Effect of hypoxia on recovery from damage induced by heat and radiation in plateau-phase CHO cells

The effect of hypoxia on the induction of and recovery from damage by radiation alone and in combination with heat has been investigated using plateau-phase Chinese hamster ovary (CHO) cells. Postirradiation hypoxia reduced the potentially lethal damage recovery (PLDR) in cells irradiated under an euoxic state and completely eliminated PLDR in cells irradiated under hypoxia. Cells which were maintained under hypoxia during both irradiation and a 4-hr recovery period and then incubated for a further period of 4 hr under euoxic conditions showed PLDR, suggesting that the inhibition of PLDR by hypoxia is reversible. Oligomycin, an inhibitor of energy metabolism, completely eliminated PLDR when present at a concentration of 1 microM during the postirradiation period. Pre- or postirradiation heat treatment at 42.5 degrees C for 30 min appreciably sensitized the cells to the induction of lethality. Thermal enhancement ratio (TER) was 1.7 for cells irradiated and heat treated under hypoxic conditions. The same heat treatment reduced the oxygen enhancement ratio (OER) associated with gamma radiation from 3.1 to 2.5. Cells subjected to this postirradiation heat treatment showed a small extent of PLDR, whereas the pre-heat-treated cells showed as much recovery as non-heat-treated cells. When hypoxic conditions prevailed during the post-treatment incubation period, PLDR was reduced in preheated cells and completely eliminated in postheated cells. The kinetics of interaction between heat and radiation damage were studied by introducing a time gap of 4 hr between the treatments. Cells maintained under euoxic conditions between the treatments showed an appreciable decrease in interaction, suggesting recovery from damage induced by the first treatment. Hypoxic conditions intervening the two treatments largely inhibited the loss of sensitization. Analysis of the results suggests that cells fail to recover from sublethal heat damage when held for 4 hr under hypoxic conditions. Cells held under hypoxic conditions partly recover from the radiation damage which subsequently interacts with sublethal heat damage, resulting in cell lethality.     

Chromatin condensation dynamics and implications of induced premature chromosome condensation

Somatic cell cycle is a dynamic process with sequential events that culminate in cell division. Several physiological activities occur in the cytoplasm and nucleus during each of the cell cycle phases which help in doubling of genetic content, organized arrangement of the duplicated genetic material and perfect mechanism for its equal distribution to the two daughter cells formed. Also, the cell cycle checkpoints ensure that the genetic material is devoid of damages thus ensuring unaltered transmission of genetic information. Two important phenomena occurring during the cell cycle are the DNA condensation and decondensation cycles in the nucleus along with the cyclic expression and functioning of certain specific proteins that help in the same. Several protein families including Cyclins, cyclin dependent kinases, condensins, cohesins and surivins ensure error free, stage specific DNA condensation and decondensation by their highly specific, controlled orchestrated presence and action. Understanding the molecular mechanisms of chromatin compaction towards formation of the structural units, the chromosomes, give us valuable insights into the cellular physiology and also direct us to techniques such as premature chromosome condensation. The techniques of inducing ‘prophasing’ of interphase cells are undergoing rapid advances which have multidimensional applications for basic research and direct applications.     

Induction of chromosome damage by methylene chloride in CHO cells

The genotoxicity of methylene chloride was determined using sister-chromatid exchange (SCE) and chromosome aberration assays in cultured Chinese hamster ovary (CHO) cells. Methylene chloride caused extensive chromosome aberrations both with and without metabolic activation. However, the results of the SCE assay were negative for methylene chloride. These results agree with previously observed genotoxic effects of methylene chloride in Salmonella typhimurium and Saccharomyces cerevisiae. The fact that methylene chloride causes chromosome aberrations without increasing the SCE level indicates that complete reliance on the induction of SCE as a test system for assessing chromosomal effects is not valid.     

Modifications in repair and expression of potentially lethal damage (alpha-PLD) as measured by delayed plating or treatment with beta-araA in plateau-phase Ehrlich ascites tumor cells after exposure to charged particles of various specific energies

The ability of Ehrlich ascites tumor cells (EAT cells) to repair potentially lethal damage (alpha-PLD) as demonstrated by either an increase in survival after delayed plating or a decrease in survival after treatment with beta-arabinofuranosyladenine (beta-araA) was investigated after exposure to protons, deuterons, 3He, 4He, and heavy ions of various specific energies. A significant amount of repair or fixation was observed after delayed plating or treatment with beta-araA, respectively, in cells that were exposed to protons of 6-21 MeV energy, reflecting mainly variations in the survival curve shoulder width. Four-hour treatment with 80 microM/liter beta-araA resulted in an exponential survival curve for all proton energies tested. A decrease in particle energy increased killing and caused a reduction in Dq without a significant change in D0. The survival curve obtained after exposure of cells to 3.4 MeV protons had only a small shoulder and was only slightly modified by either delayed plating or treatment with beta-araA, suggesting a decrease in the induction rate of alpha-PLD. Similar results were also obtained after exposure to deuterons and 4He ions. The results are interpreted as indicating the importance of the specific particle energy and the delta-electron spectrum in the induction of alpha-PLD. When the results of delayed plating of cells exposed to protons, deuterons, or helium ions were pooled, an exponential relationship between Dq and penumbra radius was indicated. After exposure to 40Ar ions of 18 MeV specific energy, a shouldered survival curve was obtained, and beta-araA significantly enhanced killing by modifying Dq as well as D0, a result that also suggests induction of repairable damage by the delta particles produced and interaction of lesions induced within the core of the ion path with penumbra lesions. Based on these results a model is proposed assuming that alpha-PLD results from interaction, during the course of repair, of pairs of DNA lesions induced within a distance di. The model assumes the existence of a critical separation distance dic, with the property that pairs of lesions induced with separation distance shorter than dic (expressed as number of base pairs) will always be expressed as lethal, and the existence of a maximum separation distance dim, with the property that pairs of lesions induced with separation distance larger than dim will not interact.(ABSTRACT TRUNCATED AT 400 WORDS)     

Mechanism of radiosensitization by halogenated pyrimidines: effect of BrdU on repair of DNA breaks, interphase chromatin breaks, and potentially lethal damage in plateau-phase CHO cells.

There is evidence suggesting that radiosensitization induced in mammalian cells by substitution in the DNA of thymidine with BrdU has a component that relies on inhibition of repair and/or fixation of radiation damage. Here, experiments designed to study the mechanism of this phenomenon are described. The effect of BrdU incorporation into DNA was studied on cellular repair capability, rejoining of interphase chromosome breaks, as well as induction and rejoining of DNA double- and single-stranded breaks (DSBs and SSBs) in plateau-phase CHO cells exposed to X rays. Repair of potentially lethal damage (PLD), as measured by delayed plating of plateau-phase cells, was used to assay cellular repair capacity. Rejoining of interphase chromosome breaks was assayed by means of premature chromosome condensation (PCC); induction and rejoining of DNA DSBs were assayed by pulsed-field gel electrophoresis and induction and rejoining of DNA SSBs by DNA unwinding. A decrease was observed in the rate of repair of PLD in cells grown in the presence of BrdU, the magnitude of which depended upon the degree of thymidine replacement. The relative increase in survival caused by PLD repair was larger in cells substituted with BrdU and led to a partial loss of the radiosensitizing effect compared to cells tested immediately after irradiation. A decrease was also observed in the rate of rejoining of interphase chromosome breaks as well as in the rate of rejoining of the slow component of DNA DSBs in cells substituted with BrdU. The time constants measured for the rejoining of the slow component of DNA DSBs and of interphase chromosome breaks were similar both in the presence and in the absence of BrdU, suggesting a correlation between this subset of DNA lesions and interphase chromosome breaks. It is proposed that a larger proportion of radiation-induced potentially lethal lesions becomes lethal in cells grown in the presence of BrdU. Potentially lethal lesions are fixed via interaction with processes associated with cell cycle progression in cells plated immediately after irradiation, but can be partly repaired in cells kept in the plateau-phase. It is hypothesized that fixation of PLD is caused by alterations in chromatin conformation that occur during normal progression of cells throughout the cell cycle.(ABSTRACT TRUNCATED AT 400 WORDS)     

Direct analysis of radiation-induced chromosome fragments and rings in unstimulated human peripheral blood lymphocytes by means of the premature chromosome condensation technique

Development of the procedure to stimulate peripheral blood lymphocytes has greatly facilitated the understanding of chromosome aberration formation and repair mechanisms in human cells. Yet, because radiation induces far more initial chromosome breaks than are observed as aberrations in metaphase, it has not been possible to examine the kinetics of primary chromosome breakage and rejoining with this procedure. An improved method to induce premature chromosome condensation in unstimulated lymphocytes has been used to study primary chromosome breakage, rejoining, and ring formation at various times after irradiation with up to 800 rad of X-rays. The dose-response relations for chromosome fragments analyzed immediately or 1, 2, or 24 h after exposure were found to be linear. Rapid rejoining of chromosome fragments, which takes place in the first 3 h after X-ray exposure, was not correlated with a simultaneous increase in the formation of rings. The yield of rings per cell scored 24 h after irradiation, however, increased significantly and fit a linear quadratic equation. Both chromosome fragment rejoining and ring formation were completed about 6 h after irradiation. The frequency distributions of rings among cells followed a Poisson distribution, whereas chromosome fragments were overdispersed.     

Heterogeneity in radiation-induced DNA damage and repair in tumor and normal cells measured using the "comet" assay

A method for measuring DNA damage to individual cells, based on the technique of microelectrophoresis, was described by Ostling and Johanson in 1984 (Biochem. Biophys. Res. Commun. 123, 291-298). Cells embedded in agarose are lysed, subjected briefly to an electric field, stained with a fluorescent DNA-binding stain, and viewed using a fluorescence microscope. Broken DNA migrates farther in the electric field, and the cell then resembles a "comet" with a brightly fluorescent head and a tail region which increases as damage increases. We have used video image analysis to define appropriate "features" of the comet as a measure of DNA damage, and have quantified damage and repair by ionizing radiation. The assay was optimized for lysing solution, lysing time, electrophoresis time, and propidium iodide concentration using Chinese hamster V79 cells. To assess heterogeneity of response of normal versus malignant cells, damage to both tumor cells and normal cells within mouse SCC-VII tumors was assessed. Tumor cells were separated from macrophages using a cell-sorting method based on differential binding of FITC-conjugated goat anti-mouse IgG. The "tail moment", the product of the amount of DNA in the tail and the mean distance of migration in the tail, was the most informative feature of the comet image. Tumor and normal cells showed significant heterogeneity in damage produced by ionizing radiation, although the average amount of damage increased linearly with dose (0-15 Gy) and suggested similar net radiosensitivities for the two cell types. Similarly, DNA repair rate was not significantly different for tumor and normal cells, and most of the cells had repaired the damage by 30 min following exposure to 15 Gy. The heterogeneity in response did not appear to be a result of differences in response through the cell cycle.     

Induction by H2O2 of DNA and interphase chromosome damage in plateau-phase Chinese hamster ovary cells.

The induction by H2O2 of DNA breaks, DNA double-strand breaks (DSBs), and interphase chromatin damage and their relationship to cytotoxicity were studied in plateau-phase Chinese hamster ovary (CHO) cells. Damage in interphase chromatin was assayed by means of premature chromosome condensation (PCC); DNA DSBs were assayed by nondenaturing filter elution (pH 9.6), and DNA breaks by hydroxyapatite chromatography. Cells were treated with H2O2 in suspension at 0 degrees C for 30 min and treatment was terminated by the addition of catalase. Concentrations of H2O2 lower than 1 mM were not cytotoxic, whereas concentrations of 40 and 60 mM reduced cell survival to 0.1 and 0.004, respectively. An induction of DNA breaks that was dependent on H2O2 concentration was observed at low H2O2 concentrations that reached a maximum at approximately 1 mM; at higher H2O2 concentrations induction of DNA breaks either remained unchanged or decreased. Damage at the chromosome level was not evenly distributed among the cells, when compared to that expected based on a Poisson distribution. Three categories of cells were identified after exposure to H2O2: cells with intact, control-like chromosomes, cells showing chromosome fragmentation similar to that observed in cells exposed to ionizing radiation, and cells showing a loss in the ability of their chromatin to condense into chromosomes under the PCC reaction. The fraction of cells with fragmented chromosomes, as well as the number of excess chromosomes per cell, showed a dose response similar to that of DNA DSBs, reaching a maximum at 1 mM and decreasing at higher concentrations. The results indicate that induction of DNA and chromosome damage by H2O2 follows a complex dependence probably resulting from a depletion of reducing equivalents in the vicinity of the DNA. Reducing equivalents are required to recycle the transition metal ions that are needed to maintain a Fenton-type reaction. The absence of cell killing at H2O2 concentrations that yielded the maximum amount of DNA and chromosome damage suggests that this damage is nonlethal and repairable. It is suggested that lethal DNA and chromosome damage is induced at higher concentrations of H2O2 where cell killing is observed by an unidentified mechanism.     

Radioadaptive response: Efficient repair of radiation-induced DNA damage in adapted cells

To verify the hypothesis that the induction of a novel, efficient repair mechanism for chromosomal DNA breaks may be involved in the radioadaptive response, the repair kinetics of DNA damage has been studied in cultured Chinese hamster V79 cells with single-cell gel electrophoresis. The cells were adapted by priming exposure with 5 cGy of γ-rays and 4-h incubation at 37°C. There were no indication of any difference in the initial yields of DNA double-strand breaks induced by challenging doses from non-adapted cells and from adapted cells. The rejoining of DNA double-strand breaks was monitored over 120 min after the adapted cells were challenged with 5 or 1.5 Gy, doses at the same level to those used in the cytogenetical adaptive response. The rate of DNA damage repair in adapted cells was higher than that in non-adapted cells, and the residual damage was less in adapted cells than in non-adapted cells. These results indicate that the radioadaptive response may result from the induction of a novel, efficient DNA repair mechanism which leads to less residual damage, but not from the induction of protective functions that reduce the initial DNA damage.     

Heterogeneity in radiation-induced DNA damage and repair in tumor and normal cells measured using the "comet" assay

A method for measuring DNA damage to individual cells, based on the technique of microelectrophoresis, was described by Ostling and Johanson in 1984 (Biochem. Biophys. Res. Commun. 123, 291-298). Cells embedded in agarose are lysed, subjected briefly to an electric field, stained with a fluorescent DNA-binding stain, and viewed using a fluorescence microscope. Broken DNA migrates farther in the electric field, and the cell then resembles a "comet" with a brightly fluorescent head and a tail region which increases as damage increases. We have used video image analysis to define appropriate "features" of the comet as a measure of DNA damage, and have quantified damage and repair by ionizing radiation. The assay was optimized for lysing solution, lysing time, electrophoresis time, and propidium iodide concentration using Chinese hamster V79 cells. To assess heterogeneity of response of normal versus malignant cells, damage to both tumor cells and normal cells within mouse SCC-VII tumors was assessed. Tumor cells were separated from macrophages using a cell-sorting method based on differential binding of FITC-conjugated goat anti-mouse IgG. The "tail moment", the product of the amount of DNA in the tail and the mean distance of migration in the tail, was the most informative feature of the comet image. Tumor and normal cells showed significant heterogeneity in damage produced by ionizing radiation, although the average amount of damage increased linearly with dose (0-15 Gy) and suggested similar net radiosensitivities for the two cell types. Similarly, DNA repair rate was not significantly different for tumor and normal cells, and most of the cells had repaired the damage by 30 min following exposure to 15 Gy. The heterogeneity in response did not appear to be a result of differences in response through the cell cycle.     

A radiation-induced inhibitor of chromosome condensation and nuclear envelope breakdown in HeLa cells.

An in vitro microscopic assay for mitosis-inducing activity in mitotic HeLa cells was developed and used to demonstrate that cells irradiated and arrested in G2 phase of the cell cycle contain an inhibitor of mitosis. This assay system has a number of advantages over other assays including the use of autologous components (HeLa nuclei and mitotic cell extracts) in contrast to the microinjection method with Xenopus oocytes and without the requirements for microinjection expertise and Xenopus oocytes. The radiation-inducible inhibitor was detected at the lowest radiation dose tested (2 Gy) with maximal activity achieved within 30 min after radiation. Inhibitor activity decayed with time after radiation (2 Gy) with no activity detected at 6 h even though the cells remained in G2 phase, suggesting that either synthesis or activation of additional components is necessary for recovery from G2 arrest. The inhibitor activity was not detected in irradiated cells treated with caffeine to induce premature recovery from G2 arrest.     

Rabl's model of the interphase chromosome arrangement tested in Chinise hamster cells by premature chromosome condensation and laser-UV-microbeam experiments

Summary In 1885 Carl Rabl published his theory on the internal structure of the interphase nucleus. We have tested two predictions of this theory in fibroblasts grown in vitro from a female Chinese hamster, namely (1) the Rabl-orientation of interphase chromosomes and (2) the stability of the chromosome arrangement established in telophase throughout the subsequent interphase. Tests were carried out by premature chromosome condensation (PCC) and laser-UV-microirradiation of the interphase nucleus. Rabl-orientation of chromosomes was observed in G1 PCCs and G2 PCCs. The cell nucleus was microirradiated in G1 at one or two sites and pulse-labelled with ³H-thymidine for 2h. Cells were processed for autoradiography either immediately thereafter or after an additional growth period of 10 to 60h. Autoradiographs show unscheduled DNA synthesis (UDS) in the microirradiated nuclear part(s). The distribution of labelled chromatin was evaluated in autoradiographs from 1035 cells after microirradiation of a single nuclear site and from 253 cells after microirradiation of two sites. After 30 to 60h postincubation the labelled regions still appeared coherent although the average size of the labelled nuclear area fr increased from 14.2% (0h) to 26.5% (60h). The relative distance dr, i.e. the distance between two microirradiated sites divided by the diameter of the whole nucleus, showed a slight decrease with increasing incubation time. Nine metaphase figures were evaluated for UDS-label after microirradiation of the nuclear edge in G1. An average of 4.3 chromosomes per cell were labelled. Several chromosomes showed joint labelling of both distal chromosome arms including the telomeres, while the centromeric region was free from label. This label pattern is interpreted as the result of a V-shaped orientation of these particular chromosomes in the interphase nucleus with their telomeric regions close to each other at the nuclear edge. Our data support the tested predictions of the Rabl-model. Small time-dependent changes of the nuclear space occupied by single chromosomes and of their relative positions in the interphase nucleus seem possible, while the territorial organization of interphase chromosomes and their arrangement in general is maintained during interphase. The present limitations of the methods used for this study are discussed.Part of this work is included in the doctoral thesis of H. Baumann to be submitted to the Faculty of Biology of the University of HeidelbergPart of this work is included in the doctoral thesis of V. Teuber to be submitted to the Faculty of Medicine of the University of Freiburg i. Br.     

Effect of somatostatin on repair of ionizing radiation-induced DNA damage in pituitary adenoma cells GH3

Ionizing radiation and somatostatin analogues are used for acromegaly treatment to achieve normalization or reduction of growth hormone hypersecretion and tumor shrinkage. In this study, we investigated a combination of somatostatin (SS14) with ionizing radiation of (60)Co and its effect on reparation of radiation-induced damage and cell death of somatomammotroph pituitary cells GH3. Doses of gamma-radiation 20-50 Gy were shown to inhibit proliferation and induce apoptosis in GH3 cells regardless of somatostatin presence. It has been found that the D(0) value for GH3 cells was 2.5 Gy. Somatostatin treatment increased radiosensitivity of GH3 cells, so that D(0) value decreased to 2.2 Gy. We detected quick phosphorylation of histone H2A.X upon irradiation by the dose 20 Gy and its colocalization with phosphorylated protein Nbs-1 in the site of double strand break of DNA (DSB). Number of DSB decreased significantly 24 h after irradiation, however, clearly distinguished foci persisted, indicating non repaired DSB, after irradiation alone or after combined treatment by irradiation and SS14. We found that SS14 alone triggers phosphorylation of Nbs1 (p-Nbs1), which correlates with antiproliferative effect of SS14. Irradiation also increased the presence of p-Nbs1. Most intensive phosphorylation of Nbs1 was detected after combined treatment of irradiation and SS14. The decrease of the number of the DSB foci 24 h after treatment shows a significant capacity of repair systems of GH3 cells. In spite of this, large number of unrepaired DSB persists for 24 h after the treatment. We conclude that SS14 does not have a radioprotective effect on somatomammotroph GH3 cells.     

The induction of chromosome damage in CHO cells by beryllium and radiation given alone and in combination

Studies were conducted to determine the effects of BeSO4 or X rays, alone and in combination, on cell cycle kinetics, cell killing, and the production of chromosome aberrations in Chinese hamster ovary (CHO) cells. The concentration of BeSO4 required to kill 50% of CHO cells exposed to BeSO4 for 20 h was determined to be 1.1 mM with 95% confidence intervals of 0.72 to 1.8 mM. During the last 2 h of the 20-h beryllium treatment (0.2 and 1.0 mM), cells were exposed to 0.0, 1.0, or 2.0 Gy of X rays. Exposure to either BeSO4 or X rays produced a change in cell cycle kinetics which resulted in an accumulation of cells in the G2/M stage of the cell cycle. However, combined exposure to both agents resulted in a block similar to that observed following exposure to X rays only. The background level of chromosome damage was 0.05 +/- 0.015 aberrations/cell in the CHO cells. Seven hours after the end of exposure to 0.2 and 1.0 mM beryllium, 0.03 +/- 0.003 and 0.09 +/- 0.02 aberrations/cell, respectively, were observed. The data for chromosome aberrations following X-ray exposure were fitted to a linear model with a coefficient of 0.14 +/- 0.01 aberrations/cell/Gy. When beryllium was combined with the X-ray exposure the interactive response was predicted by a multiplicative model and was significantly higher (P less than 0.05) than predicted by an additive model. The influence of time after radiation exposure on the interaction between beryllium and X rays was also determined. No interaction between beryllium and X-ray exposure in the induction of chromosome-type aberrations (P greater than 0.05) was detected. The frequency of chromatid-type exchanges and total aberrations was significantly higher (P less than 0.05) in the radiation plus beryllium-exposed cells when compared to cells exposed to X rays only, at both 9 and 12 h after X-ray exposure. These data suggest that the multiplicative interaction may be limited to cells in the S and G2 stages of the cell cycle.     

Premature chromosome condensation. I. Visualization of x-ray-induced chromosome damage in interphase cells

A new method is described to visualize chromosome damage in interphase cells immediately after exposure to mutagenic agents. This method involves the fusion of treated interphase cells with untreated mitotic cells which results in the induction of premature chromosome condensation (PCC). Chinese hamster ovary (CHO) cells were treated with X-rays and chromosome aberrations were scored in G2-PCC and the mitotic chromosomes. The incidence of aberrations was significantly higher in PCC than that observed in the mitotic chromosomes of the treated cells. Post-irradiation incubation for I h before fusion allowed the repair of some of the chromosome damage. Data are also presented which indicate that the extent of radiation damage visualized in PCC is inversely proportional to the degree of chromosome condensation. These results indicate that the PCC method has a greater senstivity in the detection of induced chromosome damage than the standard method of scoring metaphase chromosomes.     

Method for probing cells in radiation-induced G2 arrest: demonstration of potentially lethal damage repair

Chinese hamster ovary cells were arrested in the G2 phase of the cell cycle by X-irradiation. When subsequently treated with 5 mM caffeine the arrested population progressed into mitosis as a synchronous cohort where it was harvested by mitotic cell selection. This procedure provides a means to isolate cell populations treated in G2, for the investigation of G2 arrest. Comparisons were made of the number of cells retrieved from G2 arrest with the number suffering arrest, as determined by flow cytometry and by matrix algebraic simulations of irradiated cell progression. The retrieved population was not significantly less than expected for doses up to 3.5 Gy, indicating that the retrieval process does not favour the isolation of any population subset below this dose. Cell populations retrieved from arrest at varying intervals (0-3 h) after irradiation (0-3.5 Gy) showed an increase in survival with increase in interval, consistent with repair of potentially lethal damage. The repair curves (surviving fraction vs time) were each described by a single exponential. G2 cells that were brought to mitosis without a period of arrest exhibited the same radiation response as cells irradiated in mitosis.