Evidence for differences among the sectors of potentially lethal damage expressed by hypertonic treatment in plateau-phase V79 cells after exposure to neutrons or gamma rays: the importance of distinction between alpha and beta-PLD forms

Expotentially growing and plateau-phase V79 cells were exposed to various doses of neutrons and plated either immediately or after treatment in hypertonic medium (250-500 mM NaCl) to express radiation-induced potentially lethal damage (PLD). Postirradiation treatment of exponentially growing cells in hypertonic medium (500 mM) resulted in a decrease in both Dq and D0, whereas postirradiation treatment of plateau-phase cells in hypertonic medium (in the range between 200 to 1,500 mM) resulted mainly in a reduction of Dq. This difference in response between exponentially growing and plateau-phase cells may reflect differences in the chromatin structure in cells at various stages of the cell cycle, affecting fixation of radiation-induced damage. Exposure of plateau-phase cells to gamma rays, on the other hand, resulted in a treatment time and salt concentration-dependent decrease in Dq along with a decrease in D0. Repair of neutron-induced, hypertonic treatment-sensitive PLD, measured by delaying treatment for various periods after irradiation, was found to proceed with a t1/2 of about 1 h. This is similar to the repair kinetics obtained by delaying treatment of plateau-phase cells with 150 microM beta-D-arabinofuranosyladenine (araA) after exposure to gamma rays or neutrons and contrasts the repair kinetics observed after exposure of cells to gamma rays. In this case, hypertonic treatment was found to affect a form of PLD repaired with a t1/2 of 10-15 min (beta-PLD) and araA, a different form of PLD, repaired with a t1/2 of about 1 h (alpha-PLD). Based on these results it is hypothesized that the sector of lesions affected by hypertonic treatment and araA coincides after exposure to neutrons (effect on alpha-PLD) but only partly overlaps after exposure to gamma rays (due to the effect on beta-PLD of hypertonic treatment). The results presented, together with previously published observations, suggest a differential induction and/or fixation by hypertonic medium of the alpha- and beta-PLD forms as the LET of the radiation increases. Furthermore, they indicate that direct comparison of the effects of a postirradiation treatment, as well as of the repair kinetics obtained by its delayed application after exposure to radiations of various LET, should be made with caution.     

Effect of arabinofuranosyladenine on radiation-induced chromosome damage in plateau-phase CHO cells measured by premature chromosome condensation: implications for repair and fixation of alpha-PLD

The effect of the DNA polymerase inhibitor beta-arabinofuranosyladenine (araA) on radiation-induced damage was studied at the cell survival and chromosome level in unfed plateau-phase cultures of Chinese hamster ovary cells. At the cell survival level postirradiation treatment with araA fixed a form of radiation-induced potentially lethal damage, termed alpha-PLD. In the absence of araA treatment, repair of PLD resulted in the formation of the survival curve shoulder in immediately plated cells and in the increase in survival observed after delayed plating. The repair kinetics observed after delayed plating of plateau-phase cells or after delayed administration of 500 microM araA were similar, suggesting that both protocols assay similar lesions. AraA-mediated fixation reached a plateau at concentrations higher than 500 microM, indicating complete fixation of alpha-PLD. At the cytogenetic level, postirradiation treatment with araA at concentrations higher than 500 microM caused a complete inhibition of chromosome repair, as scored by premature chromosome condensation. In the absence of araA, the linearity of the dose-effect relationship for chromosome fragmentation obtained immediately after irradiation was preserved even after long repair times. The repair kinetics of chromosome damage measured in cells held postirradiation in the plateau phase were the mirror image of the repair kinetics for alpha-PLD. The half-time was 1 h in both cases and repair reached a plateau after about 4-6 h. AraA-mediated repair inhibition of chromosome damage was reversible, and a decrease in residual chromosome damage was observed after post-treatment incubation in araA-free conditioned medium. This persistent chromosome damage increased with increasing araA concentration and, as with PLD fixation, reached a plateau at about 500 microM. These results suggest that repair and araA-mediated fixation of alpha-PLD have their counterparts at the chromosome level as indicated by the similar repair kinetics and inhibition/fixation characteristics obtained for alpha-PLD and chromosome damage. This relationship implies a correlation between repair at the DNA and the chromosome level and suggests that DNA polymerization is required for the repair of chromosome damage.     

The influence of conditions affecting repair and fixation of potentially lethal damage on the induction of 6-thioguanine resistance after exposure of mammalian cells to X-rays

We have studied the influence of postirradiation conditions resulting in repair or fixation of X-ray-induced potentially lethal damage (PLD) on the induction of 6-thioguanine-resistant mutants in plateau phase Ehrlich ascites tumour cells. For repair of PLD cells were incubated under plateau-phase conditions for 6–8 hours after irradiation. For fixation of PLD we used either a 4-h treatment with 120 μM β-araA or a 50-min treatment in hypertonic medium (2.5 times the normal tonicity). These treatment are known to effectively reduce or eliminate the shoulder of the X-ray survival care. The mutants were allowed to form colonies in agar medium containing 1.5 μg/ml 6-thioguanine, after expression times of 6–12 days.We observed a decrease in the number of mutants induced (per 10⁵ cells) when the cells were allowed to repair PLD, as compared with that of cells processed immediately after irradiation, and an increase in their number after treatment either with β-araA or in hypertonic medium. The curves obtained for the induction of mutants as a function of the radiation dose were usually upward bending.After irradiation at low dose rate we obtained an exponential survival curve and a linear induction of mutants as a function of the dose.Based on these results we suggest that potentially lethal lesions resulting in the formation of the shoulder of the survival curve are not identical with those lesions responsible for the induction of mutants.     

Recovery from potentially lethal damage and recruitment time of noncycling clonogenic cells in 9L confluent monolayers and spheroids

Cells that have been grown as multicell tumor spheroids exhibit radioresistance compared to the same cells grown in monolayers. Comparison of potentially lethal damage (PLD) repair and its kinetics was made between 9L cells grown as spheroids and confluent monolayers. Survival curves of cells plated immediately after irradiation showed the typical radioresistance associated with spheroid culture compared to plateau-phase monolayers. The dose-modification factor for spheroid cell survival is 1.44. Postirradiation incubations in normal phosphate-buffered saline (PBS), conditioned media, or 0.5 M NaCl in PBS reduced the differences in radiosensitivity between the two culture conditions. Postirradiation treatment in PBS or conditioned medium promoted repair of potentially lethal damage, and 0.5 M NaCl prevented the removal of PLD and allowed the fixation of damage resulting in lower survival. Survival of spheroid and monolayer cells after hypertonic NaCl treatment was identical. NaCl treatment reduced Do more than it did the shoulder (Dq) of the survival curve. PLD repair kinetics measured after postirradiation incubation in PBS followed by hypertonic NaCl treatment was the same for spheroids and for plateau-phase monolayers. The kinetics of PLD repair indicates a biphasic phenomenon. There is an initial fast component with a repair half-time of 7.9 min and a slow component with a repair half-time of 56.6 min. Most of the damage (59%) is repaired slowly. Since the repair capacity and kinetics are the same for spheroids and monolayers, the radioresistance of spheroids cannot be explained on this basis. Evidence indicates that the time to return from a Go (noncycling G1 cells) state to a proliferative state (recruitment) for cells from confluent monolayers and from spheroids after dissociation by protease treatment may be the most important determinant of the degree of PLD repair that occurs. Growth curves and flow cytometry cell cycle analysis indicate that spheroid cells have a lag period for reentry into a proliferative state. Since plating efficiency remains high and unchanging during this period, one cannot account for the delay on the basis of the existence of a large fraction of Go cells which are not potentially clonogenic. The cell cycle progression begins in 6-8 h for monolayer cells and in 14-15 h for spheroids. It is hypothesized that the slower reentry of spheroid cells into a cycling phase allows more time for repair than for the rapidly proliferating monolayer cells.     

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)     

Repair and fixation of potentially lethal damage (PLD) as demonstrated by delayed plating or incubation with araA in contact inhibited refed plateau-phase C3H mouse embryo 10 T1/2 cells grown in the presence of BrdUrd

Summary OH mouse 10 T_1/2 cells showing strong inhibition of growth at confluency were grown under daily refeeding in the presence of BrdUrd (from 0 to 1 µM) and exposed to-rays either while exponentially growing or in the plateau phase. An increase in radiosensitivity was observed in both growth conditions mainly reflected by a reduction in Dq. Greater radiosensitization was observed in exponentially growing than in plateau-phase cells, and 3–4 times higher BrdUrd concentrations were required in plateau-phase cells for similar potentiation in killing. This effect could not be entirely attributed to a reduction in BrdUrd incorporation since measurements with³H-BrdUrd showed reductions in incorporation between only 17–47% in plateau-phase cells. The rate of repair of potentially lethal damage (PLD) as demonstrated by delayed plating was not affected by the incorporation of BrdUrd, but the amount of repair (measured as the relative increase in cell survival) was higher for BrdUrd-containing cells. Post-irradiation treatment of cells in the plateau-phase (no BrdUrd) with 9--d-arabinofuranosyladenine (araA) caused fixation of radiation-induced PLD. AraA treatment of cells grown in the presence of various amounts of BrdUrd also caused fixation of PLD, but resulted in survival levels similar to those observed with cells growing in BrdUrd-free medium. This result indicates that BrdUrd mediated radiosensitization cannot be observed when cells are prevented from repairing PLD by postirradiation incubation with araA. Based on these findings we propose that the mechanism of radiosensitization by BrdUrd incorporation might be, by increasing probability of fixation, mediated by the postirradiation progression of cells through the cycle, of a sector of PLD also sensitive to post-irradiation treatment with araA. For this sector of PLD the term -PLD has been proposed.This investigation was partly supported by PHS grants CA33951, CA39938 and CA42026 awarded by NCI, DHHS     

The level of induced DNA double-strand breaks does not correlate with cell killing in X-irradiated mitotic and G1-phase CHO cells

The neutral (pH 9.6) filter elution technique was used to evaluate DNA damage induced in CHO cells irradiated at mitosis or in G1-phase under various incubation and postirradiation treatment conditions. Mitotic and G1/S border cells were more sensitive to radiation than G1 cells with respect to cell killing, but showed similar (G1/S) or lower (M) DNA elution dose--response curves. Similar cell survival and DNA/elution dose--response curves were obtained with plateau-phase cultures containing mainly G1-cells, as well as with G1 cells obtained after division of mitotic cells in either fresh or conditioned medium. However, survival of plateau-phase cells could be modified substantially by delayed-plating or postirradiation treatment with araA. These results, together with previously published observations, indicate that induction of DNA dsb cannot be invoked as an explanation for the variations in radiosensitivity observed through the cycle, or as an explanation for the formation of the survival curve shoulder. It is proposed that repair and fixation of radiation-induced DNA damage, expressed at the cell survival level as repair and fixation of alpha-PLD, are responsible for these effects.     

Cell-cycle-dependent repair of potentially lethal damage in the XR-1 gamma-ray-sensitive Chinese hamster ovary cell

Repair of potentially lethal damage (PLD) was investigated in a gamma-ray-sensitive Chinese hamster cell mutant, XR-1, and its parent by comparing survival of plateau-phase cells plated immediately after irradiation with cells plated after a delay. Previous work indicated that XR-1 cells are deficient in repair of double-strand DNA breaks and are gamma-ray sensitive in G1 but have near normal sensitivity and repair capacity in late S phase. At irradiation doses from 0 to 1.0 Gy (100 to 10% survival), the delayed- and immediate-plating survival curves of XR-1 cells were identical; however, at doses greater than 1.0 Gy a significant increase in survival was observed when plating was delayed (PLD repair), approaching a 20-fold increase at 8 Gy. Elimination of S-phase cells by [3H]thymidine suicide dramatically increased gamma-ray sensitivity of plateau-phase XR-1 mutant cells and reduced by 600-fold the number of cells capable of PLD repair after a 6-Gy dose. In contrast, elimination of S-phase cells in plateau-phase parental cells did not alter PLD repair. These results suggest that the majority of PLD repair observed in plateau-phase XR-1 cells occurs in S-phase cells while G1 cells perform little PLD repair. In contrast, G1 cells account for the majority of PLD repair in plateau-phase parental cells. Thus, in the XR-1 mutant, a cell's ability to repair PLD seems to depend upon the stage of the cell cycle at which the irradiation is delivered. A possible explanation for these findings is discussed.     

Radiation-induced potentially lethal damage: DNA lesions susceptible to fixation

The various postirradiation incubation conditions reported to uncover potentially lethal damage (PLD) induced by ionizing radiation are outlined and critically discussed. The process of damage fixation is the most characteristic determinant in distinguishing between PLD and other forms of damage (lethal or non-lethal). The results compiled indicate the induction of two forms of PLD (termed alpha- and beta-PLD). Evidence is presented that repair and fixation of alpha-PLD may underlie the variation in radiosensitivity observed through the cycle. Beta-PLD appears to be sensitive only to postirradiation treatment in anisotonic sale solutions. Results obtained at the DNA and chromosome level, under conditions allowing repair or causing fixation of PLD, are reviewed and combined together to devise a qualitative model that outlines a possible sequence of events from damage fixation at the DNA level, to damage fixation at the chromosome level and, ultimately, to cell death. It is suggested that damage uncovered at the cellular level as potentially lethal, comprises DNA dsb (single, pairs or groups) and that fixation is mediated by forces transmitted to the double helix through alteration (local or general) in chromatin conformation. Changes in chromatin conformation are caused either as a result of the cell's progression through the cycle or in response to a postirradiation treatment. The fixation process leads to the induction of chromosome aberrations. The validity of the concept of PLD in in vivo systems is shown, and the possible importance of PLD repair in radiation therapy is reviewed. The concept of PLD is compared to the concept of sublethal damage, and the possibility that similar molecular lesions underlie both types of damage is discussed.     

Loss of repair capacity in density-inhibited cultures of C3H 10T1/2 cells during multifraction irradiation

Multifraction survival curves for slowly cycling, density-inhibited C3H 10T1/2 cells were shown previously to bend toward lower survival levels with increasing total dose, even for doses per fraction as small as about 2.0 Gy. In an attempt to explain this, we tested the capacity of cells to repair potentially lethal damage (PLD) as fractionation progressed. Plateau-phase cultures were exposed to repeated doses of 4.0 Gy of 137Cs gamma rays delivered at 12-hr intervals. After zero, three, five, and seven fractions, some cultures were put aside, incubated for 12 hr at 37 degrees C, irradiated with a single dose of 9.0 Gy, and subsequently returned to a 37 degrees C incubator. At 0, 2, 4, 6, and 12 hr after the 9.0 Gy dose, cultures were trypsinized and plated for a survival assay. Following three fractions of 4.0 Gy, cells were able to repair PLD as well as those receiving a single dose of 9.0 Gy without prior fractionation. Following five fractions, cells were less able to repair PLD, and after seven fractions, only a very small amount of PLD repair was detectable using this method of measurement.     

Quiescence in 9L cells and correlation with radiosensitivity and PLD repair

The onset of quiescence, changes in X-ray sensitivity, and changes in capacity for potentially lethal damage (PLD) repair of unfed plateau-phase 9L44 cell cultures have been systematically investigated. The quiescent plateau phase in 9L cells was the result of nutrient deprivation and was not a cell contact effect. Eighty-five to 90% of the plateau-phase cells had a G1 DNA content and a growth fraction less than or equal to 0.15. The cell kinetic shifts in the population were temporally correlated with a developing radioresistance, which was characterized by a larger shoulder in the survival curve of the quiescent cells (Dq = 5.71 Gy) versus exponentially growing cells (Dq = 4.48 Gy). When the quiescent plateau-phase cells were refed, an increase in radiosensitivity resulted which approached that of exponentially growing 9L cells. Delayed plating experiments after irradiation of exponentially growing cells, quiescent plateau-phase cells, and synchronized early to mid-G1-phase cells indicated that while significant PLD repair was evident in all three populations, the quiescent 9L cells had a higher PLD repair capacity. Although data for immediate plating indicated that 9L cells may enter quiescence in the relatively radioresistant mid-G1 phase, the enhanced PLD repair capacity of quiescent cells cannot be explained by redistribution into G1 phase. When the unfed quiescent plateau-phase 9L cells were stimulated to reenter the cell cycle by replating into fresh medium, the first G1 was extended by 6 h compared with the G1 of exponentially growing or refed plateau-phase 9L cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for reduced capacity for damage accumulation and repair in plateau-phase C3H 10T1/2 cells following multiple-dose irradiation with gamma rays

The effects of multiple-dose gamma irradiation on the shape of survival curves were studied with mouse C3H 10T1/2 cells maintained in contact-inhibited plateau phase. The dose-fractionation intervals included 3, 6, and 24 h. Following three fractionated doses (5 Gy per dose) of exposures, cells responded to further irradiation by displaying a survival curve with a much reduced shoulder width (Dq) compared to that of the survival curve measured in cells irradiated with single-graded doses alone. The effect on the mean lethal dose (D0) was small and appeared to be significant. The effect on reduction of Dq could not be completely overcome by lengthening the fractionation intervals from 3 to 6 h or 24 h, times in which repair of sublethal damage (SLD) measured by simple split-dose scheme and potentially lethal damage (PLD) measured by postirradiation incubation was completed. Other experiments showed that pretreatments of cells with fractionated irradiation appeared to slow down the cellular repair processes of SLD and PLD. Therefore, the observed change in the shape of survival curves after fractionation treatments may be attributed to a reduction of the cells' capacity for damage accumulation by an enhancement of the lethal expression of SLD and PLD. Although the molecular mechanism(s) is not known, the results of this study indicate that the acute graded dose-survival curve cannot be used a priori to extrapolate and reliably predict results of hyperfractionation. It is probable that for a nondividing or slowly dividing cell population, such an extrapolation may lead to an underestimation of cell killing. Furthermore, the findings of this investigation appear to support an interpretation, alternative to the high-linear energy transfer (LET) track-end postulate, for the effects on cell survival seen at low doses or low dose rates.     

Deficiency in fast repair process of potentially lethal damage induced by X-irradiation in fibroblasts derived from LEC strain rats.

The time course for the repair of PLD in LEC and WKAH rat cells irradiated at 5 Gy was examined. In the case of WKAH rat cells, the surviving fraction increased with increasing incubation times after X-irradiation. When hypertonic treatment was performed at each incubation time with 0.5 M NaCl for 20 min, increase in the surviving fractions was not shown. In contrast, no significant recovery of the surviving fraction in LEC rat cells was observed after incubation of irradiated cells with or without 0.5 M NaCl for 20 min. On dose-survival curves, hypertonic treatment with 0.5 M NaCl enhanced radiosensitivity of WKAH rat cells, but not LEC rat cells. Although the surviving fraction of the cells from backcross mice with normal radiosensitivity reduced by treatment with 0.5 M NaCl, the survival fraction was not affected in the cells from backcross mice with higher radiosensitivity by treatment with 0.5 M NaCl. When the cells were X-irradiated and incubated with or without 0.225 M NaCl, the radiosensitivities of LEC and WKAH rat cells treated with 0.225 M NaCl for 4 h were approximately two-fold higher than those of untreated cells. Treatment with caffeine also reduced the surviving fractions of both X-irradiated LEC and WKAH rat cells, compared with those of untreated cells. These results indicated that the slow repair of PLD occurred in LEC rat cells but not the fast repair of PLD.     

The relationship between radiosensitivity and repair of potentially lethal damage in human tumor cell lines with implications for radioresponsiveness

The relationship between intrinsic radiosensitivity and repair capacity was studied for 22 human tumor cell lines in vitro. The experimental material was taken from 19 published papers. Parameters from three radiobiological models were used to assess this relationship: the one-hit multitarget model (D0 and n), the linear-quadratic model (alpha and beta), and the mean inactivation dose (D). Data were obtained for cells in three stages: exponentially growing cells (exp), plateau-phase cells plated immediately after irradiation (ip), and plateau-phase cells plated after completion of PLD repair (dp). No significant difference was found between radiosensitivity of exp and ip cells. There was no correlation between repair capacity and intrinsic radiosensitivity assessed with plateau-phase cells plated immediately after irradiation. The correlation studies between intrinsic radiosensitivity or repair capacity and clinical responsiveness were achieved by assigning cell lines to one of three groups of decreasing in vivo radioresponsiveness: highly, medium, and poorly responsive. There was a significant correlation between radiosensitivity and radioresponsiveness, but no correlation between repair capacity and radioresponsiveness. The average repair capacity was about 0.6 Gy, in terms of D. Three parameters, the mean inactivation dose of exponentially growing cells, of plateau-phase cells plated immediately after irradiation, and of plateau-phase cells plated after completion of PLD repair, could be used equally to assess the relationship between in vitro data and radioresponsiveness. The present results are compared to those obtained in a similar study on a group of 48 nontransformed fibroblast cell strains.     

On the identity of the damage expressed by treatment of X-irradiated HeLa cells with different agents

To determine whether different agents that enhance the expression of potentially lethal X-ray damage (PLD) interact with the same or different lesions (or spectrum of lesions), cell killing was measured in three kinds of experiments: (1) When cells were irradiated in G1 phase and treated with caffeine or hydroxyurea at concentrations that yield maximal response, the same survival plateaus were reached. (2) Treatment of cells irradiated in G1 phase either with caffeine or with hydroxyurea so as to yield survival levels that differed twofold after 4 h incubation, followed by treatment with caffeine to allow expression of PLD in G2 phase, resulted eventually in the same level of survival. (3) When cells were irradiated and treated with caffeine, hydroxyurea, or 9-beta-D-arabinofuranosyladenine (araA) after progressively longer delays, to trace the time course of recovery from the PLD, the responses obtained with caffeine and araA were similar; sensitivity to hydroxyurea was lost more rapidly. The results are consistent with the possibility that these three agents interact with the same lesions, but that different steps in the repair process are inhibited by caffeine or araA than by hydroxyurea.     

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.     

Relationship between DNA repair and cell recovery: Importance of competing biochemical and metabolic processes

Summary The relationship between the inhibition of repair of radiation-induced DNA damage and the inhibition of recovery from radiation-induced potentially lethal damage (PLD) by hypertonic treatment was compared in 9L/Ro rat brain tumor cells. Fed plateau phase cultures were-irradiated with 1500 rad and then immediately treated for 20 min with a 37° C isotonic (0.15 M) or hypertonic (0.50 M) salt solution. The kinetics of repair of radiation-induced DNA damage as assayed using alkaline filter elution were compared to those of recovery from radiation-induced PLD as assayed by colony formation. Hypertonic treatment of unirradiated cells produced neither DNA damage nor cell kill. Post-irradiation hypertonic treatment inhibited both DNA repair and PLD recovery, while post-irradiation isotonic treatment inhibited neither phenomenon. However, by 2 h after irradiation, the amount of DNA damage remaining after a 20 min hypertonic treatment was equivalent to that remaining after a 20 min isotonic treatment. In contrast, cell survival after hypertonic treatment remained 2 logs lower than after isotonic treatment even at times up to 24 h. These results suggest that the repair of radiation-induced DNA damageper se is not causally related to recovery from radiation-induced PLD. However, the data are consistent with the time of DNA repair as an important parameter in determining cell survival and, therefore, tend to support the hypothesis that imbalances in sets of competing biochemical or metabolic processes determine survival rather than the presence of a single class of unrepaired DNA lesions.     

Importance of TP53 and RB in the repair of potentially lethal damage and induction of color junctions after exposure to ionizing radiation

Repair of potentially lethal damage (PLD) was investigated in cells with functional G1-phase arrest with wild-type TP53 and wild-type RB and in cells in which G1-phase arrest was abrogated by inactivation of TP53 or RB. Confluent cultures of cells were plated for clonogenic survival assay either immediately or 24 h after irradiation. Induction of color junctions, an exchange between a painted and unpainted chromosome, was studied in chromosomes 18 and 19 after irradiation with 4 Gy gamma rays. Significant repair of PLD was found in cells carrying both wild-type TP53 and wild-type RB. In cells in which TP53 or RB was inactivated, the survival curves from immediately plated and delayed-plated cells were not significantly different. The numbers of radiation-induced color junctions in chromosomes 18 and 19 were similar in all cell lines. From this study we conclude that a functional G1-phase arrest is important for repair of PLD and that TP53 and RB do not affect the frequencies of induction of color junctions in chromosome 18 or 19.     

Differential repair of potentially lethal damage in exponentially growing and quiescent 9L cells

The alteration of potentially lethal damage repair by postirradiation treatment with hypertonic saline (0.5 M PBS) was investigated in exponentially growing and quiescent 9L cells in vitro. A single dose of X rays (8.5 Gy) immediately followed by a 30-min treatment with hypertonic PBS at 37 degrees C reduced the survival of exponentially growing 9L cells by a factor of 13-18 compared to survival of irradiated immediately and delayed-plated cells, while the survival of quiescent cells was reduced by only a factor of 5-8. Survival curves confirmed the relative resistance of the quiescent 9L cells versus exponentially growing 9L cells to X rays plus hypertonic treatment. Both the slope and the shoulder of the survival curve were reduced to a greater extent in exponentially growing cells than in the quiescent cells by hypertonic treatment. The response of quiescent cells cannot be explained by either the duration of hypertonic treatment or the redistribution of the cells into G1 phase. We show that quiescent 9L cells can recover from hypertonically induced potentially lethal damage when incubated under conditions which have been found to delay progression through the cell cycle, and postulate that an altered chromatin structure or an enhanced repair capacity of quiescent 9L cells may be responsible for their resistance.     

Reduced repair of potentially lethal radiation damage in glutathione synthetase-deficient human fibroblasts after X-irradiation

Using a human fibroblast strain deficient in glutathione synthetase and a related proficient control strain, the role of glutathione (GSH) in repair of potentially lethal damage (PLD) has been investigated in determining survival by plating cells immediately or 24 h after irradiation. After oxic or hypoxic irradiation, both cell strains repair radiation-induced damage. However, under hypoxic conditions, the proficient cells repair PLD as well as under oxic conditions while the deficient cells repair less PLD after irradiation under hypoxic than under oxic conditions. Therefore, the oxygen enhancement ratio (o.e.r.) for proficient cells is similar whether the cells are plated immediately or 24 h later (2.0 and 2.13, respectively). In contrast, the o.e.r. for deficient cells is lower when the cells are plated 24 h after irradiation than when they are plated immediately thereafter (1.16 as compared to 1.55). The results indicate that GSH is involved in PLD repair and, in particular, in the repair of damage induced by radiation delivered under hypoxic conditions.