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.     

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.     

Recovery from sublethal and potentially lethal damage in an X-ray-sensitive CHO cell

It has been suggested that DNA strand breaks are the molecular lesions responsible for radiation-induced lethality and that their repair is the basis for the recovery of irradiated cells from sublethal and potentially lethal damage. EM9 is a Chinese hamster ovary cell line that is hypersensitive to killing by X rays and has been reported to have a defect in the rate of rejoining of DNA single-strand breaks. To establish the importance of DNA strand-break repair in cellular recovery from sublethal and potentially lethal X-ray damage, those two parameters, recovery from sublethal and potentially lethal damage, were studied in EM9 cells as well as in EM9's parental repair-proficient strain, AA8. As previously reported, EM9 is the more radiosensitive cell line, having a D0 of 0.98 Gy compared to a D0 of 1.56 Gy for AA8 cells. DNA alkaline elution studies suggest that EM9 cells repair DNA single-strand breaks at a slower rate than AA8 cells. Neutral elution analysis suggests that EM9 cells also repair DNA double-strand breaks more slowly than AA8 cells. All of these data are consistent with the hypothesis that DNA strand-break ligation is defective in EM9 cells and that this defect accounts for increased radiosensitivity. The kinetics and magnitude of recovery from sublethal and potentially lethal damage, however, were similar for both EM9 and AA8 cells. Six-hour recovery ratios for sublethal damage repair were found to be 2.47 for AA8 cells and 1.31 for EM9 cells. Twenty-four-hour recovery ratios for potentially lethal damage repair were 3.2 for AA8 and 3.3 for EM9 cells. Both measurements were made at approximately equitoxic doses. Thus, the defect in EM9 cells that confers radiosensitivity and affects DNA strand-break rejoining does not affect sublethal damage repair or potentially lethal damage repair.     

The S-phase checkpoint and its regulation in Saccharomyces cerevisiae

Cells are never more vulnerable than during DNA replication, which represents a major moment of potential genetic instability. Genotoxic insults induce many different forms of DNA damage that may interfere with the ability of cells to properly duplicate their genome. Primary damage may in turn undergo structural transformations during DNA replication, thus generating secondary lesions that may be even more dangerous. Cells experiencing replication of damaged DNA or replication blocks activate an S-phase checkpoint response that assures the fidelity and completion of DNA replication before cells enter M-phase. The S-phase checkpoint pathway regulates not only progress through the cell cycle but also DNA repair and DNA replication itself.     

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.     

Role and regulation of p53 during an ultraviolet radiation-induced G1 cell cycle arrest.

p53 can play a key role in response to DNA damage by activating a G1 cell cycle arrest. However, the importance of p53 in the cell cycle response to UV radiation is unclear. In this study, we used normal and repair-deficient cells to examine the role and regulation of p53 in response to UV radiation. A dose-dependent G1 arrest was observed in normal and repair-deficient cells exposed to UV. Expression of HPV16-E6, or a dominant-negative p53 mutant that inactivates wildtype p53, caused cells to become resistant to this UV-induced G1 arrest. However, a G1 to S-phase delay was still observed after UV treatment of cells in which p53 was inactivated. These results indicate that UV can inhibit G1 to S-phase progression through p53-dependent and independent mechanisms. Cells deficient in the repair of UV-induced DNA damage were more susceptible to a G1 arrest after UV treatment than cells with normal repair capacity. Moreover, no G1 arrest was observed in cells that had completed DNA repair prior to monitoring their movement from G1 into S-phase. Finally, p53 was stabilized under conditions of a UV-induced G1 arrest and unstable when cells had completed DNA repair and progressed from G1 into S-phase. These results suggest that unrepaired DNA damage is the signal for the stabilization of p53, and a subsequent G1 phase cell cycle arrest in UV-irradiated cells.     

Recovery from UV-induced potentially lethal damage in systemic lupus erythematosus skin fibroblasts

The repair of ultraviolet light-induced potentially lethal damage was investigated in density-inhibited skin fibroblast cell strains derived from patients with systemic lupus erythematosus. The effect of exposure to polychromatic ultraviolet light composed of environmentally relevant wavelengths or to the more commonly studied, short wavelength (254 nm) ultraviolet light was studied. Systemic lupus erythematosus cells, which are hypersensitive to ultraviolet light under growth promoting conditions, were able to repair potentially lethal damage as well as normal cells.     

Effects of pre- and post-thaw cell-to-cell contact and trypsin on survival of freeze-thaw damaged mammalian cells

When multicellular spheroids, which simulate small bits of tissue, are exposed to a freeze-thaw (FT) cycle, the survival of the individual cells in the spheroid is higher if the cells of the spheroid are trypsinized and plated as single cells immediately after thawing than if the spheroid is allowed to remain intact for 4 hr and then trypsinized for plating. The results imply either that cell-to-cell contact inhibits repair of potentially lethal damage (PLD) or that accumulation of additional lethal or sublethal damage during the post-thaw period for cells in contact is taking place. Pre- and post-FT trypsinization of single cells indicate that trypsin does not enhance repair of PLD caused by a FT cycle.     

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.     

Fixation and repair of radiation-induced potentially mutagenic damage sensitive to hypertonic treatment in human diploid fibroblasts

The effect of hypertonic salt treatment on the repair of potentially lethal damage and potentially mutagenic damage in X-irradiated asynchronous and synchronous human diploid fibroblasts (IMR91) have been studied. Resistance to 6-thioguanine was used for the mutagenic end point. When cells in late-S-phase were treated with hypertonic salt solution immediately after X-irradiation, both cell killing and mutation induction were enhanced, as compared to X-irradiation alone. This suggests that X-irradiation of cells in late S phase induces both potentially lethal damage and potentially mutagenic damage and that both are sensitive to hypertonic salt solution. When cells were allowed 2 h for repair after exposure to X-rays, both types of damage were completely repaired. Almost the same results were obtained with asynchronous cells. These results are discussed in terms of the relationship between radiation damage leading to cell lethality and mutagenesis.     

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)     

Radiosensitization in multifraction schedules. I. Evidence for an extremely low oxygen enhancement ratio

Stable monolayers of contact-inhibited C3H 10T1/2 cells were used in multifraction radiation experiments to measure the oxygen enhancement ratio (OER) at low doses/fraction under conditions where cell cycle effects (repopulation, redistribution) were minimal. Consistent with there being a dose-dependent reduction in the OER at low doses, an extremely low OER of 1.34 was measured after 20 fractions of 1.7 Gy every 12 h. The sparing effects of fractionating radiation doses were not apparent for cells irradiated under hypoxic conditions (i.e., multifraction survivals were lower than acute single-dose values) until doses exceeding 15 Gy were reached. This result suggested a deficiency in the recovery from sublethal and/or potentially lethal damage might exist after hypoxic irradiations, thereby reducing the OER. The capacity to repair potentially lethal damage was found to be nearly the same after hypoxic as compared to aerobic irradiations. However, there was an apparent absence of sublethal damage repair by 10T1/2 cells between two hypoxic irradiations which could be a major contributing factor to the extremely low OER value measured in this multifraction schedule.     

Potentially lethal damage repair is due to the difference of DNA double-strand break repair under immediate and delayed plating conditions

Cells plated immediately after irradiation on nutrient agar (immediate plating) exhibit a lower survival than cells which are kept under nongrowth conditions before plating (delayed plating). The difference between the survival curves obtained after immediate plating and delayed plating is considered to exhibit the cell's capacity to repair potentially lethal damage. In yeast evidence has been presented previously for the DNA double-strand break (DSB) as the molecular lesion involved in the repair of potentially lethal damage observed at the cellular level. Radiation-induced DSB are repaired in cells plated on nutrient agar, i.e., under growth conditions, as well as in cells kept under nongrowth conditions. In this paper DSB repair under growth and nongrowth conditions is studied with the help of the yeast mutant rad54-3 which is temperature conditional for DSB repair. It is shown that the extent of repair of potentially lethal damage can be varied by shifting the relative fractions of repair of DSB under growth conditions versus nongrowth conditions. Repair of DSB in cells plated on nutrient agar is promoted when glucose is substituted by Na-succinate as an energy source. As a result the immediate plating survival curve approaches the delayed plating survival curve, thus reducing the operationally defined repair of potentially lethal damage. We show that this reduced potentially lethal damage repair is caused, however, by a higher amount of DSB repair in cells immediately plated on succinate agar as compared to glucose agar.     

Heterogeneity in premature senescence by oxidative stress correlates with differential DNA damage during the cell cycle

The development of cellular senescence both by replication and by oxidative stress is not homogenous in cultured primary human fibroblasts. To investigate whether this is due to the heterogeneity in the susceptibility of DNA in different phases of the cell cycle, we subjected synchronised cells to oxidative stress and examined the extent of DNA damage and its long-term effects on the induction of cellular senescence. Here, we first show marked heterogeneity in DNA damage as detected by markers of double strand breaks caused by oxidative stress in an asynchronous human fibroblast culture. Cell cycle synchronization followed by oxidative stress demonstrated that DNA in S-phase is most susceptible to oxidative stress whereas DNA in the quiescent phase is most resistant. DNA repair is an ongoing process after sensing DNA damage; reparable DNA damage is repaired even in cells that contain persistent DNA damage. The extent of persistent DNA damage is tightly correlated with permanent cessation of DNA replication and SA-beta-gal activity. Oxidative stress encountered by cells in S-phase resulted in more persistent DNA damage, more permanent cell cycle arrest and the induction of premature senescence.     

A calmodulin antagonist has no effect on the repair of X-ray-induced damage in a murine mammary carcinoma cell line

The effects of the calmodulin antagonist W13 were determined on potentially lethal damage repair, sublethal damage repair, and X-ray-induced DNA damage repair following X irradiation of 67 murine mammary carcinoma cells in the proliferative and quiescent states. Studies with W13 (20 micrograms/ml) on proliferating cells showed that the cells rounded up within 2 h but stayed attached to the dishes and there was a slight transient G2 block by 6 h. Also, the proportion of S-phase cells at 12 h was reduced to 65% of control with the concurrent [3H]thymidine incorporation reduced to 62% of control. There was no detectable effect from this pharmacological dose of W13 either on PLDR in proliferating cells at 400 and 800 rad or on quiescent cells at 200 and 400 rad. Likewise, there was no measurable effect on SLDR in either proliferating or quiescent cells at equally split doses of 800 and 600 rad, respectively. In addition, for control vs W13-treated proliferating cells, no difference was detected either in the induction of DNA damage by X irradiation or in the initial rate of repair (T 1/2 approximately equal to 7 min), as measured by the alkaline filter elution assay. In contrast to uv and bleomycin-induced damage, these data suggest that calmodulin may have no major role in either the molecular or cellular recovery from X-ray-induced damage in mammalian cells.     

Quantitative aspects of repair of potentially lethal damage in mammalian cells.

Stationary cultures of Ehrlich ascites tumour cells have been irradiated with X-rays and then immediately or after a time interval trep plated to measure the survival. The increase in survival observed after delayed plating is interpreted as repair of potentially lethal damage. A cybernetic model is used to analyse these data. Three states of damage are assumed for the cells. In state A the cells can grow to macrocolonies, in state B the cells have suffered potentially lethal damage and can grow to macrocolonies only if they are allowed to repair the damage and in state C the cells are lethally damaged. A method of deriving the values of the parameters of the model from the experimental data is given. The dependence of the reaction rate constant of the repair of potentially lethal damage on the dose D is used to derive a possible mechanism for the production of the shoulder in the dose effect curve. Finally this model is compared with other models of radiation action on living cells.     

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.