Radiation-induced DNA Strand Breaks Research Articles

Impaired DNA Repair as Assessed by the “Comet” Assay in Patients with Thyroid Tumors After a History of Radiation Therapy: A Preliminary Study

Purpose: Patients with a history of head and neck irradiation in childhood are at risk to develop thyroid tumors. The aim of this study was to determine if an impairement of DNA strand breaks repair could account for this observation. Methods and Materials: Circulating unstimulated lymphocytes of a group of 13 patients who developed thyroid tumors after radiotherapy were submitted to the alkaline single-cell gel electrophoresis assay (SCGE or “comet” assay) after in vitro exposure to 2 and 5 Gy of γ-rays. A control group of 8 healthy donors and 2 cases with a history of neck irradiation who did not develop a thyroid tumor were also analysed. The immediate response was compared to that observed after 15, 30, and 60 min of postexposure incubation periods. Results: Induction of DNA strand breaks is a dose–dependent process. The SCGE assay parameters did not differ significantly between patients and controls immediately ( t = 0) after irradiation at the two doses used. As compared to healthy donors, a slower kinetics of repair was found in the patients. The proportion of residual damage at 60 min postirradiation was significantly ( p < 0.01) higher in patients than in controls, at both doses analysed. Flow cytometric analysis of apoptosis and p53 protein status studied before and after irradiation showed no apparent relationship with the repair capacity. Conclusion: This preliminary study suggests that a subgroup of patients who develop thyroid tumors after a history of irradiation are partially defective in the late restitution of in vitro radiation-induced DNA strand breaks. This deficiency could be a predisposing factor to radiation-associated thyroid tumorigenesis. Detection of susceptible individuals using the simple and rapid comet assay, especially children receiving radiotherapeutic treatment, may allow a preventive surveillance for radiation-associated epithelial thyroid tumor development.

Radiation-Induced DNA Damage in Tumors and Normal Tissues: IV. Influence of Proliferation Status and Cell Type on the Formation of Oxygen-Dependent DNA Damage in Cultured Cells

Using a variety of techniques, several laboratories have recently demonstrated the feasibility of using radiation-induced DNA strand breaks and/or DNA-protein crosslinks (DPCs) to detect and/or quantify hypoxic cells in tumors and normal tissues. However, if strand breaks and/or DPCs are to be used to estimate the hypoxic fraction or the fractional hypoxic volume of tumors and normal tissues, their formation as a function of the oxygen concentration near the DNA must be relatively independent of the biological properties of these cells. In the present study, the shape of the oxygen dependence curves and the K(m) values for radiation-induced strand breaks and DPCs were measured by alkaline elution for proliferative (P) and quiescent (Q) cells of the mouse mammary adenocarcinoma, line 66. The sigmoidal shape of the oxygen dependence curves, the K(m) for strand breaks (approximately 0.027 mM) and the K(m) for the formation of DPCs (approximately 0.020 mM) were identical for the P and Q cells of line 66. Consequently, the proliferative status of these tumor cells had no measurable influence on the oxygen-dependent formation of radiation-induced strand breaks and DPCs. In addition, the percentage of the DNA retained on the filters after approximately 24 ml of elution without proteinase K in the lysis solution, a parameter equal to the sum of the strand breaks and DPCs that has been shown to be proportional to the percentage of hypoxic cells in the sample, was not significantly different for fully oxygenated or fully hypoxic populations from five tumor cell lines that varied in species, site of origin, proliferative status and/or properties of the proteins which are intimately associated with their DNA. These data indicate that the formation of radiation-induced strand breaks and DPCs depends predominantly on the oxygen concentration in the microenvironment around the DNA, and only minimally on the biological properties of the cells.

Interaction of DNA-Dependent Protein Kinase and Poly(ADP-Ribose) Polymerase with Radiation-Induced DNA Strand Breaks

Two of the enzymes involved in the response of mammalian cells to ionizing radiation are the DNA-dependent protein kinase and poly(ADP-ribose) polymerase. These enzymes are known to be activated by binding to DNA strand breaks, but previous studies designed to look at strand break specificity have employed enzymatically generated strand breaks and not irradiated DNA. Using highly purified DNA-dependent protein kinase, we compared enzyme activation by a series of DNA substrates. Irradiated plasmid DNA activated DNA-dependent protein kinase in a dose-dependent manner. When calculated in terms of the molar concentration of double-strand breaks, the enzyme activation by irradiated DNA was comparable to that by restriction enzyme-cleaved DNA. Linear DNA purified after plasmid irradiation also activated DNA-dependent protein kinase to a comparable extent, but nicked DNA, either isolated from irradiated plasmid or generated by DNase I, failed to activate the enzyme. A comparison of the enzyme activation by plasmid molecules with different 3'- and 5'-terminal groups indicated that the chemical nature of the DNA termini at the double-strand break does not significantly influence the response of the DNA-dependent protein kinase. Similar experiments with poly(ADP-ribose) polymerase demonstrated that single- and double-strand breaks activate this enzyme with almost equal efficiency, but because of their greater number, single-strand breaks dominate the response of poly(ADP-ribose) polymerase to irradiated DNA.

Melatonin and a spin-trap compound block radiofrequency electromagnetic radiation-induced DNA strand breaks in rat brain cells.

Effects of in vivo microwave exposure on DNA strand breaks, a form of DNA damage, were investigated in rat brain cells. In previous research, we have found that acute (2 hours) exposure to pulsed (2 microseconds pulses, 500 pps) 2450-MHz radiofrequency electromagnetic radiation (RFR) (power density 2 mW/cm2, average whole body specific absorption rate 1.2 W/kg) caused an increase in DNA single- and double-strand breaks in brain cells of the rat when assayed 4 hours post exposure using a microgel electrophoresis assay. In the present study, we found that treatment of rats immediately before and after RFR exposure with either melatonin (1 mg/kg/injection, SC) or the spin-trap compound N-tert-butyl-alpha-phenylnitrone (PBN) (100 mg/kg/injection, i.p.) blocks this effects of RFR. Since both melatonin and PBN are efficient free radical scavengers it is hypothesized that free radicals are involved in RFR-induced DNA damage in the brain cells of rats. Since cumulated DNA strand breaks in brain cells can lead to neurodegenerative diseases and cancer and an excess of free radicals in cells has been suggested to be the cause of various human diseases, data from this study could have important implications for the health effects of RFR exposure.

Radiation-Induced DNA Damage in Tumors and Normal Tissues. III. Oxygen Dependence of the Formation of Strand Breaks and DNA-Protein Crosslinks

Results from several laboratories, including ours, have suggested that measurements of radiation-induced DNA strand breaks and DNA-protein crosslinks (DPCs) may be used to estimate the hypoxic fraction or fractional hypoxic volume of tumors and normal tissues. This suggestion has been predicted on both published and unpublished information that (1) the oxygen dependence of the formation of strand breaks in irradiated mammalian cells is similar to the oxygen dependence of radiation-induced cell killing, and (2) the oxygen dependence of the formation of DPCs in irradiated mammalian cells is the mirror image of the oxygen dependence of radiation-induced cell killing. However, the published studies that attempted to determine the relationship between the oxygen dependence of the formation of strand breaks and the radiation sensitivity of mammalian cells were not performed at 37 degrees C, the exact oxygen concentrations were not always known, and the results were conflicting. In addition, most of the data on the oxygen dependence of the formation of DPCs are unpublished. Consequently, we have undertaken a comprehensive investigation of one cell line, 9L/Ro rat brain tumor cells, to determine if the shape of the oxygen dependence curve and the Km value for radiation-induced strand breaks and DPCs were similar when 9L cells were irradiated under both ideal gas-liquid equilibrium conditions at 4 degrees C and nonideal gas-liquid equilibrium conditions at 37 degrees C. At 4 degrees C under ideal gas-liquid equilibrium conditions, the Km for the formation of strand breaks was approximately 0.0045 mM, and the Km for radiation sensitivity was approximately 0.005 mM. A similar comparison for the formation of DPCs at 4 degrees C could not be made, because the efficiency of the formation of DPCs was much lower at 4 degrees C than at 37 degrees C. At 37 degrees C under nonideal gas-liquid equilibrium conditions, the apparent Km for the formation of strand breaks and radiation sensitivity was approximately 0.032 mM, and the Km for the formation of DPCs was approximately 0.02 mM. The data for strand breaks are in agreement with the published data of Chapman et al. (Int. J. Radiat. Biol. 26, 383-389, 1974), and the data for DPCs are in agreement with the unpublished data of Meyn (personal communication). These results support the suggestion that measurements of radiation-induced strand breaks and/or DPCs may be used to detect hypoxic cells and estimate the hypoxic fraction or fractional hypoxic volume of tumors and normal tissues.

Chromatin structure and radiation-induced DNA strand breaks in human cells: soluble scavengers and DNA-bound proteins offer a better protection against single- than double-strand breaks.

In the present investigation we have studied the formation of DNA double-strand breaks (dsbs) in different chromatin substrates and made a comparison with our previous results on radiation-induced single-strand breaks (ssb). Removal of soluble scavengers increased the number of ssbs by a factor of 2, but increased dsbs only 1.2 times. Decondensation of the chromatin increased ssbs and dsbs to the same extent, by a factor of 3-4. Removal of DNA-bound proteins gave an additional increase in ssbs and dsbs by a factor of 14 and 5 respectively. The increase in both ssbs and dsbs was caused by OH radicals. The differential effect on ssbs and dsbs can be explained by assuming that dsbs are formed mainly by multiple hits from clusters of ionizations close to the DNA. When the DNA-bound proteins are removed, more distant radicals can reach the DNA, which favours ssb formation. Under those conditions dsbs also induced by single OH radicals become important. DNA-bound proteins protect DNA only to a limited extent by acting as scavengers of OH radicals. The largest part of the protection depends on the fact that DNA in the chromatin is made up of large, compact aggregates, where the distance between separate aggregates exceeds the effective range of the OH radicals and the amount of water inside the aggregates is too small to give a maximal contribution of OH radicals compared with when DNA is more evenly distributed in a given volume.

Determination of radiation-induced DNA strand breaks in individual cells by non-radioactive labelling of 3' OH ends.

To quantify DNA strand breaks generated by ionizing radiation in single cells with preserved morphology, the number of radiation-induced 3' OH ends of nuclear DNA was determined by enzymatic labelling. Confluent CHO-K1 cells were irradiated with doses up to 100 Gy. After fixation and permeabilization of the cell monolayer the nuclear DNA was labelled with Digoxigenin-11-dUTP using terminal transferase. The incorporated nucleotide was detected with an anti-Digoxigenin antibody conjugated with alkaline phosphatase. The phosphatase bound was quantified by a colour reaction and the integrated optical densities of the cell nuclei were measured. For doses ranging from 20 to 100 Gy a linear relationship between dose and labelling signal was obtained. Repair experiments showed a fast component of repair with a half-time of about 14 min, followed by a slower decline to background values, which were reached after 6-8 h. This method allows the measurement of radiation-induced DNA strand breaks in morphologically preserved single cells in a reproducible way, which may be of importance in the prediction of tumour response in radiotherapy.

Ionizing radiation-induced DNA strand breakage and rejoining in specific genomic regions as determined by an alkaline unwinding/Southern blotting method.

A recently developed, combined alkaline unwinding/Southern blotting assay was utilized to examine DNA damage and repair induced by ionizing radiation within specific large-scale genomic regions. Following treatment of MCF-7 breast tumour cells with 2-10-Gy gamma-rays, strand breakage and rejoining were measured in bulk DNA, in the centromeric alpha-satellite region of chromosome 17, and in the chromatin regions containing the unexpressed beta-globin gene and the expressed c-myc oncogene, which is known to be important for growth in the MCF-7 cell line. Damage in both the c-myc and beta-globin regions was markedly greater than in either alpha-satellite or bulk DNA. However, the kinetics of strand break repair were approximately the same in c-myc as in alpha-satellite or bulk DNA. Surprisingly, the radiomimetic antibiotic bleomycin, which also induces free-radical-mediated strand breakage, showed considerably less heterogeneity of DNA damage among the genomic regions examined than did radiation. The results suggest that actively transcribed genes, as well as at least some inactive genes, are surrounded by large-scale domains of radiosensitive chromatin. With no apparent enhancement of rejoining, the increased incidence of strand breaks in these regions persists until rejoining is essentially complete. Changes in the integrity of specific chromatin regions may be an important aspect of DNA damage-induced cell death.

Radiation-Induced DNA Damage in Tumors and Normal Tissues. II. Influence of Dose, Residual DNA Damage and Physiological Factors in Oxygenated Cells

Detection and quantification of hypoxic cells in solid tumors is important for many experimental and clinical situations. Several laboratories, including ours, have suggested that assays which measure radiation-induced DNA strand breaks and DNA-protein crosslinks (DPCs) might be used to detect or quantify hypoxic cells in tumors and normal tissues. Recently, we demonstrated the feasibility of using an alkaline elution assay that measures strand breaks and DPCs to detect and/or quantify hypoxic cells in tissues. For this approach to be valid, DPCs must not be formed to any great extent in irradiated oxygenated cells, and the formation and repair of strand breaks and DPCs in oxygenated cells must not be modified appreciably by physiological factors (e.g. temperature, pH and nutrient depletion) that are often found in solid tumors. To address these issues, two sets of experiments were performed. In one set of experiments, oxygenated 9L cells in tissue culture, subcutaneous 9L tumors and rat cerebella were irradiated with doses of 15 or 50 Gy and allowed to repair until the residual strand break damage was low enough to detect DPCs. In another set of experiments, oxygenated exponentially growing or plateau-phase 9L cells in tissue culture were irradiated with a dose of 15 Gy at 37 or 20 degrees C, while the cells were maintained at a pH of either 6.6 or 7.3. DNA-protein crosslinks were formed in oxygenated cells about 100 times less efficiently than in hypoxic cells. In addition, temperature, pH, nutrient depletion and growth phase did not appreciably alter the formation and repair of strand breaks or the formation of DPCs in oxygenated 9L cells. These results support the use of this DNA damage assay for the detection and quantification of hypoxic cells in solid tumors.

Caffeine potentiates or protects against radiation-induced DNA and chromosal damage in human lymphocytes depending on temperature and concentration

The effect of caffeine on raditiona-induced chromosal aberrations and DNA strand breaks in unstimulated human lymphocytes was investigated. When present prior to and during the radiation exposure, caffeine treatment was fojnd to cause either potentiation or protection agains induction of chromosal aberrations depending on the concentration and temperature. When the nucleoid sedimentation technique was applied, enhancement or reduction of radiation-induced DNA strand breaks by caffeine was also found to be dependent on temperature and caffeine concentration. It is proposed that caffeine, in addition to its suspected ability to influence DNA repair, can also influence the induction of DNA damage, leading to alterations in the yield of chromosal aberrations.

The Effect of Polyamine Depletion on the Cytotoxic Response to PUVA, Gamma Rays, and UVC in V79 Cells in Vitro

Difluoromethylornithine (DFMO) depletes cells of polyamines, sensitizing cells against the action of antineoplastic drugs and altering ability to repair radiation-induced DNA strand breaks. Others have hypothesized that the mechanism by which polyamine depletion sensitizes cells is through inhibition of DNA strand break repair or through altering the spectrum of initial DNA damage. We have compared the effect of polyamine depletion on cytotoxic effects in V79 cells for three agents that damage DNA: PUVA (8-methoxypsoralen and ultraviolet light, 365 nm), γ-rays and UVC (ultraviolet light, 254 nm) in polyamine depleted V79 cells. DFMO pretreatment sensitizes cells to PUVA and γ-ray toxicity but not to UVC. Unlike UVC photoinduction of DNA lesions, PUVA- and γ-ray-induced DNA damage is modulated by chromatin structure. Our results suggest that polyamine depletion sensitizes cells to the action of PUVA and γ-rays by mechanisms disparate from those for UVC, suggesting that the level or type of initial damage, rather than DNA repair, per se, may be the more important determinant of enhanced cytotoxicity.

Metal-ion assisted, radiation-induced conversion of B- to Z-DNA in poly (dGdC) and two natural DNAs.

Metal-ion assisted, gamma-radiation-induced B-Z conformation changes have been observed with UV and circular dichroism spectroscopy for poly (dGdC), calf thymus and herring testis DNA. These conformational changes are similar to those induced by increasing multivalent metal ion concentrations in DNA containing alternating purine and pyrimidine base sequences. In both the metal-ion-induced and the metal-ion-assisted, radiation-induced conformation changes, the conversions were from the right-handed B-DNA to the left-handed Z-DNA conformation. It is proposed that radiation-induced DNA strand breaks markedly reduce the high activation energy barrier in the metal ion-driven B-Z conformation conversion and allow much smaller metal ion concentrations to induce this conversion than in the absence of strand breaks. The biological importance of such radiation-induced conformational changes is discussed in terms of the potential significance of the Z-DNA conformation in the control of the DNA transcription process.

Poly(ADP-ribose) polymerase activity in intact or permeabilized leukocytes from mammalian species of different longevity

Poly(ADP-ribosyl)ation is a eukaryotic posttranslational protein modification catalyzed by poly(ADP-ribose) polymerase (PARP), a highly conserved nuclear enzyme which uses NAD as substrate. We have previously tested PARP activity in permeabilized mononuclear blood cells (MNC) from 13 mammalian species as a function of the species-specific life span. A direct and maximal stimulus of PARP activation was provided by including saturating amounts of a double-stranded oligonucleotide in the PARP-reaction buffer. The data yielded a strong positive correlation between PARP activities and the species' maximal life spans (r = 0.84; p << 0.001). Here, we investigated the formation of poly(ADP-ribose) in living MNC from two mammalian species with widely differing longevity (rat and man) by immunofluorescence detection of poly(ADP-ribose). The fraction of positive cells was recorded, following gamma-irradiation of intact MNC, as a semiquantitative estimation of poly(ADP-ribose) formation. Human samples displayed a significantly higher percentage of positivity than did those from rats, consistent with our previous results on permeabilized cells. While rat MNC had a higher NAD content than human MNC, the number of radiation-induced DNA strand breaks was not significantly different in the two species. Since poly(ADP-ribosyl)ation is apparently involved in DNA repair and the cellular recovery from DNA damage, we speculate that the higher poly(ADP-ribosyl)ation capacity of long-lived species might more efficiently help to slow down the accumulation of unrepaired DNA damage and of genetic alterations, as compared with short-lived species.

Establishment of Hypoxic Conditions for Cultured Monolayer Cells: Hypoxia in Five Minutes or Less

A versatile deaeration chamber for altering the gaseous environment of monolayer cell cultures has been developed. The design is such that multiple samples of radioactively labeled or unlabeled cells can be gassed simultaneously with a short period of time. Deaeration times to reduce the level of oxygen in initially aerated cellular medium to very low levels (less than 20 parts per million) were decreased at least 6- to 20-fold when compared to most other methods commonly used to achieve N2- or N2O-induced hypoxia. The application of this chamber in the investigation of radiation-induced cellular DNA strand breaks, which are very sensitive to the presence of oxygen, is described. The vessel should be useful for a variety of metabolic and physiologic experiments or cell, tumor, and radiation biology studies.

Acquisition of telomere repeat sequences by transfected DNA integrated at the site of a chromosome break.

Previous analysis of plasmid DNA transfected into 108 cell clones demonstrated extensive polymorphism near the integration site in one clone. This polymorphism was apparent by Southern blot analysis as diffuse bands that extended over 30 kb. In the present study, nucleotide sequence analysis of cloned DNA from the integration site revealed telomere repeat sequences at the ends of the integrated plasmid DNA. The telomere repeat sequences at one end were located at the junction between the plasmid and cell DNA. The telomere repeat sequences at the other end were located in the opposite orientation in the polymorphic region and were shown by digestion with BAL 31 to be at the end of the chromosome. Telomere repeat sequences were not found at this location in the plasmid or parent cell DNA. Although the repeat sequences may have been acquired by recombination, a more likely explanation is that they were added to the ends of the plasmid by telomerase before integration. Comparison of the cell DNA before and after integration revealed that a chromosome break had occurred at the integration site, which was shown by fluorescent in situ hybridization to be located near the telomere of chromosome 13. These results demonstrate that chromosome breakage and rearrangement can result in interstitial telomere repeat sequences within the human genome. These sequences could promote genomic instability, because short repeat sequences can be recombinational hotspots. The results also show that DNA rearrangements involving telomere repeat sequences can be associated with chromosome breaks. The introduction of telomere repeat sequences at spontaneous or ionizing radiation-induced DNA strand breaks may therefore also be a mechanism of chromosome fragmentation.

Acquisition of Telomere Repeat Sequences by Transfected DNA Integrated at the Site of a Chromosome Break

Previous analysis of plasmid DNA transfected into 108 cell clones demonstrated extensive polymorphism near the integration site in one clone. This polymorphism was apparent by Southern blot analysis as diffuse bands that extended over 30 kb. In the present study, nucleotide sequence analysis of cloned DNA from the integration site revealed telomere repeat sequences at the ends of the integrated plasmid DNA. The telomere repeat sequences at one end were located at the junction between the plasmid and cell DNA. The telomere repeat sequences at the other end were located in the opposite orientation in the polymorphic region and were shown by digestion with BAL 31 to be at the end of the chromosome. Telomere repeat sequences were not found at this location in the plasmid or parent cell DNA. Although the repeat sequences may have been acquired by recombination, a more likely explanation is that they were added to the ends of the plasmid by telomerase before integration. Comparison of the cell DNA before and after integration revealed that a chromosome break had occurred at the integration site, which was shown by fluorescent in situ hybridization to be located near the telomere of chromosome 13. These results demonstrate that chromosome breakage and rearrangement can result in interstitial telomere repeat sequences within the human genome. These sequences could promote genomic instability, because short repeat sequences can be recombinational hotspots. The results also show that DNA rearrangements involving telomere repeat sequences can be associated with chromosome breaks. The introduction of telomere repeat sequences at spontaneous or ionizing radiation-induced DNA strand breaks may therefore also be a mechanism of chromosome fragmentation.

Radiation-Induced DNA Single-Strand Breaks in Freshly Isolated Human Leukocytes

Single-strand breaks are a major form of DNA damage caused by ionizing radiation, and measurement of strand breaks has long been used as an index of overall cellular DNA damage. Most assays for DNA single-strand breaks in cells rely on measuring fractionated DNA samples following alkali denaturation. Quantification is usually achieved by prelabeling cells with radioactive DNA precursors; however, this is not possible in the situation of nondividing cells or freshly isolated tissue. It has previously been demonstrated that the alkali unwinding assay of DNA strand breaks can be quantified by blotting the recovered DNA on nylon membranes and hybridizing with radiolabeled sequence-specific probes. We report here improvements to the technique, which include hot alkali denaturation of DNA samples prior to blotting and the use of carrier DNA that is non-complementary to the radiolabeled probe. Our method allows both single- and double-stranded DNA to be quantified with the same efficiency, thereby improving the sensitivity and reproducibility of the assay, and allows calibration for determination of absolute levels of DNA strand breaks in cells. We also used this method to assay radiation-induced DNA strand breaks in freshly isolated human leukocytes and found them to have a strand break induction rate of 1815 strand breaks/cell/Gy.

Differential radioprotection of cultured human diploid fibroblasts and fibrosarcoma cells by WR1065

The present studies were performed to determine whether WR1065, the dephosphorylated, free-thiol active metabolite of WR2721, could provide differential radioprotection of normal and tumor cell lines in vitro and secondly to investigate potential mechanisms for the selective nature of the radioprotection at the cellular and molecular level. When 4 mM WR1065 was administered 30 min prior to and during irradiation, a protection factor of 1.9 was obtained in clonogenic assays performed with normal human diploid fibroblasts (AG1522) while no protection of fibrosarcoma cells (HT1080) was observed. Some radioprotection of fibrosarcoma cells was observed with higher drug concentrations (10–40 mM), but the increase in survival was considerably less than the plateau level reached with the diploid fibroblasts (3-fold vs 24-fold at 6 Gy). The observation of such a selective effect in vitro with WR1065 indicates that differences in tissue-specific variables such as blood flow, pH, pO 2, and drug dephosphorylation cannot solely account for the selective nature of the radioprotection afforded by WR2721 in vivo. Incubation of nucleoids with increasing concentrations of the DNA intercalating dye propidium iodide was used to titrate the ability of DNA to undergo supercoiling changes. The relaxation and rewinding of supercoiled DNA loops in isolated nucleoids serves as an indicator of both the presence of DNA damage and inherent differences in DNA loop characteristics. Fibrosarcoma cells had a much larger propidium iodide-relaxable DNA loop size than fibroblasts. The rewinding phase of the DNA supercoiling response is impaired by the presence of radiation-induced DNA strand breaks. Four mM WR1065 resulted in a significant reduction in the amount of rewinding inhibition observed after a dose of 10 Gy in diploid fibroblasts (protection factor = 1.43) but did not alter the response of irradiated fibrosarcoma cells. These results, indicating that WR1065 had a preferential radioprotective effect in vitro on both survival and the manifestation of DNA damage at the nucleoid level, are consistent with the hypothesis that cell type differences in chromatin organization and DNA-drug associations could play a role in the selective radioprotection.

DNA ligands as radiomodifiers: Studies with minor-groove binding bibenzimidazoles

An iodinated bibenzimidazole, iodoHoechst 33258, was previously reported to markedly sensitize DNA and cells to UV-A, exemplifying the potential of iodinated DNA ligands as radiosensitizers a rational extension of sensitization by halogenated pyrimidines. However, unlike the latter sensitizers, iodoHoechst 33258 is not a sensitizer of ionizing radiation, presumably due to the innate radioprotective properties of the uniodinated ligand. Experiments with purified DNA show that both Hoechst 33258 and Hoechst 33342 decrease the yield the radiation-induced DNA strand breakage. The ligands bind at discrete sites in the minor groove of DNA, and analysis on DNA sequencing gels show pronounced protection at the ligand binding sites, as well as more generalized protection. The extent of protection of strand breakage on plasmid DNA and the fact that it persists in the presence of 0.5 M NaCI (which prevents low affinity ionic binding between the high affinity sites) suggests that the protective effects of bound ligand are not confined to the high affinity binding sites in the minor groove. The mechanisms of this generalized protection is unknown, but there is some evidence indicating that the H-atom donation from the ligand may account for the site-specific protection. The extent of protection is much diminished, but still evident, in the presence of 100 mM mannitol, a known hydroxyl radical scavenger, indicating that some of the protective effects might relate to DNA damage mediated by direct action. Further evaluation of the mechanisms of protection should enable development of both more active radioprotectors and, by elimination of the radioprotective features from halogenated DNA ligands, more effective radiosensitizers.

Simultaneous measurement of cell cycle phase position and ionizing radiation-induced DNA strand breakage in single human tumour cells using laser scanning confocal imaging.

Techniques for the assessment of DNA damage and repair in individual cells are pertinent to several areas of research, in particular the study of the heterogeneity of tumour cell populations in response to anticancer agents. We describe an adaptation of an in situ alkaline denaturation assay performed on individual nuclei of lysed cells, termed nucleoids, trapped within an agarose film. A novel aspect of the technique described in the application of confocal laser scanning fluorescence microscopy for the measurement of nucleoid relaxation in response to DNA damage. The volumes of spherical nucleoids and their relative DNA contents were determined by ethidium bromide staining and the analysis of confocal sections through the equatorial planes of the nucleoids. Mean nucleoid volume increased as a linear function of X-ray dose (0.5-8 Gy) administered to intact cells prior to lysis. We provide evidence of heterogeneity, in asynchronous cultures, in the DNA unfolding/unwinding characteristics of cells irrespective of cell cycle age. Bivariate plots of relative DNA content versus nucleoid volume allowed the direct assessment of cellular repair capacity with respect to cell cycle position.