RESUMEN
DNA strand breaks produced by the decay of (125)I positioned against a specific site in plasmid DNA via a triplex-forming oligonucleotide were studied both in the immediate vicinity of the site of the decay with a single nucleotide resolution and in the whole plasmid by measuring the percentages of supercoiled, open-circular and linear forms. The localized breaks are distributed within 10 bp in each direction from the decay site with maxima in both strands just opposite the (125)I-dC residue in the triplex-forming oligonucleotide. The distributions of breaks in the two DNA strands are almost symmetrical, in agreement with the geometry of the pyrimidine motif triplex. We found that about 25% of the double-strand breaks were located outside the 90-bp fragment containing the triplex-forming oligonucleotide binding sequence. The ratio of single- to double-strand breaks in the whole plasmid was 11 for bound triplex-forming oligonucleotide compared to 26 when the triplex-forming oligonucleotide was free in solution. The number of double-strand breaks per decay of (125)I was 0.46 for bound triplex-forming oligonucleotide and 0.17 for free triplex-forming oligonucleotide. Comparing the data on the localized damage and those for the whole plasmid, we concluded that, in addition to DNA breaks that are confined to a helical turn around the (125)I atom, the decay can produce breaks hundreds of base pairs away in the plasmid molecule. This linear plasmid molecule containing radiation-induced damage at a specific DNA site should be useful in studies of the molecular mechanisms of DNA repair.
Asunto(s)
Daño del ADN , Radioisótopos de Yodo/efectos adversos , Oligodesoxirribonucleótidos/efectos de la radiación , Plásmidos/efectos de la radiación , Secuencia de Bases , Reparación del ADN , Genes MDR , Humanos , Técnicas In Vitro , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos/síntesis química , Oligodesoxirribonucleótidos/química , Plásmidos/química , Plásmidos/genéticaRESUMEN
PURPOSE: Antigene radiotherapy (AR) is based on targeting localized radiodamage to specific sites in the genome by using sequence-specific triplex-forming oligonucleotides (TFO) to carry Auger-electron-emitters (A-Ettr) such as Iodine-125 (125I) to the target gene sequence. The radiodecay of an A-Ettr produces a cascade of low-energy electrons and creates a highly positively-charged daughter atom; delivered by a TFO, it should produce double-strand breaks (dsb) localized to the specific DNA target sequence. The result should be a "knock-out" of the targeted gene. METHODS AND MATERIALS: As a model, we used the MDR1 gene amplified nearly 100 times in the human KB-V1 carcinoma cell line. Chemically modified TFO complementary to the polypurine/polypyrimidine region of the MDR1 gene were synthesized and radiolabeled with 125I-dCTP by the primer extension method. Purified plasmid and genomic DNA and extracted nuclei were treated with 125I-TFO and analyzed for sequence-specific cleavage by electrophoresis in agarose gel and Southern hybridization. RESULTS: We created 125I-TFO that could effectively recognize, bind, and cleave the target sequence in plasmid and genomic DNA. We showed that these 125I-TFO in nanomolar concentrations were able to cleave the target MDR1 gene sequence in a natural environment, i.e., within the eucaryotic nucleus. CONCLUSION: 125I-TFO can effectively introduce sequence-specific dsb to a target within the MDR1 gene, both in purified DNA and inside intact nuclei. Chemically modified TFO conjugated with nuclear localization signal appear to be a promising delivery vehicle for future in vivo trials of AR.
Asunto(s)
Daño del ADN/genética , ADN de Neoplasias/efectos de la radiación , ADN/genética , Genes MDR/genética , Radioisótopos de Yodo/metabolismo , Oligonucleótidos/metabolismo , Radiofármacos/metabolismo , ADN/metabolismo , Cartilla de ADN/genética , Cartilla de ADN/uso terapéutico , ADN de Neoplasias/genética , Electroforesis en Gel de Agar , Genes MDR/efectos de la radiación , Humanos , Radioisótopos de Yodo/uso terapéutico , Hibridación de Ácido Nucleico/métodos , Oligonucleótidos/uso terapéutico , Plásmidos/metabolismo , Radiobiología , Radiofármacos/uso terapéutico , Radioterapia/métodos , Células Tumorales CultivadasRESUMEN
Triplex-forming oligonucleotides (TFOs) show potential for sequence-specific DNA binding and inhibition of gene expression. We have applied this antigene strategy using a TFO incorporating an Auger-emitting radionucleotide, 125I, to study the production of double-strand breaks (dsb) in the rat aquaporin 5 (rAQP5) cDNA. 125I-TFO bound to the pCMVrAQP5 plasmid in vitro in a dose-dependent manner and formed stable triplexes up to 65 degrees C and in the presence of 140 mM KCl. Further, 125I-TFO resulted in a predictable dsb when analyzed by Southern hybridization. To deliver TFOs to epithelial cells, we employed 125I-TFO-polyethyleneimine-adenovirus (125I-TFO-PEI-Ad) complexes. We hypothesized that these complexes would take advantage of adenoviral characteristics to transfer 125I-TFO to the cell nucleus. Adenovirus-containing complexes brought about greater uptake and nuclear localization of TFOs compared with delivery with 125I-TFO-PEI complexes alone. No significant degradation of 125I-TFO was found after delivery into cells using PEI-Ad complexes and freezing and thawing. We next used PEI-Ad complexes to deliver 125I-TFO and pCMVrAQP5 separately to epithelial cells to determine if triplexes can form de novo within cells, resulting in the specific dsb in the rAQP5 cDNA. After delivery, cell pellets were stored at -80 degrees C for more than 60 days. Thereafter, plasmid DNA was isolated from cells and analyzed for dsb by Southern hybridization. However, none were detected. We conclude that under the experimental conditions employed, effective triplexes, with 125I-TFO and pCMVrAQP5, do not form de novo inside cells.
Asunto(s)
Acuaporinas/metabolismo , ADN Complementario/metabolismo , ADN/metabolismo , Sistemas de Liberación de Medicamentos/métodos , Proteínas de la Membrana , Conformación de Ácido Nucleico , Oligonucleótidos/metabolismo , Adenoviridae/genética , Adenoviridae/metabolismo , Animales , Acuaporina 5 , Acuaporinas/genética , Southern Blotting , Línea Celular , Daño del ADN , Células Epiteliales/metabolismo , Regulación de la Expresión Génica , Genes Reporteros , Radioisótopos de Yodo/química , Oligonucleótidos/genética , Plásmidos/genética , Plásmidos/metabolismo , Polietileneimina/metabolismo , Ratas , TransfecciónRESUMEN
Antigene radiotherapy is our approach to targeting specific sites in the genome by combining the highly localized DNA damage produced by the decay of Auger electron emitters, such as 125I, with the sequence-specific action of triplex-forming oligonucleotides (TFO). As a model, we used the multidrug resistance gene (mdr1) overexpressed and amplified nearly 100 times in the human KB-V1 carcinoma cell line. Phosphodiester pyrrazolopyrimidine dG (PPG)-modified TFO complementary to the polypurine-polypyrimidine region of the mdr1 gene were synthesized and labeled with 125I-dCTP at the C5 position of two cytosines by the primer extension method. 125I-TFO were delivered into KB-V1 cells with several delivery systems. DNA from the 125I-TFO-treated cells was recovered and analyzed for sequence-specific cleavage in the mdr1 target by Southern hybridization. Experiments with plasmid DNA containing the mdr1 polypurine-polypyrimidine region and with purified genomic DNA confirmed the ability of the designed 125I-TFO to bind to and introduce double-strand breaks into the target sequence. We showed that 125I-TFO in nanomolar concentrations can recognize and cleave a target sequence in the mdr1 gene in situ, that is, within isolated nuclei and intact digitonin-permeabilized cells. Our results demonstrate the ability of 125I-TFO to target specific sequences in their natural environment, that is, within the eukaryotic nucleus. The nearly 100-fold amplification of the mdr1 gene in KB-V1 cells affords a very useful cell culture model for evaluation of methods to produce sequence-specific DNA double-strand breaks for gene-specific radiotherapy.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/antagonistas & inhibidores , ADN/farmacología , Oligonucleótidos Antisentido/farmacología , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Núcleo Celular/efectos de los fármacos , Núcleo Celular/genética , Supervivencia Celular/efectos de los fármacos , ADN/administración & dosificación , ADN/efectos de los fármacos , ADN/genética , ADN/metabolismo , Sistemas de Liberación de Medicamentos , Marcación de Gen , Vectores Genéticos , Humanos , Radioisótopos de Yodo , Células KB , Conformación de Ácido Nucleico , Oligonucleótidos Antisentido/administración & dosificación , Oligonucleótidos Antisentido/química , Oligonucleótidos Antisentido/genética , Plásmidos/genética , TransfecciónRESUMEN
We studied the stability of a DNA triplex resulting from the binding of a 38 nt long purine motif triplex-forming oligonucleotide (TFO) to a covalently closed plasmid containing a target sequence from the human HPRT gene. Our in vitro experiments showed that the triplex formed at plasmid and TFO concentrations as low as 10(-9)M. Once formed, the triplex was remarkably stable and could withstand 10 min incubation at 65 degrees C. We next delivered these TFO-plasmid complexes into cultured human cells. To monitor the TFO-plasmid complexes inside cells we applied a new technique that we call 'radioprinting'. Because the TFO was(125)I labeled, we could quantitatively monitor the triplexes by measuring(125)I-induced DNA strand breaks in the target plasmid sequence. We found that the triplexes remain stable inside the cells for at least 48 h. Based on these findings we propose using TFO for indirect labeling of intact plasmid DNA. As a demonstration, we show that the intracellular distribution of a fluorescein-labeled TFO was different when it was liposome-delivered into cultured human cells alone or in a complex with the plasmid. In the latter case, the fluorescence was detected in nearly all the cells while detection of the plasmid by use of a marker gene (beta-galactosidase) revealed expression of the gene in only half of the cells.