Fixation of potentially lethal radiation damage by post-irradiation exposure of Chinese hamster cells to 0.5 M or 1.5 M NaCl solutions.

The effect of 0.05 M and 1.5 M NaCl treatments on CHO cells during and after irradiation has been examined. Treatment with either hypotonic or hypertonic salt solutions during and after irradiation resulted in the fixation of radiation damage which would otherwise not be expressed. The half time for fixation was 4 to 5 min, and the increased expression of the potentially lethal damage by anisotonic solutions was mainly characterized by large decreases in the shoulder of the survival curve, as well as by decreases in DO. Fixation of radiation damage at 37 degrees C occurred to a much greater extent for the hypertonic treatment than for the hypotonic treatment and was greater at 37 degrees C than at 20 degrees C. Although both the hypotonic and hypertonic treatments during and after irradiation reduced or eliminated the repair of sublethal and potentially lethal damage, treatment during irradiation only, radiosensitized the cells when the treatment was hypotonic, and radioprotected the cells when the treatment was hypertonic. These observations are discussed in relation to salt treatments and different temperatures altering competition between repair and fixation of potentially lethal lesions, the number of which depends on the particular salt treatment at the time of irradiation.     

Ataxia-telangiectasia homo- and heterozygous cells show a normal repair and fixation response to anisotonic NaCl treatment after irradiation

The effect of anisotonic NaCl treatment on fixation and repair of radiation-induced potentially lethal damage (PLD) was tested in normal human cells and in three homozygous ataxia-telangiectasia (A-T) and two heterozygous A-T cell strains. Fixation of radiation-induced PLD occurred in all cell strains exposed to 0.05, 0.5, or 1.5 mole/liter NaCl solutions immediately after irradiation. This effect was observed in both plateau-phase and exponentially growing normal and A-T cells. When an incubation period at 37 degrees C was introduced between irradiation and the subsequent anisotonic treatment, recovery was observed in both normal and A-T cells strains. These data show that A-T cells are as proficient as normal cells in repairing PLD that is sensitive to anisotonic NaCl treatment. It is proposed that two PLD repair systems may exist, one that is expressed after irradiation in proliferatively arrested cells and another that occurs in plateau-phase as well as exponentially growing cells, and is expressed by the postirradiation treatments described here and by Raaphorst and Azzam (Radiat. Res. 86, 52-66 (1981].     

Modification of cell survival and transformation by exposure to anisotonic solutions during irradiation

C3H 10T 1/2 cells were exposed to a wide range of anisotonic NaCl solutions and were irradiated during the last few minutes of the salt treatment. Radiosensitization in terms of cell killing and transformation was observed for hypotonic NaCl treatment. Hypertonic treatment with 0.5 mol/liter NaCl also caused radiosensitization for cell killing and transformation, while extreme hypertonic treatment (1.5 mol/liter) resulted in radioprotection for both end points. Radiosensitization for both end points declined as the hypotonic treatment (0.05 mol/liter) was prolonged. To a lesser extent, a decline in radioprotection by 1.5 mol/liter treatment occurred as exposure time increased.     

Fixation of radiation-induced potentially lethal damage by anisotonic treatment and its modification by DMSO or BrdUrd in V79 cells

Summary The effects of radiosensitization by bromodeoxyuridine (BrdUrd) substitution and radioprotection by dimethyl sulfoxide (DMSO) have been examined in relation to fixation and repair of radiation damage by anisotonic treatment. The fixation of radiation damage in cells exposed to 0.05 M or 1.5 M NaCl after irradiation was the same at equal survival levels irrespective of (BrdUrd) incorporation into the DNA. Also, during incubation between irradiation and a subsequent anisotonic treatment, cells containing BrdUrd repaired radiation damage to the same extents as cells without BrdUrd.DMSO treatment resulted in radiprotection. Fixation, by anisotonic salt treatment, of damage resulting from irradiation in the presence of DMSO was less extensive than from irradiation in the absence of DMSO, even though X-ray doses were adjusted to give equal survival levels. Recovery during incubation at 37° C between irradiation and a subsequent salt treatment occurred for irradiation in the presence and absence of DMSO. These data show that the alteration of DNA radiosensitivity by BrdUrd had no effect on fixation or repair of radiation damage as assessed by salt treatment, while DMSO which is an OH scavenger caused the damage to be less susceptible to fixation and this damage was repaired during incubation at 37° C.     

Enhancement of hyperthermic killing cultured mammalian cells by treatment with anisotonic NaCl or medium solutions

The exposure of cultured Chinese hamster cells (CHO) to anisotonic medium increased the cellular sensitivity to heat treatment at 42.3°C. A greater potentiation of heat killing is observed when the anisotonic solution consists of pure NaCl in water compared to growth medium made anisoltonic by dilution or by addition of NaCl. Hypertonic treatment caused greater heat sensitization than hypotonic treatment. Thermal tolerance observed in the control cells after 4–6 hours of heating in medium was also observed for cells exposed to anisotonic media during heating if the heating period was greater than 4 h. The exposure of cells to anisotonic media during heating if the heating period was greater than 4 h. The exposure of cells to anisotonic NaCl solutions during heating removed the shoulder from the heat survival curve, while the curves for cells heated in medium made anisotonic retained their shoulders. These studies suggest: (1) that either the plasma membrane is a primary target for heat inactivation of mammalian cells, or (2) that changes in intracellular ion concentrations enhance thermal damage occurring in critical intracellular structures.     

Potentially lethal radiation damage repair and its inhibition by hyperthermia in normal hamster cells, mouse cells, and transformed mouse cells

The capacity of plateau-phase Chinese hamster V79 and normal and transformed C3H-10T1/2 cells for repair of potentially lethal radiation damage (PLD) was evaluated for cells irradiated alone or given combined treatments of heat and radiation. The data show that all cell lines tested could repair PLD and that transformation to the tumorigenic state may reduce the capacity to repair PLD, especially if cells are evaluated at equal survival levels. Hyperthermia treatments before irradiation produced less sensitization than treatments after irradiation. In addition, hyperthermia treatment led to the inhibition of cellular capacity to repair PLD. This effect was the greatest for cells heated after irradiation, and repair of PLD could be completely eliminated. Several temperature isodose heat treatments were evaluated, and the lower temperature heat treatments were more effective in the inhibition of PLD than the higher temperature heat treatments; this is consistent with earlier results indicating temperature dependence in thermal radiosensitization (S. A. Sapareto et al., Int. J. Radiat. Oncol. Biol. Phys. 5, 343-347 (1979)).     

Hypertonic treatment inhibits radiation-induced nuclear translocation of the Ku proteins G22p1 (Ku70) and Xrcc5 (Ku80) in rat fibroblasts

The effects of X irradiation and hypertonic treatment with 0.5 M NaCl on the subcellular localization of the Ku proteins G22p1 (also known as Ku70) and Xrcc5 (also known as Ku80) in rat fibroblasts with normal radiosensitivity were examined using confocal laser microscopy and immunoblotting. Although these proteins were observed mainly in the nuclei of human fibroblasts, approximately 80% of the intensities of immunofluorescence from both G22p1 and Xrcc5 was observed in the cytoplasm of rat fibroblasts. When the rat cells were X-irradiated with 4 Gy, the intensities of the fluorescence derived from G22p1 and Xrcc5 in the nuclei increased from 20% to 50% of the total cellular fluorescence intensity at 20 min postirradiation. No significant differences were observed between the total intensities of the cellular fluorescence from the proteins in unirradiated and irradiated rat fibroblasts. The results showed that the proteins were translocated from the cytoplasm to the nucleus in the rat cells after X irradiation. The nuclear translocation of the proteins from the cytoplasm was inhibited by hypertonic treatment of the cells with 0.5 M NaCl for 20 min, which inhibits the fast repair process of potentially lethal damage (PLD). When the rat cells were treated with 0.5 M NaCl immediately after X irradiation, the repair of DNA DSBs was inhibited. The surviving fraction was approximately 60% of that of irradiated cells that were not treated with 0.5 M NaCl. The surviving fraction increased with incubation time in the growth medium before treatment with NaCl. The proportions of the intensities of fluorescence from G22p1 in the nuclei of X-irradiated cells also increased from 20% to 50% with increasing interval between X irradiation and treatment with NaCl. These results suggest that nuclear translocation of G22p1 and Xrcc5 is important for the fast repair process of PLD in rat cells.     

Attenuation of radiation-induced genomic instability by free radical scavengers and cellular proliferation.

To investigate the mechanisms of radiation-induced chromosomal instability, cells were irradiated in the presence of the free radical scavengers DMSO, glycerol, or cysteamine, in the presence of DMSO while frozen, or held in confluence arrest post-irradiation to permit cells to repair potentially lethal DNA damage. Clones derived from single progenitor cells surviving each treatment were then analyzed for the subsequent development of chromosomal instability. The presence of scavengers (+/- freezing) during irradiation, and the recovery from potentially lethal damage after irradiation led to an increase in cell survival that was accompanied by a decrease in the initial yield of chromosomal rearrangements. Furthermore, analysis of over 400 clones and 80,000 metaphases indicates that these same treatments reduced the incidence of instability at equitoxic doses when compared to controls irradiated in the absence of scavengers at ambient temperature. Results suggest that preventing reactive species from damaging DNA, promoting chemical repair of ionized DNA intermediates, or allowing enzymatic removal of genetic lesions, represent measures that reduce the total burden of DNA damage and reduce the subsequent onset of radiation-induced genomic instability.     

Recovery from lethal and mutagenic damage during postirradiation holding and low-dose-rate irradiations of cultured hamster cells

Survival and mutation to thioguanine resistance were measured in V79-4 hamster cells grown to plateau phase without refeeding and irradiated with 60Co gamma rays. The effects of low-dose-rate irradiation and of postirradiation holding on recovery from gamma-ray damage leading to these two responses were also studied. The responses of these plateau (extended G1)-phase cells to acute irradiation were similar to those we previously found for exponentially growing cells, including the linear relationship between induced mutant frequency and (log) surviving fraction. Irradiation at low dose rate (0.34 rad/min) considerably reduced both the lethal and mutagenic effects of given doses of gamma rays, but the linear mutation-survival relationship was approximately the same as for acute irradiation. In contrast, cells given a 5-hr holding period after acute irradiation showed the anticipated recovery from potentially lethal damage but no recovery from damage leading to mutation. These results are discussed in terms of previously proposed cellular repair processes (sublethal damage repair and potentially lethal damage repair) and the possibility that the radiation damage leading to lethality is different from mutagenic damage.     

Arrest of irradiated G1, S, or G2 cells at mitosis using nocodazole promotes repair of potentially lethal damage

The ability of synchronized Ehrlich ascites tumor cells, irradiated in G1, S, and G2 phases, to repair potentially lethal damage when arrested at mitosis by using 0.4 microgram/ml nocodazole, a specific inhibitor of microtubule polymerization, has been studied. Cells irradiated in these phases were found to repair potentially lethal damage at mitosis. The extent of this repair was similar to that observed for cells irradiated at the same stages in the cell cycle but allowed to repair potentially lethal damage by incubating in balanced salt solution for 6 hr after X irradiation.     

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.     

Response of X-ray-sensitive CHO mutant cells to gamma radiation. I. Effects of low dose rates and the process of repair of potentially lethal damage in G1 phase

X-ray-sensitive CHO mutants (xrs-5 and xrs-6) were exposed to isoleucine-deficient (IL-) medium for 24-36 h to accumulate G1-phase cells. Cells exposed to IL- medium for up to 5 days did not show significant changes in plating efficiency when returned to normal medium. Nearly confluent cultures of IL- -treated cells were irradiated with either 60Co gamma rays (75 cGy/min) or 137Cs gamma rays (2.7, 6.0, or 15.3 cGy/h). A significant reduction (approximately 2.5-fold) in the radiation sensitivity of the parental CHO K-1 cells was observed for chronic low-dose-rate radiation exposure compared to the results obtained for acute high-dose-rate exposure. However, no noticeable differences were observed in the survival curves of either xrs-5 or xrs-6 cells when low-dose-rate and acute exposures were compared. CHO K-1 cells exhibited potentially lethal damage repair while held in IL- medium after gamma irradiation, whereas no repair was observed in either of the radiation-sensitive mutant lines examined at similar survival levels.     

Mechanism of radiosensitizing effect of chloride ion on E. coli

Cells of E. coli capable of repairing DNA damage are sensitized to radiation in the presence of NaCl. However, the enhanced radiolethality was suppressed by the addition of compounds such as an amino acid to the irradiation buffer. The protective efficiencies of these compounds depend on their reactivities with Cl-.2 or OH.. ATP synthesis in the cells irradiated in the presence of NaCl was severely inhibited depending on the dose of irradiation. This reduced rate of ATP synthesis can account for the inhibition of protein, RNA and DNA synthesis in the irradiated cells with NaCl.     

Enhancement of the Over-all Lethal Effect of Ionizing Radiations on Microorganisms by Sodium Chloride

Aiming at the enhancement of total lethal effect of radiations, chemical agent actions on irradiated cells were investigated. It was found that the sensitivity of irradiated cells to NaCl was markedly increased by radiation. In other words, the over-all lethal effect of radiations on microorganisms was enhanced by the pre- or post-treatment with NaCl. Some microbiological aspects of radiation enhancement of NaCl sensitivity were described in this paper, connecting with the possible mechanism. The fact that this phenomenon of radiation enhancement was observed in the case of the cells which gave the one-hit type survival curve suggests that radiation damage responsible for this phenomenon is not restricted to biological targets. Further discussion and experimental evidences will be presented in the subsequent paper.     

Mechanism of radiosensitization by halogenated pyrimidines: effect of BrdU on radiation induction of DNA and chromosome damage and its correlation with cell killing

The effect of BrdU incorporation on cell radiosensitivity as well as on the induction of DNA double-strand breaks (DSB) and chromosome damage by radiation was studied in CHO cells. Induction of DNA DSB was measured by the nonunwinding filter elution technique and damage at the chromosome level was visualized and scored in G1 cells using the technique of premature chromosome condensation. The results indicated an increase in the radiosensitivity of cells grown in the presence of BrdU. Although sensitization was observed both in cells irradiated in the exponential phase and in cells irradiated in the plateau phase of growth, the degree of sensitization was greater in exponentially growing cells for the same degree of thymidine replacement by BrdU in the DNA. It is hypothesized that this indicates the possible importance of chromatin structure at the time of irradiation and/or the importance of chromatin conformation changes after irradiation in the expression of radiation-induced potentially lethal damage in cells containing BrdU. Incorporation of BrdU affected both the slope and the width of the shoulder of the survival curve and increased the induction of DNA and chromosome damage per unit absorbed dose. The increase observed in the slope of the survival curve was quantitatively similar to the increase observed in damage induction at the DNA and the chromosome level, suggesting a cause-effect relationship between these phenomena. Reduction in the width of the shoulder did not correlate with the increase in the induction of DNA and chromosome damage, suggesting that different phenomena, probably related to enhanced fixation of radiation-induced potentially lethal damage in cells containing BrdU, underlie its modulation.     

Interaction between radiation and drug damage in mammalian cells. III. The effect of adriamycin and actinomycin-D on the repair of potentially lethal radiation damage.

We have studied the effects of actinomycin-D (AMD) and Adriamycin (ADRM) on the repair of radiation damage in Chinese hamster cells (V79) in plateau phase growth. Suppression of potentially lethal damage repair (PLDR) was observed in the presence of non-toxic levels of AMD and minimally toxic levels of ADRM. The suppression of PLDR by AMD persisted as long as the drug was present. Removal of AMD was followed by prompt repair of potentially lethal injury suggesting that suppression of PLDR by AMD was not accompanied by fixation of injury to a non-repairable state. On the other hand, irradiated cells exposed to ADRM eventually repair potentially lethal injury in the presence of drug after an initial delay. AMD, but not ADRM, inhibited repair of sublethal radiation damage.     

Poly(ADP-ribose) metabolism in X-irradiated Chinese hamster cells: its relation to repair of potentially lethal damage

Nicotinamide-adenine dinucleotide (NAD+) is the substrate used by cells in poly(ADP-ribose) synthesis. X-irradiation of log-phase Chinese hamster cells caused a rapid decrease in NAD+ levels which was linearly dependent on radiation dose. The activity of ADP-ribosyl transferase ( ADPRT ) also increased linearly with radiation dose. The decrease of NAD+ was slower, and the increase in ADPRT activity was less pronounced, in a radiation sensitive line, V79- AL162 /S-10. An inhibitor of ADPRT , m-aminobenzamide, largely prevented the depletion of cellular NAD+ and reduced the rate at which ADPRT activity disappeared during post-irradiation incubation. Post-irradiation treatment with hypertonic buffer or with medium containing D2O--which inhibit repair of radiation-induced potentially lethal damage--enhanced the depletion of NAD+ and prevented the reduction in ADPRT activity following irradiation. The characteristics of the effects of treatment with hypertonic buffer on NAD+ metabolism were qualitatively similar to the effects that such treatment has on radiation-induced cell killing. These results suggest that poly(ADP-ribose) synthesis after irradiation plays a role in the repair of potentially lethal damage.     

The use of immunostaining to quantify x-ray and heat-induced multiple microtubule organizing centers in normal and transformed cells

Microtubule organizing centers (MTOC) in control, irradiated and heated C3H 10T1/2 mouse embryo cells and two radiation-transformed sublines, R1 and R25, were made visible by indirect immunofluorescence using antibody against tubulin. The MTOC were reformed by 5-min incubation in fresh medium after the microtubules were depolymerized with nocodazole. The R1 line had a different distribution of MTOC/cell than the parent 10T1/2 line or R25, which had similar distributions. After irradiation, multiple MTOC appeared in the normal and radiation-transformed cells irradiated to 10 Gy and incubated for 24 or 48 h. The multiple foci of microtubule reformation in the irradiated cells indicate that radiation damage is expressed in structural elements in the cytoplasm. After heat treatment of the three cell lines (43 degrees C for 93 min and 45 degrees C for 25 min), the MTOC were disrupted and many cells did not have visible organizing centers at 24 or 48 h, while others had a large number of small centers of microtubule reformation. The distribution of MTOC/cell seen in R25 cells after the treatment had similar patterns to those of the 10T1/2 line rather than to those of the other radiation-transformed line, R1. Thus, the radiation or heat response seen in the MTOC is not dependent upon cell transformation.     

The action of caffeine on X-irradiated HeLa cells. X. Depressed recovery from potentially lethal damage in cells containing 5-bromodeoxyuridine

HeLa S3 cells were sensitized to the lethal action of 220-kV X rays by partially replacing the thymidine in their DNA with 5-bromodeoxyuridine (BrdU). To examine the expression of and recovery from potentially lethal radiation damage (PLD), both BrdU-grown and control cells were treated with 4 mM caffeine for increasing times up to 2 days, either immediately after irradiation or after increasing delays up to 28 h. When the same dose of X rays (3 Gy) was applied to BrdU-grown and control cells, the difference in survival that is found in the absence of caffeine disappeared after about 30 h of incubation in its presence; when isosurvival doses were applied (BrdU-grown cells, 2.5 Gy; control cells, 4 Gy), the control cells suffered more killing. When treatment with caffeine was delayed for progressively longer times after both groups of cells received 3 Gy, the control cells achieved a higher level of survival. These results indicate that the increased radiation sensitivity of cells containing BrdU derives from a decreased ability to repair PLD.     

Radiation sensitization of Escherichia coli B/r by 2'-chloro-2'-deoxythymidine under various irradiation conditions

In order to elucidate the mechanism of sensitization of E. coli B/r cells to X-irradiation by 2'-chloro-2'-deoxythymidine (2'Cl-TdR), the survival curves of the cells in which 2'Cl-TdR was incorporated into DNA were obtained following X-irradiation under various conditions. The marked sensitization of E. coli cells by 2'Cl-TdR to the killing action of X-rays was observed, when E. coli cells labelled with 2'Cl-TdR were exposed to X-rays in the absence of oxygen as well as in the presence of oxygen. The sensitization factor calculated from inactivation constants from survival curves irradiated in the absence of O2 was about a half of that obtained in the presence of O2. Under the conditions where 2'Cl-TdR was not incorporated into the DNA of E. coli cells, the presence of 2'Cl-TdR in the cell suspension fluid at the time of irradiation caused no sensitization of the cells to X-irradiation. The sensitization factor for 2'Cl-TdR obtained under N2O was almost same as that obtained under N2. It was also observed that the sensitization factor obtained in the presence of glycerol at a concentration of 1 mol dm-3 under N2 was similar to that obtained in the absence of glycerol. These results indicated that the direct effect of ionizing radiation on DNA was closely associated with the sensitization of E. coli B/r cells by 2'Cl-TdR and that the radical at the C-2' position of the deoxyribose moiety in DNA produced by X-irradiation was transformed into lethal damage for E. coli cells even in the absence of O2. However, this transformation occurred more efficiently in the presence of O2 than in the absence of O2.