A microbeam study of DNA double-strand breaks in bystander primary human fibroblasts.

Radiation-induced bystander effect has been well documented. However, the mechanisms are poorly understood. How we incorporate this effect into the classical models of risk assessment remains an open question. Here, the induction of bystander effect was studied by assessing DNA double-strand break (DSB) formation in situ with the rapid and sensitive gamma-H2AX focus formation assay. Utilising the Columbia University single-cell microbeam system to deliver 2 or 20 individual alpha particles to selected cell nuclei in a precisely known proportion of cells in a population, the induced DNA DSB incidences were quantified 30 min and 18 h post-IR. The increase in DNA DSB incidence in bystander cells lacked of a linear dose response indicating that neither the dose of irradiation nor proportion of irradiated cells in a population, is a critical parameter. This study confirms a binary all-or-nothing model of triggering the bystander response. The delay and persistence of the bystander response suggests a different mechanism of DSB induction in bystander cells than in directly irradiated cells.

Antigene radiotherapy: targeted radiodamage with 125i-labeled triplex-forming oligonucleotides.

Antigene radiotherapy is based upon damaging selected genes by a high dose of radiation from radionuclides delivered to this gene by a sequence-specific DNA-binding molecule. Here we describe our recent trials of antigene radiotherapy using the human mdr1 gene over-expressed in KB-V1 cells as a model. As a delivery molecule, we used a triplex-forming oligonucleotide (TFO) with a binding site in intron 14 of mdr1. This TFO was labeled with an Auger-electron-emitting radionuclide 125I. Decay of 125I releases a shower of low energy electrons that produce DNA strand breaks mostly within 10 bp from the decay site. Targeting in situ was assessed by restriction enzyme digestion of the DNA recovered from the TFO-treated cells followed by Southern hybridization with DNA probes flanking the target sequence. Double-strand breaks in the target sequence were detected in purified nuclei and digitonin-permeabilized cells, but not in the intact cells when TFO were delivered with liposomes. On the basis of these observations we hypothesized that there are cytoplasmic factors that bind such TFO and deliver them into the nucleus, but do not release them inside the nucleus, thus preventing TFO from binding their genomic targets. To test this hypothesis we (i) delivered TFO along with an excess of unlabeled oligonucleotide with an arbitrary sequence ("ballast") and (ii) conjugated TFO with a nuclear localization sequence peptide (NLS). We have found that TFO/NLS conjugates cleaved the target in a concentration-dependent manner regardless of the presence of the "ballast" oligonucleotide. In contrast, TFO without NLS cleaved the target only in the presence of an excess of the "ballast." These results may provide a new insight into the mechanism of intracellular transport of oligonucleotides.

Sequence-specific gene cleavage in intact mammalian cells by 125I-labeled triplex-forming oligonucleotides conjugated with nuclear localization signal peptide.

Triplex-forming oligonucleotides (TFO) are designed to bind sequence specifically to their DNA targets without a significant disturbance of the double helix. They have been proposed to deliver DNA-reactive agents to specific DNA sequences for gene targeting applications. We suggested the use of 125I-labeled TFO for delivery of the energy of radioiodine decay to specific genes. This approach is called antigene radiotherapy. Here we demonstrate the ability of 125I-labeled TFO to produce sequence-specific breaks within a target in the human mdrl gene in cultured cells. TFO and TFO conjugated with a nuclear localization signal peptide (NLS) were delivered into cells using cationic liposomes. This was done either alone or in the presence of an excess of a "ballast" oligonucleotide with an unrelated sequence. In all cases, nuclear localization of TFO and survival of the cells after treatment has been confirmed. Breaks in the gene target were analyzed by restriction enzyme digestion of the DNA recovered from the TFO-treated cells followed by Southern hybridization with DNA probes flanking the target sequence. We have found that TFO/NLS conjugates cleave the target in a concentration-dependent manner regardless of the presence of the "ballast" oligonucleotide. In contrast, TFO without NLS cleaved the target only in the presence of an excess of the "ballast." We hypothesize that TFO and TFO/NLS are delivered into the nucleus by different pathways. These results provide a new insight into the mechanism of intracellular transport of oligonucleotides and open new avenues for improvement of the efficacy of antigene therapies.

Development of DNA-based radiopharmaceuticals carrying Auger-electron emitters for antigene radiotherapy.

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.

Use of polyethylenimine-adenovirus complexes to examine triplex formation in intact cells.

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.

Targeting the human mdr1 gene by 125I-labeled triplex-forming oligonucleotides.

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.

The stability of DNA triplexes inside cells as studied by iodine-125 radioprinting.

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.

Radiotoxicity of iodine-125-labeled oligodeoxyribonucleotides in mammalian cells.

UNLABELLED: We investigated the distribution, stability and radiotoxicity of 125I-oligodeoxyribonucleotides (125I-ODN) in human fibrosarcoma HT-1080 cells to study the radiotoxic effects of the Auger electron emitter 125I delivered to the cells by ODN. METHODS: We delivered 125I-ODN into the cells via complexing with a liposomal delivery system. To assess the intracellular distribution and stability of 125I-ODN delivered by the liposomal delivery system, we used autoradiography, fluorescent and confocal microscopy and electrophoresis. To study the radiotoxicity of the unbound 125I-ODN, we used a clonogenic assay. The radiotoxicity of 125I-ODN delivered by the liposomal delivery system was compared with that of freely diffusible 125I-antipyrine, membrane-excluded 125I-bovine serum albumin and DNA incorporated 125I-deoxyuridine (125I-UdR). RESULTS: Oligodeoxyribonucleotides accumulated in the cell nucleus within a few hours of incubation. On the basis of the number of decays at 37% survival, 125I-ODN are 2 times more radiotoxic than 125I-antipyrine, which is freely diffusible into cells, and 8 times more radiotoxic than 125I-bovine serum albumin, which remains outside cells. However, the radiotoxicity of unbound 125I-ODN is almost 3 orders of magnitude lower than that of DNA-incorporated 125I-UdR. The 125I-ODN are not significantly degraded by intracellular nucleases during the time of uptake incubation. CONCLUSION: The dramatic difference in radiotoxicity between 125I-ODN and 125I-UdR confirms that, despite the nuclear localization, 125I-ODN are not bound to or incorporated within the genomic DNA. Our data demonstrate that the radiotoxicity of Auger electron emitters is determined by the radiation dose delivered to nuclear DNA, not necessarily to the nucleus. Therefore, relatively high intracellular concentrations of unbound 125I-ODN can be achieved without causing significant cell death.

Triplex-forming oligonucleotides can modulate aquaporin-5 gene expression in epithelial cells.

Triplex-forming oligonucleotides (TFOs) may provide a useful approach to decrease gene transcription in vivo. We have identified two sequences in the rat aquaporin 5 (rAQP5) cDNA that are capable of forming a DNA triple helix. We designed four TFOs based on these sequences (a purine and a pyrimidine TFO per sequence). All four TFOs were able to bind to the rAQP5 cDNA at varying efficiencies in vitro as measured by using gel mobility shift assays. The TFOs were delivered to intact MDCK epithelial cells via adenovirus-polylysine complexes. Experiments with fluorescein-isothiocyanate-labeled oligonucleotides delivered in this way showed primarily a nuclear localization. Three of the four TFOs internalized by adenovirus-polylysine complexes were capable of decreasing rAQP5 expression in intact MDCK cells infected with a recombinant adenovirus encoding rAQP5. These data show that adenovirus-polylysine-TFO complexes can result in TFO delivery to the nucleus in intact epithelial cells and that TFOs may provide a useful way to selectively modulate rAQP5 gene expression.