Repair of radiation-induced DNA strand breaks does not occur preferentially in transcriptionally active DNA.

Certain DNA base lesions induced by ionizing radiation or oxidative stress are repaired faster from the transcribed strand of active genes compared to the genome overall. In this study, it was investigated whether radiation-induced DNA strand breaks are preferentially repaired in active genes compared to the genome as a whole in CHO cells. The alkaline unwinding technique coupled to slot-blot hybridization with specific DNA probes was used to study the induction and repair of DNA strand breaks in defined DNA sequences. Results using this technique showed a linear dose response for the formation of radiation-induced DNA strand breaks in the dihydrofolate reductase (DHFR) gene. Furthermore, the half-life of radiation-induced strand breaks was less than 5 min in the DHFR gene, in the ribosomal genes, and in the genome as a whole. These results suggest that the repair of DNA strand breaks is fast and uniform in the genome of mammalian cells.     

X-ray induced DNA double strand break production and repair in mammalian cells as measured by neutral filter elution.

This work presents a neutral filter elution method for detecting DNA double strand breaks in mouse L1210 cells after X-ray. The assay will detect the number of double strand breaks induced by as little as 1000 rad of X-ray. The rate of DNA elution through the filters under neutral conditions increases with X-ray dose. Certain conditions for deproteinization, pH, and filter type are shown to increase the assay's sensitivity. Hydrogen peroxide and Bleomycin also induce apparent DNA double strand breaks, although the ratios of double to single strand breaks vary from those produced by X-ray. The introduction of double strand cuts by HpA I restriction endonuclease in DNA lysed on filters results in a rapid rate of elution under neutral conditions, implying that the method can detect double strand breaks if they exist in the DNA. The eluted DNA bands with a double stranded DNA marker in cesium chloride. This evidence suggests that the assay detects DNA double strand breaks. L1210 cells are shown to rejoin most of the DNA double strand breaks induced by 5-10 krad of X-ray with a half-time of about 40 minutes.     

The effect of mitotic inhibitors on DNA strand size and radiation-associated break repair in Down syndrome fibroblasts

The effect of mitotic inhibitors on formation and repair of DNA breaks was studied in cultured fibroblasts from patients with Down syndrome in order to investigate the hypothesis that the karyotyping procedure itself may play a role in the increased chromosome breakage seen in these cells after gamma radiation exposure. Using the nondenaturing elution and alkaline elution techniques to examine fibroblasts from Down syndrome patients and from controls, no specific abnormalities in Down syndrome cells could be detected after exposure to mitotic inhibitors, including rate and extent of elution of DNA from filters as well as repair of radiation-induced DNA breaks. In both normal and Down syndrome cell strains, however, exposure to mitotic inhibitors was associated with a decrease in cellular DNA strand size, suggesting the presence of drug-induced DNA strand breaks. The mechanism of increased chromosome sensitivity of Down syndrome cells to gamma radiation remains unknown.     

Radiosensitization of Chinese hamster V79 cells by bromodeoxyuridine substitution of thymidine: enhancement of radiation-induced toxicity and DNA strand break production by monofilar and bifilar substitution

Bromodeoxyuridine (BrdU) competes with thymidine (TdR) for incorporation into DNA of exponentially growing V79-171 cells. Such cells show an enhancement of the radiation response as determined by clonogenic survival and DNA damage measured by filter elution techniques after doses up to 15 Gy. The degree of radiosensitization for both survival and rates of alkaline and neutral elution are dependent on percentage BrdU substitution and independent of whether BrdU is in one strand only (monofilar) or both strands (bifilar) of the DNA duplex: e.g., for 16% BrdU substitution distributed either monofilarly or partially bifilarly, there is an enhancement factor for Do of 1.55. At this percentage substitution, the enhancement factor for the rate of alkaline elution is 1.75 and that for the rate of neutral elution is 1.54. The greater the percentage BrdU substitution, the larger was the enhancement ratio for survival and radiation-induced strand breaks in both monofilarly and bifilarly substituted cells. The increase in cell radiosensitivity caused by BrdU substitution shows a better correlation with the increase in radiation-induced double-strand breaks than with the increase in radiation-induced single-strand breaks.     

Physical basis for detection of DNA double-strand breaks using neutral filter elution

Results using neutral filter elution are difficult to explain if this method detects only DNA double-strand breaks (DSBs). In an attempt to understand neutral filter elution, the size of DNA pieces eluted from filters was measured using pulsed-field gel electrophoresis. Contrary to expectation, the size of the pieces was independent of radiation dose and time of elution, and much smaller (approximately 460 kb) than anticipated based on the expected number of DSBs induced. Shearing of the DNA molecule, the presence of nonspecific nucleases, and the influence of DNA-associated proteins were examined but could not explain our results. Consequently, we propose that cell lysis causes swelling of the DNA gel, and the exposed fraction of DNA on the surface of the gel is then sheared as the elution solution flows through the filter. We suggest that the rate of DNA elution measured using neutral filter elution is dependent upon the number of DSBs present, the composition of the eluting solution, especially with regard to the presence of molecules which can influence chromatin swelling on the filter, and the conformation or "packaging" of DNA before lysis.     

Fractionation of DNA from mammalian cells by alkaline elution.

The method of alkaline elution provides a sensitive measure of DNA single-strand length distribution in mamalian cells and is applicable to a variety of problems concerning DNA damage, repair, and replication. The physical basis of the elution process was studied. The kinetics of elution above the alkaline transition pH were found to occur in two phases: an initial phase in which single-strand length is rate limiting, followed by a phase in which elution is accelerated due to the accumulation of alkali-induced strand breaks. The range of DNA single-strand lengths that can be discriminated by elution above the alkaline transition pH was estimated by calibration relative to the effects of x ray, and was found to be 5 X 10(8)-10(10) daltons. Shorter DNA strands elute within the pH transition zone, which extended from pH 11.3 to 11.7 when tetrapropylammonium hydroxide was used as base. This elution was relatively rapid, but was sharply limited by pH, according to the length of the strands: the length of the strands eluted increased with increasing pH. Alkaline elution was inhibited by treatment of cells with low concentrations of nitrogen mustard, a bifunctional alkylating known to cross-link DNA. On investigation of the possibility that DNA subclasses may differ in their elution behavior, satellite L strands were found to elute more slowly from cells exposed to a low dose of x ray than did the bulk DNA.     

Eumelanin causes DNA strand breaks and kills cells

Synthetic eumelanin prepared by autooxidation of D,L-DOPA causes DNA strand breaks, as determined by alkaline elution after cell lysis with detergent and proteolysis, in B16CL4 mouse melanoma cells. The melanin is toxic to the cells in the range of doses that causes strand breaks. When the melanin was incubated with the cells at 37 degrees C in tissue culture medium, it was maximally effective after 15 to 20 min at causing strand breaks in the DNA. The extent of damage is concentration dependent, but the effect plateaus at 1 mg/ml. The nature of the interaction of the cellular DNA with melanin is consistent with strand breaks, not DNA-DNA crosslinks. The strand break damage is repaired, even in the continued presence of melanin, but repair is more rapid if the cells are washed and the melanin is removed. The form of the melanin is important for obtaining the effect. Sonication for 3 min abrogates the effect to a considerable extent, and repeated cycles of sonication can completely destroy the activity. Lost activity returns slowly with storage at 4 degrees C. Melanin is more effective at damaging DNA in a protein-free medium. It is also DNA-damaging at 4 degrees C, but less so than at 37 degrees C. Preliminary studies indicate that the strand breaks caused by melanin are additive with those caused by ionizing radiation. The extent of DNA strand breaks and alkali-labile sites caused by several other melanins was also determined. Some melanins did not cause frank strand breaks, but were active in causing alkali-labile sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Radiosensitization by hypoxic pretreatment with misonidazole: an interaction of damage at the DNA level

Prolonged exposures to misonidazole (MISO) in vitro under hypoxic conditions result in radiosensitization which is characterized by a decrease in the size of the radiation survival curve shoulder for cells irradiated under hypoxic or aerobic conditions after drug removal. Although intracellular glutathione (GSH) was depleted during hypoxic exposures to MISO, this could not account for the dose-additive radiosensitization (decrease in shoulder size) since GSH depletion by diethylmaleate had no effect on the sensitivity of cells irradiated in air. The alkaline elution assay was used to measure DNA strand breaks and their repair after exposure to MISO, graded doses of X rays, and the combination of MISO pretreatment with X rays. The elution rate of DNA from irradiated cells increased linearly with X-ray dose, with and without MISO pretreatment. However, the DNA elution rates measured after MISO pretreatment were greater by a constant amount at all X-ray doses greater than 1 Gy. In terms of both cell survival and DNA elution rate, MISO-pretreated cells behaved as though they had received an extra 1.5 Gy. Although the initial damage after X rays was greater in MISO-pretreated cells, there was no effect of MISO pretreatment on the rate of repair of radiation-induced DNA strand breaks. The agreement between the differences in survival levels and DNA elution rates for irradiated control and MISO-pretreated cells and absence of an effect on DNA repair rates suggest that the pretreatment sensitization is due to an additive interaction of damage at the DNA level.     

Elaboration of cellular DNA breaks by hydroperoxides.

Cellular damage produced by ionizing radiation and peroxides, hydrogen peroxide (HOOH) and the organic peroxides tert-butyl (tBuOOH) or cumene hydroperoxide (CuOOH) were compared. DNA breaks, toxicity, malondialdehyde production, and the rate of peroxide disappearance were measured in a human adenocarcinoma cell line (A549). The alkaline and neutral filter elution assays were used to quantitate the kinetics of single and double strand break formation and repair (SSB and DSB), respectively. Peroxides, at 0.01-1.0 mM, produce multiphasic dose response curves for both toxicity and DNA SSBs. Radiation, 1-6 Gy, produced a shouldered survival curve, and both DNA SSB and DSBs produced in cells x-rayed on ice were nearly linear with dose. The peroxides produced more SSBs than radiation at equitoxic doses. X-ray induced DNA single strand breaks were rejoined rapidly by cells at 37 degrees C with approximately 80% of initial damage repaired in 20 min. Peroxide induced SSBs were maximal after 15 min at 37 degrees C. Rejoining proceeded thereafter, but at a rate less than for x-ray induced strand breaks. Significant DNA DSBs could not be achieved by peroxides even at concentrations 50-fold higher than required to produce SSBs. HOOH treatment of DNA on filters following cell lysis and proteolysis produced SSBs. CuOOH and tBuOOH produced no SSBs in lysed cell DNA. None of the peroxides produced DSBs when incubated with lysed cell DNA. Malondialdehyde was released from cells incubated with organic hydroperoxides, but not HOOH, nor up to 40 Gy of x-rays. HOOH was metabolized three times faster than the organic peroxides. The overall results demonstrate the necessity for a metabolically active cell environment to elaborate maximal DNA strand breaks and cell death at hydroperoxide concentrations of 10(-4) or greater, but prevent strand breaks and stimulate cell growth at 10(-5) M.     

Isolation and purification of large quantities of DNA replication intermediates by pH step alkaline elution

The alkaline elution technique has been modified to be used in the isolation of DNA replication intermediates and in the study of the process of DNA replication. In this procedure pulse labeled CHO cells are layered onto a membrane filter, lysed with detergent, and the nascent DNA eluted in step-wise fashion with tetrapropylammonium hydroxide at pH 11.0, 11.3, 11.5 and 12.1. Alkaline sucrose sedimentation of the eluted DNA shows that the pH 11.0 material consists of < 9S fragments consistant with those described by Okazaki and others. DNA eluting at pH 11.3 has a molecular weight of 8–12 million daltons, DNA which elutes at pH 11.5 sediments with a molecular weight of 20–30 million daltons. Two independent lines of evidence suggest that the pH 11.3 material includes DNA sequences synthesized at replicon origins. (1) Exposure of cells to low doses of X-ray prior to pulse labeling reduces the pH 11.3 fraction by 40–50% while there is little change in the other fractions. (2) Synchronization of cells by inhibiting DNA synthesis with FdU, followed by a 2 min pulse label, yields approximately 50% of the incorporated ³H-thymidine in the pH 11.3 fraction. The pH step elution technique has the following advantages: 1. Intermediates of high specific activity can be isolated from 10⁶ cells per filter; 2. By lysing cells on a filter, proteins, nucleases, and other cellular materials are eliminated; 3. DNA in the lysate is never handled, thus eliminating shearing; 4. Eluted DNA may be instantaneously neutralized by collecting into a buffer to protect it from alkaline degradation.     

Measurement of DNA-protein crosslinks in mammalian cells without X-irradiation

To study the mechanisms of formation and repair of DNA-protein crosslinks in mammalian cells, the best general method to assay these lesions is the Kohn membrane alkaline elution procedure. Use of this sensitive technique requires the introduction of random strand breaks in the DNA by X-irradiation to reduce the very high molecular weight so that it elutes off the filter at an appropriate rate. This report describes an alternative method for fragmenting the DNA in the absence of X-irradiation equipment. Convenient reproducible elution rates of DNA from various mouse and human cells in culture without X-irradiation result from elution through polyvinyl chloride filters with 75 mM sodium hydroxide (0.33 ml/min) instead of the standard 20 mM EDTA-tetrapropylammonium hydroxide, pH 12.2 (0.03 to 0.04 ml/min). Dose-dependent retardation of the DNA elution was observed over the range 0 to 30 microM trans-platinum(II)diamminedichloride, and proteinase K treatment during cell lysis restored the elution rate to that of the untreated control cell DNA. In the absence of X-irradiation, this elution method measures DNA-protein crosslinks with higher sensitivity and equivalent reproducibility as the air-burst procedure.     

125I-induced DNA double strand breaks: use in calibration of the neutral filter elution technique and comparison with X-ray induced breaks

The neutral filter elution assay, for measurement of DNA double strand breakage, has been calibrated using mouse L cells and Chinese hamster V79 cells labelled with [125I]dUrd and then held at liquid nitrogen temperature to accumulate decays. The basis of the calibration is the observation that each 125I decay, occurring in DNA, produces a DNA double strand break. Linear relationships between 125I decays per cell and lethal lesions per cell (minus natural logarithm survival) and the level of elution, were found. Using the calibration data, it was calculated that the yield of DNA double strand breaks after X-irradiation of both cell types was from 6 to 9 X 10(-12) DNA double strand breaks per Gy per dalton of DNA, for doses greater than 6 Gy. Neutral filter elution and survival data for X-irradiated and 125I-labelled cells suggested that the relationships between lethal lesions and DNA double strand breakage were significantly different for both cell types. An attempt was made to study the repair kinetics for 125I-induced DNA double strand breaks, but was frustrated by the rapid DNA degradation which occurs in cells that have been killed by the freezing-thawing process.     

Attempted separation of transcriptively active and inactive chromatin by hydroxylapatite thermal chromatography

Chromatin and purified DNA were fractionated by hydroxylapatite thermal chromatography. Fractions of varying thermal stability were tested for the proportions of transcribed sequences and repetitive sequences relative to the unfractionated genome. The first 80--85% of either total chromatin or purified DNA eluted from hydroxylapatite contained the same proportion of hybridizable sequences as total DNA. The remaining 15--20% of chromatin eluting at the highest temperatures was depleted of transcribed sequences. Analysis of the 20% highest melting fraction of purified DNA showed that, while the first two-thirds of this fraction contained the same proportion of transcribed sequences as unfractionated DNA, the last third, comprising about 6% of total DNA, was depleted of active sequences. Although no major differences were detected in nonrepetitive sequence complexity of chromatin fractions, there was a correlation between relative thermal stability and repetitive sequence content in fractions of both chromatin and DNA separated by thermal chromatography. Fragments eluting at higher temperatures contained a greater proportion of repetitive sequences, as indicated by a rapidly renaturing component. Most likely, the latest eluting fractions from both chromatin and purified DNA were enriched for a nontranscribed, highly reiterated, G+C rich satellite component of the chicken genome.     

Quiescent human lymphocytes do not contain DNA strand breaks detectable by alkaline elution

On the basis of qualitative assays, quiescent lymphocytes have previously been reported to have numerous DNA strand breaks, which are thought to be repaired after mitogenic stimulation by a process associated with poly(ADP-ribosyl)ation. Using alkaline elution, a very sensitive assay for quantifying DNA single-strand breakage, we found no evidence for a high frequency of DNA strand breaks in unstimulated human peripheral blood lymphocytes. No differences in elution profiles were observed between unstimulated lymphocytes and lymphocytes 4 or 48 h after addition of the mitogen phytohemagglutinin (PHA). Furthermore, addition of 3-aminobenzamide (3AB), an inhibitor of poly(ADP-ribose) synthetase, or aphidicolin, an inhibitor of DNA polymerase alpha, did not increase the amount of DNA eluting from the filter after PHA stimulation. In contrast to reported studies of mouse splenic lymphocytes, we found that human lymphocytes were able to replicate and divide in the presence of the ADP-ribosylation inhibitor. Human lymphocytes were also capable of proliferating in nicotinamide-free medium, with or without 3AB, indicating that ADP-ribosylation is not a requirement for lymphocyte differentiation. We therefore consider it unlikely that peripheral human lymphocytes contain significant numbers of strand breaks that play any role in their stimulation or differentiation in response to PHA.     

Gamma radiation-induced single strand breaks in DNA and their repair in spheroplasts and nuclei of light-grown and dark-grown Euglena cells

Exposure of light-grown and dark-grown Euglena cells to gamma radiation causes single strand breaks in nuclear DNA as assessed by sedimentation analysis in alkaline sucrose density gradients. The number of radiation-induced single strand breaks in nuclear DNA of light-grown cells is found to be less than that in dark-grown cells. Post-irradiation incubation of both types of cells in 0 . 1 M phosphate buffer, pH 7 . 0 at 25 degrees C for 1 hour results in restitution of the strand breaks in DNA. Light-grown cells (cells with chloroplasts) are able to rejoin all the single strand breaks in DNA produced by gamma irradiation at D50 and D5 doses. On the other hand, dark-grown cells (cells devoid of chloroplasts) are unable to rejoin all the strand breaks caused by irradiation at either of the doses. The rate of DNA repair in dark-grown cells is also much slower than that in light-grown cells. Radiation-induced single strand breaks in DNA and their repair in nuclei from both types of cells is found to be similar to that observed in the spheroplasts. It is suggested that some factor(s) elaborated by chloroplasts may contribute towards the efficiency of nuclear DNA repair in Euglena cells.     

Radiation-induced damage to DNA in drug- and radiation-resistant sublines of a human breast cancer cell line.

An Adriamycin-resistant subline of a human breast cancer cell line, MCF-7 ADRR, has been shown to exhibit radioresistance associated with an increase in the size of the shoulder on the radiation survival curve. In the present study, damage to DNA of MCF-7 sublines WT and ADRR by 60Co gamma radiation was measured by filter elution techniques. The initial amount of DNA damage, measured by both alkaline and neutral filter elution, was lower in ADRR cells, suggesting that these cells are resistant to radiation-induced single- and double-strand DNA breaks. In the case of double-strand breaks the difference between WT and ADRR cells was significant only at the lower radiation doses studied (up to 100 Gy). In cells depleted of glutathione (GSH) by L-buthionine sulfoximine (BSO) treatment, ADRR cells were sensitized to radiation-induced DNA damage, while WT cells were unaffected. The rate of repair of single- and double-strand DNA breaks following radiation was the same for both sublines, and repair of radiation damage was not affected by BSO treatment in either cell line. The relative resistance of ADRR cells to initial DNA damage by radiation is the only difference so far detected at the molecular level which reflects radiation survival, and it is possible that other factors are involved in the resistance of ADRR cells to killing by radiation. Sensitization of ADRR cells to radiation-induced DNA damage by GSH depletion, although not likely to involve inhibition of GSH-dependent detoxification enzymes per se (irradiation was done at 4 degrees C), suggests that at the molecular level radioresponse in this subline is related to maintenance of GSH/GSSG redox equilibrium.     

DNA damage repair in quiescent murine mammary carcinoma cells in culture

Murine mammary carcinoma cells (line 67) were grown in unfed cultures for up to 9 days. In cultures (day 2-3) in which cells were proliferatively active and in day 3-5 (transition) cells, a large fraction of nuclear DNA was retained on polycarbonate filters when assayed by the alkaline filter elution technique. In contrast, the fraction of DNA retained on filters was significantly reduced for nonproliferating (Q, quiescent) cells from unfed 7-9 day cultures. The increase in endogenous DNA breaks followed both the decrease in proliferative state and clonogenicity in these cells. When day 7 Q cells were refed these endogenous DNA breaks were removed with a half-time of about 2.5 h. When the cells were exposed to X-irradiation and the integrity of their nuclear DNA measured by the alkaline filter elution assay, as much as a 2-fold greater frequency of radiation-induced DNA breaks was produced in Q versus P cells. DNA breaks were also removed from irradiated Q cells at a rate which was 0.23 that observed in P cells. We suggest that the depressed capacity for DNA damage removal in Q cells is responsible for their greater radiosensitivity, and the impaired DNA damage repair is probably due to a reduced level of energy sources in these unfed Q cell cultures.     

Fractionation of mechanically sheared chromatin on ECTHAM-cellulose

Chromatography of chromatin on the weak ion-exchange resin ECTHAM-cellulose was re-examined using the combined salt-pH elution conditions of Stratling, W.H., Van, N.T. and O'Malley, B.W. (1976) Eur. J. Biochem. 66, 423-433. When mechanically sheared rat liver chromatin was chromatographed on ECTHAM-cellulose the histone composition of eluted fractions was very similar, whereas early eluting fractions were enriched in non-histone proteins, including certain high mobility group proteins, and in hnRNP particles, containing newly synthesised RNA. Later eluting fractions were depleted in all of these components. The majority of hnRNP particles in early eluting chromatin were shown to be physically associated with chromatin by centrifugation in metrizamide. Hen erythrocyte chromatin contained no early eluting material. Size of DNA fragments was not a significant factor in determining the elution position of chromatin fragments. Early eluting material was not generated by endogenous nuclease and protease action. The conditions of chromatin preparation, and of elution of early chromatin fractions caused no gross disruption of chromatin structure, or dissociation of chromatin proteins, although some nucleosome sliding may have occurred. The conditions required for elution of some of the later fractions are sufficient to cause dissociation of protein, and alteration of chromatin conformation.     

Fluid Mechanics of DNA Double-Strand Filter Elution

Measurement of infrequent DNA double-strand breaks (DSB) in mammalian cells is essential for the understanding of cell damage by ionizing radiation and many DNA-reactive drugs. One of the most important assays for measuring DSB in cellular DNA is filter elution. This study is an attempt to determine whether standard concepts of fluid mechanics can yield a self-consistent model of this process. Major assumptions of the analysis are reptation through a channel formed by surrounding strands, with only strand ends captured by filter pores. Both viscosity and entanglement with surrounding strands are considered to determine the resistance to this motion. One important result is that the average elution time of a strand depends not only on its length, but also on the size distribution of the surrounding strands. This model is consistent with experimental observations, such as the dependence of elution kinetics upon radiation dose, but independence from the size of the DNA sample up to a critical filter loading, and possible overlap of elution times for strands of different length. It indicates how the dependence of elution time on the flow rate could reveal the relative importance of viscous and entanglement resistance, and also predicts the consequences of using different filters.