RESUMEN

In the ubiquitin-proteasome system, protein substrates are degraded via covalent modification by a polyubiquitin chain. The polyubiquitin chain must be assembled rapidly in cells, because a chain of at least four ubiquitins is required to signal for degradation, and chain-editing enzymes in the cell may cleave premature polyubiquitin chains before achieving this critical length. The ubiquitin-conjugating enzyme Cdc34 and ubiquitin ligase SCF are capable of building polyubiquitin chains onto protein substrates both rapidly and processively; this may be explained at least in part by the atypically fast rate of Cdc34 and SCF association. This rapid association has been attributed to electrostatic interactions between the acidic C-terminal tail of Cdc34 and a feature on SCF called the basic canyon. However, the structural aspects of the Cdc34-SCF interaction and how they permit rapid complex formation remain elusive. Here, we use protein cross-linking to demonstrate that the Cdc34-SCF interaction occurs in multiple conformations, where several residues from the Cdc34 acidic tail are capable of contacting a broad region of the SCF basic canyon. Similar patterns of cross-linking are also observed between Cdc34 and the Cul1 paralog Cul2, implicating the same mechanism for the Cdc34-SCF interaction in other members of the cullin-RING ubiquitin ligases. We discuss how these results can explain the rapid association of Cdc34 and SCF.

Asunto(s)

Proteínas Cullin/metabolismo , Proteínas Quinasas Asociadas a Fase-S/metabolismo , Proteínas Ligasas SKP Cullina F-box/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitinación , Secuencia de Aminoácidos , Ciclosoma-Complejo Promotor de la Anafase , Proteínas Cullin/química , Humanos , Modelos Moleculares , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Estructura Terciaria de Proteína , Proteínas Quinasas Asociadas a Fase-S/química , Proteínas Ligasas SKP Cullina F-box/química , Proteínas Ligasas SKP Cullina F-box/genética , Ubiquitina/química , Ubiquitina/genética , Ubiquitina/metabolismo , Enzimas Ubiquitina-Conjugadoras/química

RESUMEN

Weak protein interactions between ubiquitin and the ubiquitin-proteasome system (UPS) enzymes that mediate its covalent attachment to substrates serve to position ubiquitin for optimal catalytic transfer. We show that a small-molecule inhibitor of the E2 ubiquitin-conjugating enzyme Cdc34A, called CC0651, acts by trapping a weak interaction between ubiquitin and the E2 donor ubiquitin-binding site. A structure of the ternary CC0651-Cdc34A-ubiquitin complex reveals that the inhibitor engages a composite binding pocket formed from Cdc34A and ubiquitin. CC0651 also suppresses the spontaneous hydrolysis rate of the Cdc34A-ubiquitin thioester without decreasing the interaction between Cdc34A and the RING domain subunit of the E3 enzyme. Stabilization of the numerous other weak interactions between ubiquitin and UPS enzymes by small molecules may be a feasible strategy to selectively inhibit different UPS activities.

Asunto(s)

Aminoácidos/química , Compuestos de Bifenilo/química , Enzimas Ubiquitina-Conjugadoras/antagonistas & inhibidores , Ubiquitina/química , Aminoácidos/farmacología , Sitios de Unión , Compuestos de Bifenilo/farmacología , Complejos de Coordinación/química , Cristalografía por Rayos X , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Concentración 50 Inhibidora , Modelos Moleculares , Unión Proteica , Estabilidad Proteica/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología

RESUMEN

Together with ubiquitin ligases (E3), ubiquitin-conjugating enzymes (E2) are charged with the essential task of synthesizing ubiquitin chains onto protein substrates. Some 75% of the known E2s in the human proteome contain unique insertions in their primary sequences, yet it is largely unclear what effect these insertions impart on the ubiquitination reaction. Cdc34 is an important E2 with prominent roles in cell cycle regulation and signal transduction. The amino acid sequence of Cdc34 contains an insertion distal to the active site that is absent in most other E2s, yet this acidic loop (named for its four invariably conserved acidic residues) is critical for Cdc34 function both in vitro and in vivo. Here we have investigated how the acidic loop in human Cdc34 promotes ubiquitination, identifying two key molecular events during which the acidic loop exerts its influence. First, the acidic loop promotes the interaction between Cdc34 and its ubiquitin ligase partner, SCF. Second, two glutamic acid residues located on the distal side of the loop collaborate with an invariably conserved histidine on the proximal side of the loop to suppress the pKa of an ionizing species on ubiquitin or Cdc34 which greatly contributes to Cdc34 catalysis. These results demonstrate that insertions can guide E2s to their physiologically relevant ubiquitin ligases as well as provide essential modalities that promote catalysis.

Asunto(s)

Proteínas Ligasas SKP Cullina F-box/química , Saccharomyces cerevisiae/genética , Enzimas Ubiquitina-Conjugadoras/química , Ubiquitinación/genética , Secuencia de Aminoácidos , Secuencia de Bases , Catálisis , Dominio Catalítico , Ciclo Celular/genética , Humanos , Proteolisis , Proteínas Ligasas SKP Cullina F-box/genética , Proteínas Ligasas SKP Cullina F-box/metabolismo , Saccharomyces cerevisiae/enzimología , Transducción de Señal , Ubiquitina/biosíntesis , Enzimas Ubiquitina-Conjugadoras/genética , Enzimas Ubiquitina-Conjugadoras/metabolismo , Complejos de Ubiquitina-Proteína Ligasa/química , Complejos de Ubiquitina-Proteína Ligasa/genética , Complejos de Ubiquitina-Proteína Ligasa/metabolismo

RESUMEN

We report in this study an intrinsic link between pyrimidine metabolism and liver lipid accumulation utilizing a uridine phosphorylase 1 transgenic mouse model UPase1-TG. Hepatic microvesicular steatosis is induced by disruption of uridine homeostasis through transgenic overexpression of UPase1, an enzyme of the pyrimidine catabolism and salvage pathway. Microvesicular steatosis is also induced by the inhibition of dihydroorotate dehydrogenase (DHODH), an enzyme of the de novo pyrimidine biosynthesis pathway. Interestingly, uridine supplementation completely suppresses microvesicular steatosis in both scenarios. The effective concentration (EC(50)) for uridine to suppress microvesicular steatosis is approximately 20 µM in primary hepatocytes of UPase1-TG mice. We find that uridine does not have any effect on in vitro DHODH enzymatic activity. On the other hand, uridine supplementation alters the liver NAD(+)/NADH and NADP(+)/NADPH ratios and the acetylation profile of metabolic, oxidation-reduction, and antioxidation enzymes. Protein acetylation is emerging as a key regulatory mechanism for cellular metabolism. Therefore, we propose that uridine suppresses fatty liver by modulating the liver protein acetylation profile. Our findings reveal a novel link between uridine homeostasis, pyrimidine metabolism, and liver lipid metabolism.

Asunto(s)

Hígado/metabolismo , Pirimidinas/metabolismo , Uridina/metabolismo , Animales , Células Cultivadas , Ácidos Grasos/metabolismo , Metabolismo de los Lípidos/efectos de los fármacos , Hígado/efectos de los fármacos , Masculino , Ratones , Ratones Transgénicos , Triglicéridos/metabolismo , Uridina Fosforilasa/genética , Uridina Fosforilasa/metabolismo

RESUMEN

Hepatic microvesicular steatosis is a hallmark of drug-induced hepatotoxicity and early-stage fatty liver disease. Current histopathology techniques are inadequate for the clinical evaluation of hepatic microvesicular steatosis. In this paper, we explore the use of multimodal coherent anti-Stokes Raman scattering (CARS) microscopy for the detection and characterization of hepatic microvesicular steatosis. We show that CARS microscopy is more sensitive than Oil Red O histology for the detection of microvesicular steatosis. Computer-assisted analysis of liver lipid level based on CARS signal intensity is consistent with triglyceride measurement using a standard biochemical assay. Most importantly, in a single measurement procedure on unprocessed and unstained liver tissues, multimodal CARS imaging provides a wealth of critical information including the detection of microvesicular steatosis and quantitation of liver lipid content, number and size of lipid droplets, and lipid unsaturation and packing order of lipid droplets. Such information can only be assessed by multiple different methods on processed and stained liver tissues or tissue extracts using current standard analytical techniques. Multimodal CARS microscopy also permits label-free identification of lipid-rich non-parenchymal cells. In addition, label-free and non-perturbative CARS imaging allow rapid screening of mitochondrial toxins-induced microvesicular steatosis in primary hepatocyte cultures. With its sensitivity and versatility, multimodal CARS microscopy should be a powerful tool for the clinical evaluation of hepatic microvesicular steatosis.

Asunto(s)

Hígado Graso/patología , Hígado/irrigación sanguínea , Hígado/patología , Microscopía/métodos , Microvasos/patología , Espectrometría Raman/métodos , Coloración y Etiquetado , Animales , Compuestos Azo/metabolismo , Células Cultivadas , Hepatocitos/metabolismo , Hepatocitos/patología , Procesamiento de Imagen Asistido por Computador , Metabolismo de los Lípidos , Ratones , Ratones Endogámicos C57BL , Mitocondrias/metabolismo

RESUMEN

Abrogation of uridine phosphorylase (UPase) leads to abnormalities in pyrimidine metabolism and host protection against 5-fluorouracil (5-FU) toxicity. We elucidated the effects on the metabolism and antitumor efficacy of 5-FU and capecitabine (N(4)-pentyloxycarbonyl-5'-deoxy-5-fluorocytidine) in our UPase knockout (UPase(-/-)) model. Treatment with 5-FU (85 mg/kg) or capecitabine (1,000 mg/kg) five days a week for four weeks caused severe toxicity and structural damage to the intestines of wild-type (WT) mice, but not in UPase(-/-) animals. Capecitabine treatment resulted in a 70% decrease in blood cell counts of WT animals, with only a marginal effect in UPase(-/-) mice. UPase expressing colon 38 tumors implanted in UPase(-/-) mice revealed an improved therapeutic efficacy when treated with 5-FU and capecitabine because of the higher maximum tolerated dose for fluoropyrimidines achievable in UPase(-/-) mice. (19)F-MRS evaluation of capecitabine metabolism in tumors revealed similar activation of the prodrug in UPase(-/-) mice compared with WT. In WT mice, approximately 60% of capecitabine was transformed over three hours into its active metabolites, whereas 80% was transformed in tumors implanted in UPase(-/-) mice. In UPase(-/-) mice, prolonged retention of 5'dFUR allowed a proportional increase in tumor tissue. The similar presence of fluorinated catabolic species confirms that dihydropyrimidine dehydrogenase activity was not altered in UPase(-/-) mice. Overall, these results indicate the importance of UPase in the activation of fluoropyrimidines, the effect of uridine in protecting normal tissues, and the role for tumor-specific modulation of the phosphorolytic activity in 5-FU or capecitabine-based chemotherapy.

Asunto(s)

Fluorouracilo/uso terapéutico , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Uridina Fosforilasa/genética , Animales , Antimetabolitos Antineoplásicos/metabolismo , Antimetabolitos Antineoplásicos/uso terapéutico , Capecitabina , Línea Celular Tumoral , Desoxicitidina/análogos & derivados , Desoxicitidina/uso terapéutico , Evaluación Preclínica de Medicamentos , Resistencia a Antineoplásicos/genética , Fluorouracilo/análogos & derivados , Fluorouracilo/metabolismo , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/fisiología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neoplasias/enzimología , Neoplasias/metabolismo , Profármacos/metabolismo , Profármacos/uso terapéutico , Pirimidinas/metabolismo , Pirimidinas/uso terapéutico , Resultado del Tratamiento , Uridina Fosforilasa/metabolismo , Uridina Fosforilasa/fisiología

RESUMEN

Uridine phosphorylase (UPP) catalyzes the reversible conversion of uridine to uracil and ribose-1-phosphate and plays an important pharmacological role in activating fluoropyrimidine nucleoside chemotherapeutic agents such as 5-fluorouracil and capecitabine. Most vertebrate animals, including humans, possess two homologs of this enzyme (UPP1 & UPP2), of which UPP1 has been more thoroughly studied and is better characterized. Here, we report two crystallographic structures of human UPP2 (hUPP2) in distinctly active and inactive conformations. These structures reveal that a conditional intramolecular disulfide bridge can form within the protein that dislocates a critical phosphate-coordinating arginine residue (R100) away from the active site, disabling the enzyme. In vitro activity measurements on both recombinant hUPP2 and native mouse UPP2 confirm the redox sensitivity of this enzyme, in contrast to UPP1. Sequence analysis shows that this feature is conserved among UPP2 homologs and lacking in all UPP1 proteins due to the absence of a necessary cysteine residue. The state of the disulfide bridge has further structural consequences for one face of the enzyme that suggest UPP2 may have additional functions in sensing and initiating cellular responses to oxidative stress. The molecular details surrounding these dynamic aspects of hUPP2 structure and regulation provide new insights as to how novel inhibitors of this protein may be developed with improved specificity and affinity. As uridine is emerging as a promising protective compound in neuro-degenerative diseases, including Alzheimer's and Parkinson's, understanding the regulatory mechanisms underlying UPP control of uridine concentration is key to improving clinical outcomes in these illnesses.

Asunto(s)

Uridina Fosforilasa/química , Secuencia de Aminoácidos , Animales , Dominio Catalítico , Cristalografía por Rayos X , Cistina/química , Pruebas de Enzimas , Humanos , Enlace de Hidrógeno , Ratones , Modelos Moleculares , Oxidación-Reducción , Unión Proteica , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Uracilo/análogos & derivados , Uracilo/química , Uridina Fosforilasa/antagonistas & inhibidores

RESUMEN

Myelodysplastic syndromes (MDSs) represent a group of incurable stem-cell malignancies which are predominantly treated by supportive care. Epigenetic silencing through promoter methylation of a number of genes is present in poor-risk subtypes of MDS and often predicts transformation to acute myelogenous leukemia (AML). Azacitidine and decitabine, two FDA-approved DNA methyltransferase (DNMT) inhibitors, are able to improve overall response although their oral bioavailability complicates their clinical use. This study evaluated 2', 3', 5'-triacetyl-5-azacitidine (TAC) as a potential prodrug for azacitidine. The prodrug demonstrated significant pharmacokinetic improvements in bioavailability, solubility, and stability over the parent compound. In vivo analyses indicated a lack of general toxicity coupled with significantly improved survival. Pharmacodynamic analyses confirmed its ability to suppress global methylation in vivo. These data indicate that esterified nucleoside derivatives may be effective prodrugs for azacitidine and encourages further investigation of TAC into its metabolism, activity, and possible clinical evaluation.

RESUMEN

BACKGROUND: Obesity is associated with increased tumorigenesis. Previously, we demonstrated that inflammation in obesity caused cancer fighting cells to display greater surface receptor levels, predisposing them to early cell death. We measured the inflammatory tumor growth factor levels to determine whether inflammation in obesity increases expression of these factors, potentially predisposing these patients to greater rates of neoplasia. METHODS: A total of 24 patients undergoing weight loss surgery had samples collected preoperatively and at 6 and 12 months after surgery. The growth factors analyzed included tumor necrosis factor (TNF)-α, granulocyte-macrophage colony-stimulating factor, interferon-γ, interleukin (IL)-1b, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, vascular endothelial growth factor, hepatocyte growth factor, TNF-receptor I (TNF-RI), TNF-RII, death receptor 5, leptin, and adiponectin. Control samples were obtained from 10 healthy, normal weight volunteers. RESULTS: The tumor growth factors TNF-α, TNF-RI, TNF-RII, vascular endothelial growth factor, hepatocyte growth factor, interferon-γ, IL-2, IL-5, and IL-6 all decreased significantly (P <.05) compared with the preoperative values. The IL-4, IL-8, leptin, death receptor 5, adiponectin, and granulocyte-macrophage colony-stimulating factor levels did not change significantly over time. The IL-1b and IL-10 levels were less than the detection limit at all points. When obese patient serum was compared with healthy volunteer pooled serum, we found that the leptin, death receptor 5, hepatocyte growth factor, vascular endothelial growth factor, TNF-RI, TNF-RII, TNF-α, IFN-γ, granulocyte-macrophage colony-stimulating factor, IL-4, IL-5, IL-6, and IL-8 levels were all 2-37 times greater than the levels in the controls at baseline. The concentrations of these same growth factors had decreased levels only 1-3.5 times greater than those of the controls at 12 months postoperatively. CONCLUSION: Many inflammatory tumor growth factors are present in greater concentrations in obese individuals. This could explain the greater prevalence of neoplasia in the morbidly obese population.

Asunto(s)

Neoplasias/metabolismo , Obesidad/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Pérdida de Peso , Adiponectina/metabolismo , Adulto , Susceptibilidad a Enfermedades , Femenino , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Factor de Crecimiento de Hepatocito/metabolismo , Humanos , Inflamación/metabolismo , Interferón gamma/metabolismo , Interleucinas/metabolismo , Leptina/metabolismo , Masculino , Neoplasias/etiología , Obesidad/complicaciones , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Receptores del Factor de Necrosis Tumoral/metabolismo , Factores de Riesgo , Factor A de Crecimiento Endotelial Vascular/metabolismo

RESUMEN

Uridine phosphorylase (UPase) has been shown to be induced in various human and murine tumors and could potentially serve as a specific target for the modulation of tumor-selectivity of fluoropyrimidines. However, the signaling mechanisms underlying the regulation of UPase gene expression have not been determined. In this study, we investigated the effects of IFN-gamma on the regulation of TNF-alpha-induced UPase activity and have uncovered the molecular mechanisms of this potentiation, utilizing murine EMT6 breast cancer cells. Our data has shown that IFN-gamma can significantly increase UPase mRNA expression and the enzymatic activity induced by TNF-alpha in a dose-dependent manner, resulting in an enhanced sensitivity to 5-fluorouracil (5-FU) and 5'-Deoxy-5-fluorouridine (5'DFUR). We have previously shown that TNF-alpha activates NF-kappaB through increased translocation of NF-kappaB p65 from the cytoplasm into the nuclei. Exposure to IFN-gamma mainly affects nuclear IRF-1 and STAT1 in EMT6, but inhibits NF-kappaB p65 activity, indicating that the cooperative stimulation was the result of the independent activation of NF-kappaB, STAT1 and IRF-1 transcriptional factors through binding to their unique sites in the UPase promoter. Notably, the activation of NF-kappaB and STAT1 in human breast tissues is consistent with UPase activity; signifying their role in the up-regulation of the UPase gene expression in human tumors.

Asunto(s)

Factor 1 Regulador del Interferón/metabolismo , Interferón gamma/farmacología , FN-kappa B/metabolismo , Factor de Transcripción STAT1/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Uridina Fosforilasa/metabolismo , Animales , Western Blotting , Ensayo de Cambio de Movilidad Electroforética , Activación Enzimática/efectos de los fármacos , Femenino , Expresión Génica/efectos de los fármacos , Humanos , Técnicas In Vitro , Ratones

RESUMEN

We evaluated the nuclear uptake of fluorescently labeled peptide nucleic acids and measured the binding of unlabeled peptide nucleic acids (PNAs) to the endogenous HER-2/neu promotor in digitonin-permeabilized SK-BR-3 cells. Fluorescently labeled PNAs readily enter the nucleus of digitonin-permeabilized cells, and binding to the chromosomal target sequence was detected with a bis-PNA. Nuclear uptake and target sequence binding were inhibited by N-ethylmaleimide (NEM) and GTPgammaS. We conclude that PNAs are transported into the nucleus through an energy-dependent process involving the nuclear pore complex.

Asunto(s)

Transporte Activo de Núcleo Celular , Núcleo Celular/metabolismo , Ácidos Nucleicos de Péptidos/farmacocinética , Secuencia de Bases , Línea Celular , Cromosomas/metabolismo , Colorantes Fluorescentes , Guanosina 5'-O-(3-Tiotrifosfato)/farmacología , Humanos , Poro Nuclear , Regiones Promotoras Genéticas , Receptor ErbB-2/genética

RESUMEN

Peptide nucleic acids (PNAs) are promising tools for gene regulation. One of the challenges of using PNAs as gene regulators is the need to optimize the efficiency of interaction with critical sequences of DNA. To improve the efficiency of binding between PNAs and the HER-2/neu promoter, mono- and bis-pyrimidine-rich PNAs were conjugated to a nitrogen mustard at either the amino or carboxy terminus. Gel shift analysis demonstrated that conjugation to an alkylating agent slowed PNA binding and favored PNA:DNA:DNA triplex helix formation while preserving a high binding affinity. Sites of DNA alkylation were visualized by piperidine cleavage and showed PNA binding first by Hoogsteen bond formation with the target duplex to form a stable PNA:DNA:DNA triplex structure which is later converted to a PNA:DNA:PNA triple helix by strand invasion and Watson-Crick base pairing by a second PNA molecule. In this way, PNA-directed DNA alkylation was used to deduce the mode of PNA binding. Transient transfection experiments demonstrated that the PNA-nitrogen mustard conjugates suppressed HER-2/neu expression by up to 80%. In comparison with an unmodified mono-PNA or a bis-PNA, these results indicate that the covalent adducts stabilized PNA binding in cells and suggest that the conjugation of PNAs to nitrogen mustards is a robust strategy for developing antigene PNA oligonucleotides to prevent transcription.

Asunto(s)

Clorambucilo/farmacología , Compuestos de Mostaza Nitrogenada/farmacología , Ácidos Nucleicos de Péptidos/farmacología , Receptor ErbB-2/metabolismo , Antineoplásicos Alquilantes/química , Antineoplásicos Alquilantes/farmacología , Clorambucilo/química , ADN/química , Regulación hacia Abajo , Regulación de la Expresión Génica/efectos de los fármacos , Genes Reporteros , Células HeLa , Humanos , Compuestos de Mostaza Nitrogenada/química , Ácidos Nucleicos de Péptidos/química , Ácidos Nucleicos de Péptidos/metabolismo , Plásmidos/genética , Regiones Promotoras Genéticas , Receptor ErbB-2/genética , Transfección

RESUMEN

Artificial control of gene expression has great potential in the treatment of many human diseases, and peptide nucleic acids (PNAs) offer several potential advantages for silencing gene expression in mammalian cells. The pseudopeptide backbone of the PNA makes it resistant to enzymatic degradation, and PNAs bind complementary DNA and RNA with high affinity and specificity. PNAs are potentially leading agents for antigene and antisense therapeutics, but the application of PNAs in the in vivo setting is hampered by their poor intracellular delivery. This problem has been addressed by PNA conjugation to lipophilic moieties, peptides, and cell-specific receptor ligands. The biological activity of PNAs can also benefit from conjugation to DNA interactive compounds like intercalators and alkylators. Here we review the most interesting literature concerning PNA conjugation with small molecules, emphasizing synthetic approaches, properties and applications of the PNA conjugates.

Asunto(s)

Ácidos Nucleicos de Péptidos/química , Alquilantes/química , Colorantes Fluorescentes/química , Conformación de Ácido Nucleico , Ácidos Nucleicos de Péptidos/síntesis química

RESUMEN

Antigene oligonucleotides have the potential to regulate gene expression through site-specific DNA binding. However, in vivo applications have been hindered by inefficient cellular uptake, degradation, and strand displacement. Peptide nucleic acids (PNAs) address several of these problems, as they are resistant to degradation and bind DNA with high affinity. We designed two cationic pyrimidine bis-PNAs (cpy-PNAs) to target the polypurine tract of the HER-2/neu promoter and compared them to an unmodified phosphodiester triplex-forming oligonucleotide (TFO1) and a TFO-nitrogen mustard conjugate (TFO2). PNA1 contains a + 2 charge and bound two adjacent 9-bp target sequences with high affinity and specificity, but only at low pH. PNA2 contains a +5 charge and bound one 11-bp target with high affinity up to pH 7.4, but with lower specificity. The PNA:DNA:PNA triplex formed by these cpy-bis-PNAs presented a stable barrier to DNA polymerase extension. The cpy-bis-PNAs and the TFO-alkylator conjugate prevented HER-2/neu transcription in a reporter gene assay (TFO2 = PNA1 > PNA2 >> TFO1). Both PNAs and TFOs were effective at binding the target sequence in naked genomic DNA, but only the TFO-alkylator (TFO2) and the more cationic PNA (PNA2) were detected at the endogenous HER-2/neu promoter in permeabilized cells. This work demonstrates the potential for preventing HER-2/neu gene expression with cpy-bis-PNAs in tumor cells.

Asunto(s)

Regulación de la Expresión Génica , Técnicas Genéticas , Regiones Promotoras Genéticas , Receptor ErbB-2/genética , Receptor ErbB-2/fisiología , Alquilantes/farmacología , Secuencia de Bases , Southern Blotting , Cationes , Línea Celular Tumoral , ADN/química , Cartilla de ADN/química , ADN Polimerasa Dirigida por ADN/química , ADN Polimerasa Dirigida por ADN/metabolismo , Digitonina/farmacología , Relación Dosis-Respuesta a Droga , Células HeLa , Humanos , Concentración de Iones de Hidrógeno , Mecloretamina/farmacología , Datos de Secuencia Molecular , Oligonucleótidos/química , Ácidos Nucleicos de Péptidos/química , Plásmidos/metabolismo , Unión Proteica , Receptor ErbB-2/metabolismo , Transfección

RESUMEN

In most cases, unmodified oligonucleotides designed as antigene molecules are incapable of binding to DNA with sufficient stability to prevent gene expression. To stabilize binding to a polypurine tract in the HER-2/neu promoter, a triplex forming oligonucleotide (TFO) was conjugated to a pyrrolo[1,4]benzodiazepine (PBD), desmethyltomaymycin, and site-specific DNA binding was evaluated. An activated ester of the PBD moiety was conjugated by an acylation reaction to a free primary amine on a 50-atom aliphatic linker at the 5' end of the TFO. This long aliphatic linker was designed to provide a bridge from the major groove binding site of the TFO to the minor groove binding site of the PBD. Triplex formation by the resulting TFO-PBD conjugate occurred more slowly and with a nearly 30-fold lower affinity compared to an unconjugated TFO. PBD binding to the triplex target was demonstrated by protection from restriction enzyme digestion, and covalent binding to the exocyclic amino group of guanine was inferred by substituting specific guanines with inosines. Although the binding of the TFO was less efficient, this report demonstrates that in principle, TFOs can be used to direct the binding of a PBD to specific location. Further optimization of TFO-PBD conjugate design, likely involving optimization of the linker and perhaps placing a PBD at both ends of the TFO, will be needed to make gene modification robust.

Asunto(s)

Benzodiazepinas/síntesis química , ADN/química , Oligonucleótidos/síntesis química , Pirroles/síntesis química , ADN/genética , Evaluación Preclínica de Medicamentos/métodos , Células HeLa , Humanos , Oligonucleótidos/genética

RESUMEN

Triplex forming oligonucleotides (TFOs) have the ability to site specifically modulate gene expression through the formation of triple helix DNA. The HER-2/neu promoter contains a strategically located triplex target sequence, and has been successfully targeted in vitro, with little success in vivo. A TFO was conjugated at both its 5' and 3' ends to an alkylating agent (phenylacetate mustard) in an attempt to stabilize the triple helix intracellularly. In vitro assays demonstrated that the bis-conjugate bound the duplex and alkylated the target guanine residues with high efficiency. The bis-conjugate suppressed promoter activity by 60-70% in cancer cells using a plasmid with a preformed triple helix, and the suppression was minimal when the nitrogen mustard was conjugated at only one end. Helicase assays demonstrated that helicase activity can unwind the TFO at the unalkylated end of the triple helix, which may leave the unwound oligonucleotide susceptible to nuclease degradation or ineffective at inhibiting transcription initiation. Our findings indicate that dual alkylation of the target sequence is required to suppress the intracellular activity of a reporter plasmid with a preformed triple helix, likely due to greater stability of the triple helix within cells and inhibition of helicase activity.

Asunto(s)

ADN Helicasas/antagonistas & inhibidores , Regulación de la Expresión Génica/efectos de los fármacos , Genes Reporteros/efectos de los fármacos , Oligodesoxirribonucleótidos/farmacología , Alquilación , ADN/química , ADN/metabolismo , Cinética , Conformación de Ácido Nucleico , Desnaturalización de Ácido Nucleico , Oligodesoxirribonucleótidos/química , Plásmidos , Regiones Promotoras Genéticas , Transfección