Radiation-induced Strand Breaks Research Articles

Mutation of yeast Ku genes disrupts the subnuclear organization of telomeres.

The mammalian Ku70 and Ku86 proteins form a heterodimer that binds to the ends of double-stranded DNA in vitro and is required for repair of radiation-induced strand breaks and V(D)J recombination [1,2]. Deletion of the Saccharomyces cerevisiae genes HDF1 and HDF2--encoding yKu70p and yKu80p, respectively--enhances radiation sensitivity in a rad52 background [3,4]. In addition to repair defects, the length of the TG-rich repeat on yeast telomere ends shortens dramatically [5,6]. We have shown previously that in yeast interphase nuclei, telomeres are clustered in a limited number of foci near the nuclear periphery [7], but the elements that mediate this localization remained unknown. We report here that deletion of the genes encoding yKu70p or its partner yKu80p altered the positioning of telomeric DNA in the yeast nucleus. These are the first mutants shown to affect the subnuclear localization of telomeres. Strains deficient for either yKu70p or yKu80p lost telomeric silencing, although they maintained repression at the silent mating-type loci. In addition, the telomere-associated silencing factors Sir3p and Sir4p and the TG-repeat-binding protein Rap1p lost their punctate pattern of staining and became dispersed throughout the nucleoplasm. Our results implicate the yeast Ku proteins directly in aspects of telomere organization, which in turn affects the repression of telomere-proximal genes.

Evidence of the involvement of p53 in gamma -radiation-induced DNA repair in human lymphoblasts

Purpose: To determine the involvement of p53 in ionizing radiation-induced excision and recombination repair. Materials and methods: Shuttle vector pZ189 containing radiationinduced single strand breaks plus base damage (ocDNA), ultraviolet-radiation damage (uvDNA), or a restriction enzymeproduced double strand break (linDNA) were processed in unirradiated or irradiated p53 wt and p53mut lymphoblasts. Mutation frequencies in the sup F-tRNA target gene and survival of plasmids processed in p53 wt and p53mut hosts were compared. Results: Mutation frequencies of oc-, uv- or linDNA were similar after processing in unirradiated p53 wt and p53mut hosts. However, the mutation frequency of ocDNA and uvDNA decreased 50 % when processed in irradiated p53 wt hosts but was unaltered in irradiated p53 mut hosts. In contrast, linDNA mutation frequencies varied similarly whether processed in irradiated p53 wt or p53mut hosts: mutation frequency decreased twofold when linDNA was transfected immediately after host irradiation but increased twofold when transfection was delayed by 2 h. Double strand break rejoining capacity, determined by the ratio of the number of progenies from linDNA to that from undamaged pZ189, differed both qualitatively and quantitatively in irradiated p53 wt and p53mut hosts. Conclusions: These studies show induction of DNA repair in mammalian cells by ionizing radiation and indicate the involvement of p53 in the modulation of excision repair fidelity and double strand break rejoining capacity.

Radiosensitivity as support for the structure of a unimolecular DNA quadruplex

Purpose: To search for possible variations of radiosensitivity along a unimolecular DNA quadruplex and to use them as support for choosing one of the two possible structures proposed by NMR studies. Materials and methods: The probability of radiation-induced frank strand breaks (FSB) and of alkali-revealed breaks (ARB) at each nucleotide site along the oligomer AATTCGTGGAGCACC (T G) , was determined by sequencing gel electrophoresis as 4 4 4 has been previously described in the literature. The oligomer was irradiated as a unimolecular quadruplex (QS) or paired to its complementary oligonucleotide in a double-stranded B-helix (DS). Results: The oligomer globally presents the same radiosensitivity in the quadruplex or in the double-stranded helix. FSB occur with the same probability at each nucleotide site in both QSand DS-DNA. More ARB are induced at T- and less at G-sites in QS-DNA. The probability of ARB induction in the second T-run is lower than in the first and the third one. Conclusion: Intrastrand radiosensitivity variations support the folding of the studied oligomer into a quadruplex with two lateral loops and one central loop of T-runs spanning the diagonal of the terminal G-quartet.

Modulation of radiation-induced strand break repair by cisplatin in mammalian cells

Purpose: To investigate the repair of ionizing radiation-induced DNA lesions in human skin fibroblasts in the presence of cisplatin-DNA adducts and to determine the persistence of DNA repair inhibition by cisplatin. Materials and methods: Normal human fibroblasts (AG1522) treated with cisplatin were exposed to 4 Gy 60Co gamma-radiation and assayed for repair of radiation-induced damage under growth-permissive conditions. DNA damage was measured by the fluorescence analysis of DNA unwinding (FADU) and cytokinesis-blocked micronucleus assays. Results: Rejoining of strand breaks caused by 4 Gy radiation in cells without cisplatin pre-treatment appeared to be biphasic with an initial fast component (up to 15 min of repair time) followed by a slower component, and was completed by 90 min. Cisplatin treatment (10 mu g/ml, 30 min) immediately before irradiation had no effect on the fast rejoining component, but inhibited the slow component (p 0.01). The same cisplatin treatment 24 h prior to irradiation inhibited both slow and fast components (p 0.01). In contrast, decreasing the cisplatin exposure to 1.0 mu g/ml for 30 min, 24 h prior to irradiation, resulted in an increased amount of strand break repair at each time point measured compared with irradiated control cells. This mild cisplatin treatment (95% survival) also resulted in a reduction of radiation-generated micronuclei indicating an adaptive response. Conclusions: Cisplatin used in combination with ionizing radiation can produce differential cellular responses depending upon the severity of the cisplatin treatment and the time interval between cisplatin and radiation exposures.

Effect of Polyamine-Induced Compaction and Aggregation of DNA on the Formation of Radiation-Induced Strand Breaks: Quantitative Models for Cellular Radiation Damage

The yield of DNA single-strand breaks, G(SSB), upon gamma irradiation of SV40 DNA and SV40 minichromosomes in aqueous solution under aerobic conditions was determined at physiological ionic strength in the presence of various potential radioprotective agents. Putrescine (PUT), spermidine (SPD), glutathione, trans-4,5-dihydroxy-1,2-dithiane, 2-mercaptoethyl disulfide and cystamine, all at 0.1-10 mM, spermine (SPM, 0.1-1 mM) and WR-33278 (WRSSWR, 0.1-2 mM) lowered G(SSB) of SV40 DNA. These results were expected from the ability of these agents to scavenge OH radical in the bulk solution. However, SPD, above 10 mM, and SPM and WRSSWR, each above 2 mM, produced dramatic radioprotection attributed to polyamine-induced compaction and aggregation of the DNA (PICA effect). The DNA of SV40 minichromosomes was inherently less radiosensitive and was subject to a PICA effect at lower polyamine concentrations, i.e. approximately 5 mM SPD, approximately 0.6 mM SPM and approximately 0.5 mM WRSSWR. The PICA effect decreased G(SSB) for SV40 DNA and minichromosomes by one to two orders of magnitude, depending upon the scavenging capacity of the medium. The final yields were similar for SV40 DNA and minichromosomes and were comparable to the corresponding yield determined for cells. Results for the yield of double-strand breaks indicated that the yield of double-strand breaks, G(DSB), for DNA and minichromosomes is subject to a PICA effect by SPM and SPD comparable to that measured for G(SSB). Values of G(SSB) for SV40 DNA and minichromosomes subjected to the PICA effect were well approximated by calculations based upon a 30-nm cylinder assumed to model their condensed states. The results indicate that a major fraction of the formation of SSBs in condensed DNA and minichromosomes results from nonscavengeable radical intermediates. Minichromosomes subjected to the PICA effect of 2 mM SPM were protected against formation of radiation-induced SSBs 1.5-fold by 20 mM DTT but 5-fold by 10 mM DTT plus 10 mM WR-1065 relative to 2 mM SPM alone. Thus WR-1065 is capable of providing marked protection of compacted and aggregated minichromosomes, a protection ascribed to the chemical repair of DNA radicals by WR-1065.

Radioprotection against the Formation of DNA Double-Strand Breaks in Cellular DNA but Not Native Cellular Chromatin by the Polyamine Spermine

The complexing of histones with DNA and the resulting condensation of chromatin protect mammalian cell DNA from radiation-induced strand breakage. In recent studies of SV40 DNA and minichromosomes, marked radioprotection was afforded by spermine through polyamine-induced compaction and aggregation (Newton et al., Radiat. Res. 145, 776-780, 1996). To evaluate the contribution of polyamines to the radioprotection of cellular chromatin, intact V79 cells, nuclei (native chromatin) and chromatin that was partially or completely stripped of histones were treated with spermine or putrescine and gamma-irradiated while embedded in agarose plugs, and induction of double-strand breaks was determined by pulsed-field gel electrophoresis. In the absence of added spermine, the order of radiosensitivity was: dehistonized chromatin (DNA loops anchored to the nuclear matrix) > chromatin depleted of histone H1 > chromatin partially depleted of histone H1 > native chromatin > intact cells. Spermine at concentrations below 1 mM was without effect on strand breakage in any of the preparations, except for limited radioprotection of H1-depleted chromatin. Increasing radioprotection with increasing concentration (1-10 mM) was provided to dehistonized chromatin by spermine but not by putrescine, a polyamine that does not compact DNA or chromatin. Significant radioprotection by spermine was also found for H1-depleted relaxed chromatin at concentrations > or = 1 mM. In contrast, no radioprotection by spermine (up to 10 mM) was observed for any of the chromatin preparations containing all histones. These observations support the hypothesis proposed by Newton et al. that spermine protects DNA against radiation damage via polyamine-induced compaction and aggregation. With removal of histone H1, the exposed chromatin develops the ability to be protected by spermine. However, the absence of radioprotection of native chromatin by spermine is consistent with a role for histones as the major radioprotectors of cellular DNA and the differential radiosensitivity of decondensed compared to condensed cellular chromatin resulting from the effects of factors other than polyamines.

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.

Sequence-dependent variations of DNA structure modulate radiation-induced strand breakage

. Using a 80 base pair DNA fragment, the sequencedependence was compared for: (i) the probability of fast neutrons induced strand breakage, (ii) the accessibility of the H4- and H5-atoms to OH attack, (iii) the width of the minor groove, and (iv) the probability of OH reactions with H4- or H5- atoms. The probability of strand breakage was measured using sequencing gel electrophoresis. The accessibility and the probability of reaction were calculated for the energy-minimized modelled DNA fragment. A Monte-Carlo simulation was used for calculating the probabilities of H-atom abstraction by OH. It was observed that reduced breakage occurs in sequences exhibiting low accessibility of H4 and H5 2 and low probability of H-atom abstraction by OH, due to a narrow, minor groove. This shows that the breakage probability at a given nucleotide site is not determined by the chemical nature of the nucleotide (A, T, G or C), but mainly by the local sequence-modulated intrinsic structure. Fitting the experimental results with the calculated probabilities of reaction suggests that a C4-centered radical evolves towards a strand break three times more efficiently than the C5 one, and that half of the breaks occur via the 4-path and half via the 5-path.

DNA-damage processing in blood lymphocytes of head-and-neck-squamous-cell-carcinoma patients is dependent on tumor site.

It has been reported that an intrinsic susceptibility to cancer is related to the way an individual responds to DNA-damaging agents. The aim of this study was to evaluate whether, in addition to bleomycin-induced chromosomal instability, radiation-induced initial DNA damage and subsequent repair is associated with the development of head-and-neck squamous-cell carcinoma. In this study, 2 assays were performed to measure DNA damage in human peripheral-blood lymphocytes. One was a chromosomal aberration assay which determines sensitivity to chromatid breaks induced by bleomycin, the other an elegant immunochemical assay which measures the level of radiation-induced strand breaks as well as subsequent repair. Age, smoking and alcohol-drinking behavior did not influence the number of chromatid breaks, initial DNA damage or repair capacity. As has been found in previous studies, the mean number of chromatid breaks per cell was significantly different between patients (n = 18) and control persons (n = 19), whereas the amount of initial DNA damage was not. No correlation was found between the outcome of the 2 assays in the subject groups. In contrast to laryngeal-carcinoma patients, oral-cavity-carcinoma patients showed significantly slower repair capacity than controls. Our hypothesis is that the way DNA damage is processed by the patients determines at which site cancer develops in the head and neck area.

Modification of Radiation-Induced Strand Breaks by Glutathione: Comparison of Single- and Double-Strand Breaks in SV40 DNA

A number of investigations have suggested that the widely observed oxygen enhancement of radiation-induced cell killing or intracellular DNA damage requires the presence of glutathione (GSH) or other thiols. We have adapted an in vitro model system to investigate the effects of GSH on radiation-induced DNA double-strand breaks (DSBs), lesions felt to be critical to cell death. Superhelical SV40 DNA, 25 micrograms/ml, was irradiated in air or nitrogen in the presence of 0-20 mM GSH and single-strand breaks (SSBs) and DSBs were measured using neutral gel electrophoresis/ethidium bromide fluorescence. Control experiments demonstrated that a substantial concentration of free SH was still present after irradiation. Dose-response curves for SSBs and DSBs in air or nitrogen were predominantly linear at all GSH concentrations tested from 0-20 mM, except for 20 mM GSH in nitrogen, indicating that both SSB and DSB formation are predominantly by one-hit mechanisms under these conditions. Dose-response curves for both SSBs and DSBs in nitrogen at 20 mM GSH closely tracked the corresponding linear curves in air for doses up to about 200 Gy, then reached a plateau at higher doses. Induction efficiencies in 20 mM GSH, calculated from these initial slopes for both SSBs and DSBs in nitrogen, were unexpectedly higher than the corresponding efficiencies in 5 mM GSH, suggesting additional damage, rather than the expected additional protection. The possible mechanism for a damaging effect from GSH is discussed. Oxygen enhancement ratios (OERs) were calculated from the slopes of dose-response curves. The OERs for SSBs did not differ substantially from those for DSBs at the same [GSH], contrary to the observations of Prise et al. (Radiat. Res. 134, 102-106, 1993). The OERs for SSBs and DSBs peaked at 6.5 and 8, respectively, at 5 mM GSH. These similarities suggest that the much lower OERs (2.5-3.0) generally reported for radiation killing of cells, which also typically contain about 5 mM GSH, cannot be accounted for by differences in OER between lethal lesions, represented by DSBs, and nonlethal lesions, represented by SSBs. In view of the present results, another possible explanation, that intracellular compounds other than reduced thiols are important in the chemical modification of the response of DNA to radiation, seems to be much more likely.

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.

Influence of Nucleic Acid Base Composition on Radiation-induced Strand Breakage in Single Stranded DNA: A Time Resolved Study

The following study investigates the pathways involved in the induction of single strand breaks (ssb) in various samples of single stranded (ss) DNA (calf thymus, Micrococcus lysodeikticus, Clostridium perfringens) with differing nucleic acid base composition. The time scale for the induction of ssb was determined from changes in the light scattering intensity following pulse irradiation of aqueous solutions containing these ssDNA samples at pH7.8 under either aerated or deaerated conditions. The induction of ssb under these conditions is predominantly by the hydroxyl radical and shows various kinetically distinct components. The immediate ssb (t < 0.02 s) account for approximately 40-60% of the total yield of ssb. The majority of these ssb are suggested to arise from the 'common' initial attack of the hydroxyl radicals at the sugar phosphate backbone for each of the three DNA samples. Furthermore, slower components for ssb formation (t > 0.02 s) were observed and are suggested to occur through base radical mediated H-atom abstraction from the sugar moiety. The half lives for formation of the majority of ssb, formed through this base radical-mediated H-atom abstraction(s), are in the range of 20-43 ms. The yields of these 'base-mediated' ssb vary markedly (under both aerobic and anaerobic conditions) and reflect the base composition of the DNA sample. It is suggested from these studies that the OH-induced base radicals of guanine/cytosine are more effective precursors for strand breakage than those from adenine/thymine in ssDNA.

Radiation-chemical discussion on inverse dose-rate effect observed in radiation-induced strand breaks of plasmid DNA.

Experimental results of inverse dose-rate effect, so-called Kada Effects, which was published by Takakura and her coworkers on radiation-induced strand breaks of plasmid DNA in aerated aqueous solution, have been kinetically analyzed and discussed on the basis of radiation chemistry, the kinetic analysis indicates that there are two possible mechanisms; 1) equilibrium mixture of O2- and HO2 is responsible for strand breaks of DNA, and 2) peroxyl radical produced from citrate is effective for the strand breaks. However, the detailed kinetic analysis revealed that the latter is improbable because unimolecular decay of the peroxyl radical must be assumed to be negligible for its participation despite fast decay of analogous organic peroxyl radicals. The analysis has also given 9.93 +/- 0.10 dm3 mol-1 s-1 per nucleotide unit, which corresponds to 7.62 x 10(4) dm3 mol-1 s-1 per DNA molecule, as the rate constant for the reaction of the equilibrium mixture with plasmid pBR 322 DNA. Furthermore the probability that the reaction of the mixture with a nucleotide unit of DNA leads to strand breaks was obtained to be 3.36 x 10(-3) for gamma-irradiated system and 1.98 x 10(-3) for beta-irradiated system, respectively.

Radiation-Induced DNA Damage in Tumors and Normal Tissues: I. Feasibility of Estimating the Hypoxic Fraction

It is well known that the type and quantity of DNA damage produced by ionizing radiation depend on the oxygen concentration around the DNA. For example, in irradiated mammalian cells, both a decrease in the DNA strand break efficiency and the induction of DNA-protein crosslinks (DPCs) occur as the extracellular oxygen concentration is decreased below 1%. In the study reported here, the feasibility of estimating the hypoxic fraction of irradiated tumors and normal tissues was investigated by measuring the single-strand scission factor, the DNA-protein crosslink factor, and the amount of DNA remaining on polycarbonate filters after elution with approximately 24 ml of tetrapropylammonium hydroxide at pH 12.3 without proteinase K (PK) in the lysis solution. In anesthetized air-breathing Fisher 344 rats, no radiation-induced DPCs were detected in either cerebellar neurons or cells of subcutaneous (sc) 9L tumors when the DNA was assayed at approximately one half-time of repair after doses < or = 15 Gy. Within 10 min after anesthetized rats were killed, the maximum decrease in the radiation-induced strand break efficiency and the maximum formation of radiation-induced DPCs occurred in both cerebellar neurons and sc 9L tumors. When irradiated cerebellar neurons or sc 9L tumor cells from air-breathing and dead rats were mixed to simulate hypoxic fractions of 0, 10, 25, 50, 75, and 100%, only the percentage of the DNA retained on the filter after approximately 24 ml of elution without PK in the lysis solution was a linear function of the simulated hypoxic fraction after doses of both 15 and 2 Gy. At 15 Gy, the linear function was identical for 9L cells in tissue culture, sc 9L tumor cells, and cerebellar neurons. In addition, the slope, but not the intercept, of the linear function appeared to be independent of dose from 2 to 15 Gy. Consequently, if the dose and the amount of strand break repair are kept relatively constant, the linear function appears to depend primarily on radiation chemistry events, rather than the biological properties of the irradiated cells. Moreover, the data suggest that this assay can measure a hypoxic fraction of < or = 10% after a conventional radiotherapy dose of 2 Gy, provided sufficient material is available for analysis.(ABSTRACT TRUNCATED AT 400 WORDS)

Pre-irradiation with low doses has no detectable effect on the repair of radiation-induced double strand breaks in DNA of Chinese hamster V79 cells

When cells are exposed to preliminary low doses of ionising radiation prior to much higher doses, the overall extent of cell damage is reduced by an effect known as the adaptive response. We have extended such studies to include measurements of double strand breaks (DSB) in the DNA of Chinese hamster V79 cells, but, surprisingly, pretreatment with low doses had no effect on the numbers of DSB. This contrast is discussed in terms of DNA repair mechanisms.

Effect of heat on induction and repair of DNA strand breaks in X-irradiated CHO cells.

Chinese hamster ovary cells were exposed to various heat treatments followed by X-irradiation, and the induction and repair of DNA strand breaks was studied using the alkaline unwinding technique. Heat treatments alone were found to cause DNA strand breakage only for temperatures greater than or equal to 43 degrees C, whereas the number of radiation-induced strand breaks was unaffected by additional heating. Strand break repair was studied for irradiated cells preheated at temperatures ranging from 42 degrees C to 45 degrees C. The total repair curve could be separated into three phases, a fast (t = 0-15 min), an intermediate (t = 15-120 min) and a slow (t greater than or equal to 120 min) phase. All phases were altered when cells were heated either prior to or after irradiation. The fast and the intermediate phase could be well interpreted by the assumption that irradiation leads to both primary and secondary single-strand breaks, the latter being generated by enzymatic incision at sites of damaged bases. For irradiation alone, the ratio of all secondary strand breaks to all primary breaks was fsec = 1.5 +/- 0.5. This ratio was not altered by preceding heat treatments (mean fsec = 1.7 +/- 0.2). The main effect of heating on the repair kinetics of single-strand breaks was an increase in the repair half-time of primary and secondary breaks (maximum increase by a factor of 3.4), whereas the generation of secondary breaks was only slightly retarded (factor 1.3). The slow repair phase, which is assumed to represent the repair of DNA double-strand breaks, was best described by a single exponential component. The half-time of this component was found to increase from tau slow = 170 +/- 70 min for non-heated cells to tau slow = 345 +/- 80 min for cells heated at 45 degrees C for 20 min, indicating that heat inhibited the repair of double-strand breaks. For irradiation alone, the initial fraction of the slow component was fslow = 0.065 +/- 0.004. This fraction was enhanced by additional heating, with a maximum increase by a factor of 2.7 for cells heated at 45 degrees C for 20 min. This elevation cannot be the result of an enhanced induction of double-strand breaks, but must be associated with an additional formation of slowly repaired strand breaks during repair incubation. These additional strand breaks must arise from strand breaks which in non-heated cells are repaired during the fast or intermediate phase.(ABSTRACT TRUNCATED AT 400 WORDS)

The Influence of Chromatin Structure on the Frequency of Radiation-Induced DNA Strand Breaks: A Study Using Nuclear and Nucleoid Monolayers

To assess the influence of chromatin structure on the frequency of radiation-induced DNA strand breaks, the alkaline unwinding technique was applied to nuclear and nucleoid monolayers. These chromatin substrates were prepared by treating human fibroblasts grown as monolayers with the nonionic detergent Triton X-100 and varying concentrations of cations. The chromatin structure was modified either by a stepwise removal of DNA-bound proteins by extraction in increasing concentrations of monovalent salt, or by the addition or deletion of mono- and divalent cations to condense or decondense the chromatin, respectively. It was found that the stepwise removal of DNA-bound proteins from the chromatin dramatically increased the frequency of radiation-induced DNA strand breaks. The DNA-bound proteins showed a qualitative difference in their ability to protect the DNA where proteins removed by salt concentrations above 1.0 M exerted the greatest protection. Furthermore, the frequency of radiation-induced DNA strand breaks was found to be 6 times lower in condensed chromatin than in decondensed chromatin and about 80 times lower than in protein-depleted chromatin. It is concluded that the presence of DNA-bound proteins and the folding of the chromatin into higher-order structures protect the DNA against radiation-induced strand breaks.

Kinetics of Radiation-induced Strand Break Formation in Single-stranded Pyrimidine Polynucleotides in the Presence and Absence of Oxygen; a Time-resolved Light-scattering Study

Time-resolved reductions in the light-scattering intensity (LSI) of aqueous oxic and anoxic solutions of poly-C and poly-U at pH 7.8, following pulse-irradiation, have been studied as indices of strand break formation. With doses of 3-24 Gy per pulse, a number of kinetically distinct strand breakage components have been detected. A comparison of the LSI responses obtained from irradiations conducted under N2O with those conducted under air or O2 show no marked difference in the overall extent of LSI change. However, the immediate and fast (t 1/2 less than or equal to 50 microseconds) reduction in LSI, accounting for about 18-19% of the pyrimidine polynucleotide's total LSI response in oxic solution, is reduced in the absence of oxygen, to about 12% of the total LSI response found with poly-C and to about 9% for poly-U. For poly-C there is a five-fold enhancement in the rate of major strand breakage under anoxia [k1(N2O) = 7.9s-1] whereas for poly-U a more modest enhancement (about two-fold) is observed. These enhanced rates are mirrored by those for the losses of the principal optical anoxic absorptions (observed pulse radiolytically) that are assigned to the pyrimidine 6-yl base radicals. Such findings support a proposal that the rate-limiting step of major strand breakage for pyrimidine polynucleotides is a base radical mediated hydrogen atom abstraction reaction (Lemaire et al. 1987, Hildenbrand and Schulte-Frohlinde 1989). Irradiation of poly-C and poly-U in N2O/O2 (4:1, v/v) saturated solutions yields LSI changes much larger than those noted under N2O and air (or O2), which are in turn approximately double the responses observed under N2. This indicates that the major strand breaking species of water radiolysis is the OH-radical and that there is an oxygen enhancement of single strand breakage of about 1.9 for poly-C and 1.6 for poly-U.

Cisplatin and radiation: Interaction probabilities and therapeutic possibilities

This paper examines the probability of interactions occurring between drug lesions and radiation lesions in DNA for the cytotoxic and radiosensitizing agent cisplatin. The number of cisplatin-induced DNA adducts and radiation-induced strand breaks after a given dose of each agent are known for given cell systems, from which the probability that these lesions will interact can be estimated. Results of these calculations indicate that the probability of interaction could be high, depending on the distance over which two lesions can interact and the probability of repair of the interaction lesion. Calculated lesion numbers have been compared with known data on radiation modification, including illustrations of inconsistencies. In the second part of the paper, ways in which combined therapy with cisplatin and radiation can be improved are described. Development of methods to predict which types of tumor and which individual tumors within a given type are sensitive to the cytotoxic and radiosensitizing effects of the drug would aid rational selection of patients for combination treatments. Immunocytochemical methods sensitive enough to monitor cisplatin-DNA interactions in patients are available and may be useful in this context. The delivery and maintenance of higher tumour concentrations of radiosensitizer offers a further possibility for improvement. Studies of intratumoral injection of cisplatin have shown promise for achieving this goal while limiting normal tissue toxicity.

Constraints to DNA Unwinding near Radiation-Induced Strand Breaks in Ewing's Sarcoma Cells

Ewing's sarcoma cell lines were compared to other cell lines for induction of DNA strand breaks by ionizing radiation and their ability to repair those breaks. The alkali-unwinding assay and alkaline sucrose gradient analysis were used for these studies. The alkali-unwinding assay revealed that the amount of DNA unwound per strand break in Ewing's sarcoma cells was less than for other cells and was not influenced by high-salt denaturation conditions. Ewing's sarcoma cells had similar induction and repair rates for strand breaks compared with other cell lines. The kinetics of unwinding suggests there are constraints to DNA unwinding in the chromatin of Ewing's sarcoma cells, possibly related to high levels of poly(ADP-ribose) polymerase in these cells.