelles. Herein, we demonstrate that, when the nanoparticles were functionalized with the protein transduction domain of human immunodeficiency virus type 1 Tat protein (HIV Tat PTD), the smaller, spherical nanoparticles had a higher rate of cell entry into Chinese hamster ovary (CHO) cells than did the larger, cylindrical nanoparticles. It was also found that nanoparticles were released after internalization and that the rate of cell exit was dependent on both the nanoparticle shape and the amount of surface-bound PTD.

Asunto(s)

Productos del Gen tat/metabolismo , VIH-1/química , Nanopartículas/química , Nanotecnología/métodos , Secuencia de Aminoácidos , Animales , Células CHO , Cricetinae , Cricetulus , Productos del Gen tat/química , Humanos , Micelas , Datos de Secuencia Molecular , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción

RESUMEN

We have recently shown that an MCF-7 tumor can be imaged in a mouse by PET with 64Cu-labeled Peptide nucleic acids (PNAs) tethered to the permeation peptide Lys4 that recognize the uniquely overexpressed and very abundant upstream of N-ras or N-ras related gene (unr mRNA) expressed in these cells. Herein we describe how the high affinity antisense PNAs to the unr mRNA were identified and characterized. First, antisense binding sites on the unr mRNA were mapped by an reverse transcriptase random oligonucleotide library (RT-ROL) method that we have improved, and by a serial analysis of antisense binding sites (SAABS) method that we have developed which is similar to another recently described method. The relative binding affinities of oligodeoxynucleotides (ODNs) complementary to the antisense binding sites were then qualitatively ranked by a new Dynabead-based dot blot assay. Dissociation constants for a subset of the ODNs were determined by a new Dynabead-based solution assay and were found to be 300 pM for the best binders in 1 M salt. PNAs corresponding to the ODNs with the highest affinities were synthesized with an N-terminal CysTyr and C-terminal Lys4 sequence. Dissociation constants of these hybrid PNAs were determined by the Dynabead-based solution assay to be about 10 pM for the highest affinity binders.

Asunto(s)

Antineoplásicos/química , Proteínas de Unión al ADN/genética , Oligonucleótidos Antisentido/química , Ácidos Nucleicos de Péptidos/química , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Antineoplásicos/síntesis química , Sitios de Unión , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/metabolismo , Femenino , Humanos , Hibridación de Ácido Nucleico/métodos , Oligodesoxirribonucleótidos Antisentido/química , Oligonucleótidos Antisentido/síntesis química , Ácidos Nucleicos de Péptidos/síntesis química , Reacción en Cadena de la Polimerasa/métodos , ARN Mensajero/química , Proteínas de Unión al ARN/antagonistas & inhibidores , Proteínas de Unión al ARN/metabolismo

RESUMEN

As more becomes known about the expression profiles of normal and cancerous cells, it should become possible to design antisense-based imaging agents for the early detection of cancer noninvasively. In this report, we rationally designed and synthesized three antisense and one sense hybrid PNA (peptide nucleic acid) to the unr mRNA that is highly overexpressed in a breast cancer cell line (MCF-7). The conjugates had a four-lysine tail at the carboxy terminus for cell permeation and a DOTA (1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid) chelating moiety at the amino terminal end for chelating (64)Cu for biodistribution and microPET imaging studies. Biodistribution of two (64)Cu-labeled conjugates with antisense and sense sequences (PNA50 and PNA50S) showed high uptake and long retention in kidney and low uptake and efficient clearance in blood and muscle in normal balb/c mice when administered intravenously or intraperitoneally. Intraperitoneal administration, however, gave a much slower release rate. MCF-7 tumors (100-320 mg) in CB-17 SCID mice were imaged with all four (64)Cu-labeled PNA conjugates by microPET, but the image contrast varied with different time points and different conjugates. Of the conjugates studied, (64)Cu-DOTA-Y-PNA50-K4 showed the best tumor image quality at all time points with a tumor/muscle ratio of 6.6 +/- 1.1 at 24 h postinjection, which is among the highest reported for radiolabeled oligonucleotides. Our work further strengthens the potential of antigene and antisense PNAs to be utilized as specific molecular probes for early detection of cancer and ultimately for patient specific radiotherapy.

Asunto(s)

Radioisótopos de Cobre , Neoplasias Experimentales/diagnóstico , Ácidos Nucleicos de Péptidos , ARN Neoplásico/metabolismo , Animales , Línea Celular Tumoral , Permeabilidad de la Membrana Celular , Radioisótopos de Cobre/farmacocinética , Compuestos Heterocíclicos con 1 Anillo/farmacocinética , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones SCID , Oligonucleótidos Antisentido/farmacocinética , Ácidos Nucleicos de Péptidos/farmacocinética , Distribución Tisular