The saturated repair kinetics of Chinese hamster V79 cells suggests a damage accumulation--interaction model of cell killing

The aim of this study is to determine whether the repair process in log-phase Chinese hamster V79 cells exposed to X rays is unsaturated, saturable, or saturated. The kinetics of recovery from damage induced by 2 to 14 Gy of 250 kVp X rays was studied by treating cells with 0.5 M hypertonic saline for 20 min at different postirradiation repair intervals. From the kinetic data, the repair half-time (t1/2), the repair time (time needed to attain maximal survival), and the recovery ratio were calculated. The results show that the t1/2 (1.42 min/Gy) and the repair time (6.04 min/Gy) increase linearly with dose, the logarithm of the recovery ratio increases linear-quadratically with dose, and the D0 increases linearly with repair interval at a rate of 2.4 cGy/min. From these results we suggest a model: the repair of damage (undefined lesions) necessary for cell survival is effected by a repair process (t 1/2 of 1.42 min/Gy) which is saturated at doses as low as 2.4 cGy; repair saturation leads to a dose-dependent accumulation of repairable lesions; and interaction among accumulated repairable lesions results in the induction of irreparable (lethal) lesions. We call this the accumulation-interaction model of cell killing by low-LET radiation.     

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.     

DNA damage and repair in relation to cell killing in neocarzinostatin-treated HeLa cells.

To elucidate the mechanism of the cell killing activity of neocarzinostatin on mammalian cells, the drug-induced damage of DNA and its repair were examined. Very low doses of neocarzinostatin, at which high survival of cells was observed, clearly produced single-strand breaks of DNA and decomposition of the 'DNA complex', but these damages appeared to be repaired almost completely. At higher doses of neocarzinostatin, single-strand breaks were repaired to a considerable extent while double-strand breaks seemed not to be repaired. The number of non-repairable single-strand breaks was about twice that of double-strand breaks. This implies that single-strand breaks are repaired except for those constituting double-strand breaks. Although at low levels of neocarzinostatin repair of double-strand breaks may occur, the correlation existing between the colony-forming ability of cells treated with neocarzinostatin and non-repairable DNA breakage suggests that production of a small number of critical non-repairable double-strand breaks per cell may be responsible for the cell killing activity of the drug.     

DNA repair after gamma irradiation in lymphocytes exposed to low-frequency pulsed electromagnetic fields

The effect of exposure to extremely low-frequency pulsed electromagnetic fields (EMFs) on DNA repair capability and on cell survival in human lymphocytes damaged in vitro with gamma rays was studied by two different micromethods. In the first assay, which measures DNA repair synthesis (unscheduled DNA synthesis, UDS), lymphocyte cultures were stimulated with phytohemagglutinin (PHA) for 66 h and then treated with hydroxyurea (which blocks DNA replication), irradiated with 100 Gy of 60Co, pulsed with [3H]thymidine ([3H]TdR), and then exposed to pulsed EMFs for 6 h (the period in which cells repaired DNA damage). In the second assay, which measures cell survival after radiation or chemical damage, lymphocytes were first irradiated with graded doses of gamma rays or treated with diverse antiproliferative agents, and then stimulated with PHA, cultured for 72 h, and pulsed with [3H]TdR for the last 6 h of culture. In this case, immediately after the damage induced by either the radiation or chemicals, cultures were exposed to pulsed EMFs for 72 h, during which cell proliferation took place. Exposure to pulsed EMFs did not affect either UDS or cell survival, suggesting that this type of nonionizing radiation--to which humans may be exposed in the environment, and which is used for both diagnostic and therapeutic purposes--does not affect DNA repair mechanisms.     

Ribbon Synapse Plasticity in the Cochleae of Guinea Pigs after Noise-Induced Silent Damage

Noise exposure at low levels or low doses can damage hair cell afferent ribbon synapses without causing permanent threshold shifts. In contrast to reports in the mouse cochleae, initial damage to ribbon synapses in the cochleae of guinea pigs is largely repairable. In the present study, we further investigated the repair process in ribbon synapses in guinea pigs after similar noise exposure. In the control samples, a small portion of afferent synapses lacked synaptic ribbons, suggesting the co-existence of conventional no-ribbon and ribbon synapses. The loss and recovery of hair cell ribbons and post-synaptic densities (PSDs) occurred in parallel, but the recovery was not complete, resulting in a permanent loss of less than 10% synapses. During the repair process, ribbons were temporally separated from the PSDs. A plastic interaction between ribbons and postsynaptic terminals may be involved in the reestablishment of synaptic contact between ribbons and PSDs, as shown by location changes in both structures. Synapse repair was associated with a breakdown in temporal processing, as reflected by poorer responses in the compound action potential (CAP) of auditory nerves to time-stress signals. Thus, deterioration in temporal processing originated from the cochlea. This deterioration developed with the recovery in hearing threshold and ribbon synapse counts, suggesting that the repaired synapses had deficits in temporal processing.     

Evasion of early cellular response mechanisms following low level radiation-induced DNA damage

DNA damage that is not repaired with high fidelity can lead to chromosomal aberrations or mitotic cell death. To date, it is unclear what factors control the ultimate fate of a cell receiving low levels of DNA damage (i.e. survival at the risk of increased mutation or cell death). We investigated whether DNA damage could be introduced into human cells at a level and frequency that could evade detection by cellular sensors of DNA damage. To achieve this, we exposed cells to equivalent doses of ionizing radiation delivered at either a high dose rate (HDR) or a continuous low dose rate (LDR). We observed reduced activation of the DNA damage sensor ataxia-telangiectasia mutated (ATM) and its downstream target histone H2A variant (H2AX) following LDR compared with HDR exposures in both cancerous and normal human cells. This lack of DNA damage signaling was associated with increased amounts of cell killing following LDR exposures. Increased killing by LDR radiation has been previously termed the "inverse dose rate effect," an effect for which no clear molecular processes have been described. These LDR effects could be abrogated by the preactivation of ATM or simulated in HDR-treated cells by inhibiting ATM function. These data are the first to demonstrate that DNA damage introduced at a reduced rate does not activate the DNA damage sensor ATM and that failure to activate ATM-associated repair pathways contributes to the increased lethality of continuous LDR radiation exposures. This inactivation may reflect one strategy by which cells avoid accumulating mutations as a result of error-prone DNA repair and may have a broad range of implications for carcinogenesis and, potentially, the clinical treatment of solid tumors.     

Radiosensitization of GL261 glioma cells by tetraiodothyroacetic acid (tetrac)

We studied effects of tetrac (tetraiodothyroacetic acid) on survival of GL261, a murine brain tumor cell line, following single doses of 250 kVp x-rays and on repair of damage (sublethal and potentially lethal damage repair; SLDR, PLDR) in both exponential and plateau phase cells. Cells were exposed to 2 μM tetrac (1 h at 37oC) prior to x-irradiation. At varying times after irradiation, cells were re-plated in medium without tetrac. Two weeks later, colonies were counted and results analyzed using either the linear-quadratic (LQ) or single-hit, multitarget (SHMT) formalisms. Tetrac sensitized both exponential and plateau phase cells to x-irradiation, as shown by a decrease in the quasi-threshold dose (Dq), leading to an average tetrac enhancement factor (ratio of SF2 values) of 2.5. Tetrac reduced SLDR in exponential cells by a factor of 1.8. In plateau phase cells there was little expression of SLDR, but tetrac produced additional cell killing at 1-4 h after the first dose. For PLDR expression in exponential cells, tetrac inhibited PLDR by a factor of 1.9, and in plateau phase cells, tetrac decreased PLDR expression by a factor of 3.4. These data show that the decreased Dq value seen after single doses of x-rays with tetrac treatment is also accompanied by a significant decrease in recovery from sublethal and potentially lethal damage.     

Role of nitric oxide-induced mtDNA damage in mitochondrial dysfunction and apoptosis

An increasing body of evidence suggests that nitric oxide (NO) can be cytotoxic and induce apoptosis. NO can also be genotoxic and cause DNA damage and mutations. It has been shown that NO damages mitochondrial DNA (mtDNA) to a greater extent than nuclear DNA. Previously, we reported that conditional targeting of the DNA repair protein hOGG1 into mitochondria using a mitochondria targeting sequence (MTS) augmented mtDNA repair of oxidative damage and enhanced cellular survival. To determine whether enhanced repair resulting from augmented expression of hOGG1 could also protect against the deleterious effects of NO, we used HeLa TetOff/MTS-OGG1-transfected cells to conditionally express hOGG1 in mitochondria. The effects of additional hOGG1 expression on repair of NO-induced mtDNA damage and cell survival were evaluated. These cells, along with vector transfectants, in either the presence or absence of doxycycline (Dox), were exposed to NO produced by the rapid decomposition of 1-propanamine, 3-(2-hydroxy-2-nitroso-1-propylhydrazino) (PAPA NONOate). Functional studies revealed that cells expressing recombinant hOGG1 were more proficient at repairing NO-induced mtDNA damage, which led to increased cellular survival following NO exposure. Moreover, the results described here show that conditional expression of hOGG1 in mitochondria decreases NO-induced inhibition of ATP production and protects cells from NO-induced apoptosis.     

Relationship between the recovery from sublethal X-ray damage and the rejoining of chromosome breaks in normal human fibroblasts

Using plateau-phase cultures of AG1522 normal human fibroblasts, we examined relationships between the breakage and rejoining of chromosomes and the induction and repair of sublethal damage (SLD) following fractionated doses of X rays. The rate constant for the rejoining of breaks in prematurely condensed interphase chromosomes, measured previously, accurately predicts both the rate of change in survival due to potentially lethal damage (PLD) repair and the rate of change in survival for dose fractionation due to SLD repair. Further, changes in the frequency of chromosome-type deletions and asymmetrical exchange aberrations measured in the first postirradiation mitosis corresponded closely with changes in cell killing when doses were fractionated, and a dose-fractionation- or dose-rate-independent alpha component of damage was similar for aberration and cell killing end points. These results substantiate the hypothesis that sublethal damage repair results from the rejoining of breaks in interphase chromatin produced by a first dose so they no longer are capable of interacting with those produced by a second dose. The fact that the repair of potentially lethal damage is also readily explained on the basis of chromosome break rejoining (M. N. Cornforth and J. S. Bedford, Radiat. Res. 111, 385-405 (1987)) strongly suggests that PLD and SLD repair are different manifestations of the same basic process operating on the same basic lesions.     

Mechanistic analysis of the contributions of DNA and protein damage to radiation-induced cell death

Protein oxidation can contribute to radiation-induced cell death by two mechanisms: (1) by reducing the fidelity of DNA repair, and (2) by decreasing cell viability directly. Previously, we explored the first mechanism by developing a mathematical model and applying it to data on Deinococcus radiodurans . Here we extend the model to both mechanisms, and analyze a recently published data set of protein carbonylation and cell survival in D. radiodurans and Escherichia coli exposed to gamma and ultraviolet radiation. Our results suggest that similar cell survival curves can be produced by very different mechanisms. For example, wild-type E. coli and DNA double-strand break (DSB) repair-deficient recA- D. radiodurans succumb to radiation doses of similar magnitude, but for different reasons: wild-type E. coli proteins are easily oxidized, causing cell death even at low levels of DNA damage, whereas proteins in recA- D. radiodurans are well protected from oxidation, but DSBs are not repaired correctly even when most proteins are intact. Radioresistant E. coli mutants survive higher radiation doses than the wild-type because of superior protection of cellular proteins from radiogenic oxidation. In contrast, wild-type D. radiodurans is much more radioresistant than the recA- mutant because of superior DSB repair, whereas protein protection in both strains is similar. With further development, the modeling approach presented here can also quantify the causes of radiation-induced cell death in other organisms. Enhanced understanding of these causes can stimulate research on novel radioprotection strategies.     

Discrepancies between patterns of potentially lethal damage repair in the RIF-1 tumor system in vitro and in vivo

Repair of potentially lethal damage (PLD) was studied in the RIF-1 tumor system in several different growth states in vivo and in vitro. Exponentially growing, fed plateau, and unfed plateau cells in cell culture as well as small and large subcutaneous or intramuscular tumors were investigated. Large single doses of radiation followed by variable repair times as well as graded doses of radiation to generate survival curves immediately after irradiation or after full repair were investigated. All repair-promoting conditions studied in vitro (delayed subculture, exposure of cells to depleted growth medium after irradiation) increased surviving fraction after a single dose. The D0 of the cell survival curve was also increased by these procedures. No PLD repair was observed for any tumors irradiated in vivo and maintained in the animal for varying times prior to assay in vitro. The nearly 100% cell yield obtained when this tumor is prepared as a single-cell suspension for colony formation, the representative cell sample obtained, and the constant cell yield per gram as a function of time postirradiation suggest that this discrepancy is not an artifact of the assay system. The most logical explanation of these data and information on radiocurability of this neoplasm is that PLD repair, which is so frequently demonstrated in vitro, may not be a major factor in the radioresponse of this tumor when left in situ.     

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.     

Ionizing-radiation resistance in the desiccation-tolerant cyanobacterium Chroococcidiopsis

The effect of X-ray irradiation on cell survival, induction, and repair of DNA damage was studied by using 10 Chroococcidiopsis strains isolated from desert and hypersaline environments. After exposure to 2.5 kGy, the percentages of survival for the strains ranged from 80 to 35%. In the four most resistant strains, the levels of survival were reduced by 1 or 2 orders of magnitude after irradiation with 5 kGy; viable cells were recovered after exposure to 15 kGy but not after exposure to 20 kGy. The severe DNA damage evident after exposure to 2.5 kGy was repaired within 3 h, and the severe DNA damage evident after exposure to 5 kGy was repaired within 24 h. The increase in trichloroacetic acid-precipitable radioactivity in the culture supernatant after irradiation with 2.5 kGy might have been due to cell lysis and/or an excision process involved in DNA repair. The radiation resistance of Chroococcidiopsis strains may reflect the ability of these cyanobacteria to survive prolonged desiccation through efficient repair of the DNA damage that accumulates during dehydration.     

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.     

Lethal and potentially lethal lesions induced by radiation--a unified repair model

A model of radiation action is described which unifies several of the major existing concepts which have been applied to cell killing. Called the lethal and potentially lethal (LPL) model, it combines the ideas of lesion interaction, irreparable lesions caused by single tracks, linear lesion fixation, lesion repair via first-order kinetics, and binary misrepair. Two different kinds of lesions are hypothesized: irreparable (lethal) and repairable (potentially lethal) lesions. They are tentatively being identified with DNA double-strand breaks of different severity. Two processes compete for depletion of the potentially lethal lesions: correct repair following first-order kinetics, and misrepair following second-order kinetics. Fixation of these lesions can also occur. The model applies presently only to plateau (stationary)-phase cells. Radiobiological phenomena described include effects of low dose rate, high LET, and repair kinetics as measured with repair inhibitors such as hypertonic solution and beta-arabinofuranosyladenine (beta-araA). One consequence of the model is that repair of sublethal damage and the slow component of the repair of potentially lethal damage are two manifestations of the same repair process. Hypertonic treatment fixes a completely new class of lesions which normally repair correctly. Another consequence of the model is that the initial slope of the survival curve depends on the amount of time available for repair after irradiation. The "dose-rate factor" occurring in several linear-quadratic formulations is shown to emerge when appropriate low-dose and long-repair-time approximations are made.     

Ionizing-Radiation Resistance in the Desiccation-Tolerant Cyanobacterium Chroococcidiopsis

The effect of X-ray irradiation on cell survival, induction, and repair of DNA damage was studied by using 10 Chroococcidiopsis strains isolated from desert and hypersaline environments. After exposure to 2.5 kGy, the percentages of survival for the strains ranged from 80 to 35%. In the four most resistant strains, the levels of survival were reduced by 1 or 2 orders of magnitude after irradiation with 5 kGy; viable cells were recovered after exposure to 15 kGy but not after exposure to 20 kGy. The severe DNA damage evident after exposure to 2.5 kGy was repaired within 3 h, and the severe DNA damage evident after exposure to 5 kGy was repaired within 24 h. The increase in trichloroacetic acid-precipitable radioactivity in the culture supernatant after irradiation with 2.5 kGy might have been due to cell lysis and/or an excision process involved in DNA repair. The radiation resistance of Chroococcidiopsis strains may reflect the ability of these cyanobacteria to survive prolonged desiccation through efficient repair of the DNA damage that accumulates during dehydration.     

Degradation and repair of rat hepatoma cell DNA following exposure to gamma rays and methylnitrosourea]

Single-strand breaks induced in DNA of ascitic hepatoma cells by gamma-rays and N-methyl-N-nitrosourea (MNU), resp., may be effectively repaired. Double-strand breaks of DNA from MNU-treated hepatoma cells are also effectively repairable in vivo. Only a small part of double-strand breaks induced by gamma-rays in DNA of these cells is repaired in the postradiation period. The combined action of gamma-rays and MNU on the hepatoma cells causes a complete inhibition of repair of DNA and its further degradation. Under these conditions, inhibition of the repair of DNA synthesis and repression of DNA polymerase I activity is observed.     

Decay of γ-H2AX foci correlates with potentially lethal damage repair and P53 status in human colorectal carcinoma cells

The influence of p53 status on potentially lethal damage repair (PLDR) and DNA double-strand break (DSB) repair was studied in two isogenic human colorectal carcinoma cell lines: RKO (p53 wild-type) and RC10.1 (p53 null). They were treated with different doses of ionizing radiation, and survival and the induction of DNA-DSB were studied. PLDR was determined by using clonogenic assays and then comparing the survival of cells plated immediately with the survival of cells plated 24 h after irradiation. Doses varied from 0 to 8 Gy. Survival curves were analyzed using the linear-quadratic formula: S(D)/S(0) = exp-(αD+βD²). The γ-H2AX foci assay was used to study DNA DSB kinetics. Cells were irradiated with single doses of 0, 0.5, 1 and 2 Gy. Foci levels were studied in non-irradiated control cells and 30 min and 24 h after irradiation. Irradiation was performed with gamma rays from a ¹³⁷Cs source, with a dose rate of 0.5 Gy/min. The RKO cells show higher survival rates after delayed plating than after immediate plating, while no such difference was found for the RC10.1 cells. Functional p53 seems to be a relevant characteristic regarding PLDR for cell survival. Decay of γ-H2AX foci after exposure to ionizing radiation is associated with DSB repair. More residual foci are observed in RC10.1 than in RKO, indicating that decay of γ-H2AX foci correlates with p53 functionality and PLDR in RKO cells.     

UV light-induced DNA damage and tolerance for the survival of nucleotide excision repair-deficient human cells

DNA damage can cause cell death unless it is either repaired or tolerated. The precise contributions of repair and tolerance mechanisms to cell survival have not been previously evaluated. Here we have analyzed the cell killing effect of the two major UV light-induced DNA lesions, cyclobutane pyrimidine dimers (CPDs) and 6-4 pyrimidine-pyrimidone photoproducts (6-4PPs), in nucleotide excision repair-deficient human cells by expressing photolyase(s) for light-dependent photorepair of either or both lesions. Immediate repair of the less abundant 6-4PPs enhances the survival rate to a similar extent as the immediate repair of CPDs, indicating that a single 6-4PP lesion is severalfold more toxic than a CPD in the cells. Because UV light-induced DNA damage is not repaired at all in nucleotide excision repair-deficient cells, proliferation of these cells after UV light irradiation must be achieved by tolerance of the damage at replication. We found that RNA interference designed to suppress polymerase zeta activity made the cells more sensitive to UV light. This increase in sensitivity was prevented by photorepair of 6-4PPs but not by photorepair of CPDs, indicating that polymerase zeta is involved in the tolerance of 6-4PPs in human cells.