Biomedical subjects

R D Neumann

Publications and source records attributed to R D Neumann.

At least 19 recordsLinked to original sources

PubMed2006

Microarray analysis of differentially expressed genes after exposure of normal human fibroblasts to ionizing radiation from an external source and from DNA-incorporated iodine-125 radionuclide.

Exposure of cells to ionizing radiation (IR) produces changes in the expression level of a large number of genes. However, less is known of gene-expression changes caused by local radiation exposure from radionuclides within cells. We studied changes in the genome-wide gene expression induced by decay of 125I incorporated into DNA as [125I]-iododeoxyuridine (125I-IUdR) in normal IMR-90 human lung fibroblasts and compared them with the changes produced by external gamma-radiation delivered at high (HDR) or low (LDR) dose rate. We found that more than 2000 genes were consistently up- or down-regulated following HDR and LDR gamma-radiation. The profiles of differentially expressed genes following HDR and LDR shared about 64% (up) and 74% (down) genes in common, with many genes identified as radiation-responsive for the first time. In contrast, in all only 206 genes changed their expression level in the 125I-IUdR-treated cells, even though the total number of DNA double-strand breaks (DSB) produced by 125I-IUdR exceeded that produced by the gamma-radiation. With few exceptions, the expression levels of 125I-IUdR-responsive genes were also altered following gamma-irradiation. Therefore, nuclear DNA-localized decays of 125I produce 10 times fewer differentially expressed genes than whole-cell exposure to gamma-radiation of comparable dose. These results suggest that the effect of IR on the changes in global gene expression depends on the distribution of energy depositions within the cell. In contrast to cell survival, DNA DSB may not be the major factor modulating changes in gene expression following irradiation.

Cell Line↗

PubMed2006

The issue of risk in complex adaptive systems: the case of low-dose radiation induced cancer.

Living systems exist in hierarchical levels of biological organization, ascending from the basic atomic-molecular level, to the cellular level, the tissue-organ level, and the whole organism. All levels and elements at each level communicate with each other though intricate intra- and intercellular signaling through many specified molecular interactions. These regulate homeostasis between the system levels and their individual elements. The probability of a defined effect at the basic atomic-molecular level per impact increment of a toxic agent, such as ionizing radiation, at that level appears constant at low doses, even if the probability constant may change as a consequence of a previous exposure. Thus, at a given state of the system, the incidence of effect at the atomic-molecular level increases linearly with the number of impact increments in terms of energy deposition events. Primary effects may amplify to damage and there are immediate attempts at repairing the damage from an effect. Amplification and propagation of damage at, and from, the basic to higher levels of biological organization meets resistance, the degree of which per impact increment is not constant. It changes with the number of impact increments. This resistance encompasses both physico-chemical and biochemical reactions. The corresponding biochemical reactions express the physiological system's capacity to respond to perturbations of homeostasis at and between the various levels. Types and degrees of these responses depend on the system and the degree of homeostatic perturbation. At relatively mild to moderate degrees of perturbation, protective responses appear with a delay of hours and may last for months, shield also against endogenous non-radiogenic damage, and in doing so may prevail over radiogenic damage. With increasing degrees of homeostatic perturbation, damage eventually overwhelms adaptive protection. Thus, systems do not respond in a linear function of impact increments at the lowest level of biological organization. For assessing the probability of radiation damage per absorbed dose, i.e., risk, in complex adaptive systems, both damaging and protecting responses need attention, and to exclude one for the other is scientifically unjustified and misleading.

Adaptation, Physiological↗

PubMed2005

Physics must join with biology in better assessing risk from low-dose irradiation.

This review summarises the complex response of mammalian cells and tissues to low doses of ionising radiation. This thesis encompasses induction of DNA damage, and adaptive protection against both renewed damage and against propagation of damage from the basic level of biological organisation to the clinical expression of detriment. The induction of DNA damage at low radiation doses apparently is proportional to absorbed dose at the physical/chemical level. However, any propagation of such damage to higher levels of biological organisation inherently follows a sigmoid function. Moreover, low-dose-induced inhibition of damage propagation is not linear, but instead follows a dose-effect function typical for adaptive protection, after an initial rapid rise it disappears at doses higher than approximately 0.1-0.2 Gy to cells. The particular biological response duality at low radiation doses precludes the validity of the linear-no-threshold hypothesis in the attempt to relate absorbed dose to cancer. In fact, theory and observation support not only a lower cancer incidence than expected from the linear-no-threshold hypothesis, but also a reduction of spontaneously occurring cancer, a hormetic response, in the healthy individual.

Adaptation, Physiological↗

PubMed2003

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.

Animals↗

PubMed2002

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.

Amino Acid Sequence↗

PubMed2002

Simulation of 125I induced DNA strand breaks in a CAP-DNA complex.

The E. coli catabolite gene activator protein (CAP)-DNA complex with 125I located at the position of the H5 atom of the cytosine near the centre was incorporated into the PARTRAC track structure code. DNA strand breaks due to irradiation were calculated by track structure and radical attack simulations; strand breaks due to neutralisation of the highly charged 125Te ion were derived from a semi-empirical distribution. According to the calculations, the neutralisation effect dominates the strand breakage frequency at 2 bases away from the 125I decay site on both strands. The first breakage distribution counted from a 32P labelled end on the strand with 125I agreed well with experimental data, but on the opposite strand, the calculated distribution is more concentrated around the decay site and its yield is about 20% larger than the measured data.

Base Pairing↗

PubMed2001

In vitro repair of complex unligatable oxidatively induced DNA double-strand breaks by human cell extracts.

We describe a new assay for in vitro repair of oxidatively induced DNA double-strand breaks (DSBs) by HeLa cell nuclear extracts. The assay employs linear plasmid DNA containing DNA DSBs produced by the radiomimetic drug bleomycin. The bleomycin-induced DSB possesses a complex structure similar to that produced by oxidative processes and ionizing radiation. Bleomycin DSBs are composed of blunt ends or ends containing a single 5'-base overhang. Regardless of the 5'-end structure, all bleomycin-induced DSBs possess 3'-ends blocked by phosphoglycolate. Cellular extraction and initial end joining conditions for our assay were optimized with restriction enzyme-cleaved DNA to maximize ligation activity. Parameters affecting ligation such as temperature, time, ionic strength, ATP utilization and extract protein concentration were examined. Similar reactions were performed with the bleomycin-linearized substrate. In all cases, end-joined molecules ranging from dimers to higher molecular weight forms were produced and observed directly in agarose gels stained with Vistra Green and imaged with a FluorImager 595. This detection method is at least 50-fold more sensitive than ethidium bromide and permits detection of </=0.25 ng double-stranded DNA per band in post-electrophoretically stained agarose gels. Consequently, our end-joining reaction requires </=100 ng substrate DNA and >/=50% conversion of substrate to product is achieved with simple substrates such as restriction enzyme-cleaved DNA. Using our assay we have observed a 6-fold lower repair rate and a lag in reaction initiation for bleomycin-induced DSBs as compared to blunt-ended DNA. Also, end joining reaction conditions are DSB end group dependent. In particular, bleomycin-induced DSB repair is considerably more sensitive to inhibition by increased ionic strength than repair of blunt-ended DNA.

Adenosine Triphosphate↗

PubMed2001

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.

DNA↗

PubMed2001

A prospective study of 2-[18F] fluoro-2-deoxy-D-glucose/positron emission tomography scan, 99mTc-labeled arcitumomab (CEA-scan), and blind second-look laparotomy for detecting colon cancer recurrence in patients with increasing carcinoembryonic antigen levels.

BACKGROUND: An increasing carcinoembryonic antigen (CEA) level in the absence of disease on imaging studies can present a diagnostic challenge. We evaluated 2-[18F] fluoro-2-deoxy-D-glucose and positron emission tomography (FDG-PET) scan and CEA scan before second-look laparotomy as a means of localizing recurrent colorectal cancer. METHODS: Patients underwent computed tomography scan, bone scan, colonoscopy, and magnetic resonance imaging, and those without evidence of disease or resectable disease in the abdomen had FDG-PET and CEA scans. At second-look laparotomy, a surgeon blinded to the results of the FDG-PET and CEA scans performed an exploration and mapped findings. A second surgeon, with knowledge of the FDG-PET and CEA scans, then explored the patient; all lesions were biopsied or resected for pathology. RESULTS: In 28 patients explored, disease was found at operation in 26 (94%). Ten had unresectable disease. FDG-PET scans predicted unresectable disease in 90% of patients. CEA scans failed to predict unresectable disease in any patient. In 16 patients found to have resectable disease or disease that could be treated with regional therapy, FDG-PET scan predicted this in 81% and CEA scan in 13%. CONCLUSIONS: FDG-PET scan can predict those patients who would likely benefit from a laparotomy. If the FDG-PET scan indicates resectable disease, laparotomy can be considered. However, if the findings predict unresectable disease or the absence of disease, the patient should pursue systemic therapy or continued observation.

Adult↗

PubMed2001

Strand breaks in whole plasmid dna produced by the decay of (125)I in a triplex-forming oligonucleotide.

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.

Base Sequence↗

PubMed2000

Radioprobing of a RecA-three-stranded DNA complex with iodine 125: evidence for recognition of homology in the major groove of the target duplex.

A fundamental problem in homologous recombination is how homology between DNAs is recognized. In all current models, a recombination protein loads onto a single strand of DNA and scans another duplex for homology. When homology is found, a synaptic complex is formed, leading to strand exchange and a heteroduplex. A novel technique based on strand cleavage by the Auger radiodecay of iodine 125, allows us to determine the distances between (125)I on the incoming strand and the target sugars of the duplex DNA strands in an Escherichia coli RecA protein-mediated synaptic complex. Analysis of these distances shows that the complex represents a post-strand exchange intermediate in which the heteroduplex is located in the center, while the outgoing strand forms a relatively wide helix intertwined with the heteroduplex and located in its minor groove. The structure implies that homology is recognized in the major groove of the duplex.

Base Sequence↗

PubMed2000

Distribution of DNA strand breaks produced by iodine-123 and indium-111 in synthetic oligodeoxynucleotides.

Antigene radiotherapy, a procedure based on delivery of short-range Auger-electron-emitting radioisotopes to target genes via sequence-specific triplex-forming oligonucleotides, has been successfully demonstrated in vitro using the well-studied radionuclide 125I. To proceed with in vivo trials, Auger electron emitters with shorter half-lives than 125I are required. Here we report a study of the efficiency and distribution of sequence-specific DNA strand breaks produced by decay of 123I and mIIn. 123I and 111In were introduced into triplex-and duplex-forming oligodeoxyribonucleotides (ODNs) through carbohydrate linkers of various lengths. Labeling with radioiodine was performed through tributylstannylbenzamide intermediates while 111In was attached via DTPA. The Auger-emitter-labeled ODNs were hybridized to a single-stranded DNA target, to form duplexes. After decay accumulation, the target DNA samples were assayed for strand breaks using a sequencing gel-electrophoresis technique. For the first time, we observed footprints of DNA strand breaks produced by 123I and 111In. Most of the breaks were located within 10 nucleotides from the decay site. The yield of strand breaks per decay varies; decay of 111In breaks DNA almost 10 times more effectively than decay of 123I. Both 123I and 111In are less effective in breaking DNA strands than 121I, which reflects the higher total energy of the Auger decay process of 125I.

Binding Sites↗

PubMed2000

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.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2000

Pulmonary lymphangioleiomyomatosis: correlation of ventilation-perfusion scintigraphy, chest radiography, and CT with pulmonary function tests.

PURPOSE: To determine the findings on ventilation-perfusion (V-P) scintigrams, computed tomographic (CT) scans, and chest radiographs and correlate them with pulmonary function test results in patients with lymphangioleiomyomatosis. MATERIALS AND METHODS: V-P scintigraphy, chest radiography, conventional and thin-section CT, and pulmonary function tests were performed in 39 patients. The images were graded on a scale of 0 (normal) to 3 (severely abnormal). RESULTS: Imaging abnormalities were found on 92% of ventilation scintigrams, 92% of perfusion scintigrams, 79% of chest radiographs, 100% of CT scans, and 100% of thin-section CT scans. On ventilation scintigrams, 28 (72%) patients demonstrated a speckling pattern. On CT scans, all patients had pulmonary cysts. Univariate analysis showed that extent of disease on chest radiographs and CT scans, cyst size, V-P abnormalities, and degree of speckling were inversely correlated with forced expiratory volume in one second (FEV(1)), diffusing capacity of lung for carbon monoxide, and the ratio of FEV(1) to forced vital capacity (FVC) (P <.01) but not with FVC and total lung capacity. Larger cyst size correlated with extent of disease at CT, but not significantly (P =.056). CONCLUSION: Scintigraphic and radiologic abnormalities are seen in a majority of patients with lymphangioleiomyomatosis. On ventilation scintigrams, a frequently seen speckling pattern may be related to accumulation of radionuclide in pulmonary cysts-a hallmark of the disease at CT. Findings with each imaging modality correlate with certain pulmonary functions.

Adult↗

PubMed2000

DNA cleavage by 111In-labeled oligodeoxyribonucleotides.

UNLABELLED: We studied the fine structure of DNA damage produced by the decay of 111In incorporated into duplex and triplex DNA strands to evaluate the usefulness of this radionuclide for sequence-specific DNA cleavage. METHODS: Oligodeoxyribonucleotides (ODNs) were prepared with 111In attached by diethylenetriaminepentaacetic acid (DTPA) at the 5' end or 3' end through a long chemical linker or to an internal nucleotide position through a short linker. Subsequent formation of DNA duplexes and triplexes was confirmed by gel electrophoresis. The 111In-induced breaks were assayed in denaturing polyacrylamide gel electrophoresis with a single-nucleotide resolution. RESULTS: 111In-labeled oligonucleotides of high specific activity (740-1554 TBq/mmol) were synthesized. The presence of the bulky 111In-DTPA group did not impede duplex or triplex formation. Localized DNA breaks were observed in all duplexes and triplexes formed. The majority of DNA breaks in duplex formations were located within +/- 10 nucleotides from the site of attachment of the 111In-bearing linker. The yield of DNA breaks per decay was 0.38 in a duplex with internally modified ODNs. This is nearly 2 times less than the yield of DNA breaks in the same duplex with 1251 attached through the same linker. The yield of DNA breaks in the pyrimidine and purine strands of DNA triplexes with 111In attached to the triplex-forming ODNs through the linkers of different length varied from 0.05 to 0.10. The distribution of DNA breaks was wider in comparison with the duplex experiment. The lower yields of breaks per 111In decay compared with 125I may be not only the result of lower deposited energy but also of the ionic repulsion of the negatively charged 111In-DTPA group from the DNA strands. CONCLUSION: We have shown that decay of 111In produces highly localized DNA breaks. 111In introduced into triplex- and duplex-forming ODNs through hydrocarbon linkers produces sequence-specific DNA strand breaks with an efficiency nearly comparable with that of 1251. These findings are supportive of our proposed use of 111In-ODNs for gene-specific radiotherapy.

DNA↗

PubMed2000

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

Targeting of radiation damage to specific DNA sequences is the essence of antigene radiotherapy. This technique also provides a tool to study molecular mechanisms of DNA repair on a defined, single radiodamaged site. We achieved such sequence-specific radiodamage 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). TFO complementary to polypurine-polypyrimidine regions of human genes were synthesized and labeled with 125I-dCTP by the primer extension method. 125I-TFO were delivered into cells with several delivery systems. In addition, human enzymes capable of supporting DNA single-strand-break repair were isolated and assessed for their role in the repair of this lesion. Also, the mutagenicity and repairability of 125I-TFO-induced double strand breaks (DSB) were assessed by repair of a plasmid possessing a site-specific DSB lesion. Using plasmids containing target polypurine-polypyrimidine tracts, we obtained the fine structure of sequence-specific DNA breaks produced by decay of 125I with single-nucleotide resolution. We showed that the designed 125I-TFO in nanomolar concentrations could bind to and introduce double-strand breaks into the target sequences in situ, i.e., within isolated nuclei and intact digitonin-permeabilized cells. We also showed 125I-TFO-induced DSB to be highly mutagenic lesions resulting in a mutation frequency of nearly 80%, with deletions comprising the majority of mutations. The results obtained demonstrate the ability of 125I-TFO to target specific sequences in their natural environment--within eucaryotic nucleus. Repair of 125I-TFO-induced DNA damage should typically result in mutagenic gene inactivation.

Animals↗

PubMed1999

Gene targeted DNA double-strand break induction by (125)I-labeled triplex-forming oligonucleotides is highly mutagenic following repair in human cells.

A parallel binding motif 16mer triplex-forming oligonucleotide (TFO) complementary to a polypurine-polypyrimidine target region near the 3'-end of the SupF gene of plasmid pSP189 was labeled with [5-(125)I]dCMP at position 15. Following triplex formation and decay accumulation, radiation-induced site-specific double-strand breaks (DSBs) were produced in the pSP189 SupF gene. Bulk damaged DNA and the isolated site-specific DSB-containing DNA were separately transfected into human WI38VA13 cells and allowed to repair prior to recovery and analysis of mutants. Bulk damaged DNA had a relatively low mutation frequency of 2.7 x 10(-3). In contrast, the isolated linear DNA containing site-specific DSBs had an unusually high mutation frequency of 7.9 x 10(-1). This was nearly 300-fold greater than that observed for the bulk damaged DNA mixture, and >1.5 x 10(4)-fold greater than background. The mutation spectra displayed a high proportion of deletion mutants targeted to the(125)I binding position within the SupF gene for both bulk damaged DNA and isolated linear DNA. Both spectra were characterized by complex mutations with mixtures of changes. However, mutations recovered from the linear site-specific DSB-containing DNA presented a much higher proportion of complex deletion mutations.

Base Sequence↗

PubMed1999

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.

Base Sequence↗