Exogenous lactate modifies the repair of potentially lethal damage in three human tumor cell lines irradiated in vitro

Lactate is one of several pathophysiological factors accumulating in the micromilieu of tumors under both hypoxic and well-oxygenized conditions, and thus may affect the recovery of irradiated tumor cells in vivo. In the present study, we investigated the effects of postirradiation incubation with exogenous lactate during confluent holding recovery on the repair of potentially lethal damage in three human tumor cell lines. Recovery was either unaffected or enhanced by low concentrations of exogenous lactate (2-5 mM), whereas it was suppressed by higher concentrations (10-50 mM). With high concentrations, survival in all three cell lines was lower at the end of the confluent holding period than at the beginning, yielding recovery ratios of less than 1.0. The effects differed quantitatively among the three tumor cell lines, and between the tumor cells and the normal diploid fibroblasts (AG 1522) studied previously.     

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.     

Bleomycin-induced potentially lethal damage and its repair

The dependence of the survival of V79 Chinese hamster cells which were treated with bleomycin on a post-treatment with anisotonic phosphate-buffered saline and/or conditioned medium was examined. In qualitative similarity to the effects of these post-treatments on survival following exposure to X rays, it was found that: anisotonic saline, both hypo- and hypertonic, significantly enhanced cell killing; the degree of enhancement increased with time and temperature; and incubation in conditioned medium resulted in the rescue of cells whether or not they were challenged with hypertonic saline after treatment with bleomycin. Based upon the qualitative similarity of these findings to those which we have observed following X irradiation, and observations of others on the site of action of bleomycin in cells and the ability that it has to break DNA, we propose that both bleomycin and radiation (X rays and fast neutrons) act on similar sensitive sites which are probably contained in or close to the envelope and/or the protein matrix of the nucleus.     

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.     

Potentially lethal damage repair in cell lines of radioresistant human tumours and normal skin fibroblasts

Radiation cell survival data were obtained in vitro for three cell lines isolated from human tumours traditionally considered to be radioresistant--two melanomas and one osteosarcoma--as well as from a diploid skin fibroblast cell line. One melanoma cell line was much more radioresistant than the other, while the osteosarcoma and fibroblast cell lines were more radiosensitive than either. For cells growing exponentially, little potentially lethal damage repair (PLDR) could be demonstrated by comparing survival data for cells in which subculture was delayed by 6 h with those sub-cultured immediately after treatment. For the malignant cells in plateau phase, which in these cells might be better termed 'slowed growth phase', since an appreciable fraction of the cells are still cycling, a small amount of PLDR was observed, but not as much as reported by other investigators in the literature. The normal fibroblasts, which achieved a truer plateau phase in terms of noncycling cells, showed a significantly larger amount of PLDR than the tumour cells.     

Novobiocin inhibits the repair of potentially lethal damage but not the repair of sublethal damage

Because of the critical role of the DNA topoisomerases in the synthesis and conformation of DNA, and the well-known observation that radiation inhibits replicative DNA synthesis, we have examined the possibility that inhibitors of these enzymes might influence radiation lethality. In particular, using protocols involving the administration of either fresh or conditioned medium, we examined the ability of intercalative and nonintercalative inhibitors to affect the expression of potentially lethal damage and/or sublethal damage. The inhibitors examined were amsacrine, teniposide, etoposide, and novobiocin; only the latter compound was clearly effective in a selective way at nontoxic concentrations, and this was observed specifically in reference to the repair of potentially lethal damage effected by incubation in conditioned medium. These results are another example of differences between the repair of sublethal versus potentially lethal damage that further support distinctions between the two. At a mechanistic level, these and other data suggest that the property of novobiocin that is relevant in the foregoing is its metabolic inhibition of replicative DNA synthesis, a process which may be more important in the repair of potentially lethal damage as opposed to sublethal damage.     

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.     

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.     

Sublethal and potentially lethal damage repair on thermal neutron capture therapy

Tonicity shock or caffeine postirradiation treatment makes evident fast-type potentially lethal damage (PLD). Caffeine expresses fast-type PLD more efficiently than tonicity shock in X-irradiated B-16 mouse melanoma cells, compared with V79 Chinese hamster cells. The survival curves of thermal neutrons for either V79 or B-16 cells exhibit no shoulder. Neither V79 nor B-16 cells show the sublethal damage (SLD) repair of thermal neutrons. Caffeine-sensitive fast-type PLD repairs exist in X-irradiated B-16 cells, as well as V79 cells. The fast-type PLD repair of B-16 cells exposed to thermal neutrons alone is rather less than that of X-irradiated cells. Furthermore, an extremely low level of fast-type PLD repair of B-16 cells with 10B1-paraboronophenylalanine (BPA) preincubation (20 hours) followed by thermal neutron irradiation indicated that 10B(n,alpha)7Li reaction effectively eradicates actively growing melanoma cells. The plateau-phase B-16 cells are well able to repair the slow-type PLD of X-rays. However, cells can not repair the slow-type PLD induced by thermal neutron irradiation with or without 10B1-BPA preincubation. These results suggest that thermal neutron capture therapy can effectively kill radioresistant melanoma cells in both proliferating and quiescent phases.     

Age-dependent modification by lactate of repair of potentially lethal damage in normal human diploid fibroblasts

We investigated age-related changes in normal human diploid fibroblasts (strain AG 1522) followed throughout their lifespan in vitro. Radiosensitivity and the capacity to repair potentially lethal radiation damage (PLD) remained constant until senescence was reached. However, the effects of incubation with lactate on PLD repair were age dependent and complex. Low millimolar concentrations of lactate increased the capacity to repair PLD, whereas higher concentrations suppressed it in a concentration-dependent manner. With increasing in vitro age, the cells became less sensitive to the modifying effects of lactate; in presenescent cells, the modifying effects of lactate on PLD repair had completely vanished.     

Evidence for the repair of potentially lethal damage in irradiated bone marrow

Summary The effects on cell survival of maintaining bone marrow cells (CFU-S) in situ following irradiation and before assay by transplantation was investigated. When the CFU-S cells are maintained in situ following irradiation survival drops and plateaus at about 9 h post-irradiation. Evidence is presented that this decrease in survival may be due to potentially lethal damage repair (PLD) inhibition caused by post-irradiation in situ holding. This effect on PLD repair is different than that usually found in cells in vitro and in vivo tumors in that it mainly alters the shoulder rather than the slope of the survival curve of CFU-S cells. It is different than PLDR found in vivo for normal mammary and thyroid gland epithelial cells because in situ holding decreases rather than increases the survival of CFU-S cells. Evidence is also presented that the radiation survival curve for in situ bone marrow cells (CFU-S) may not have a shoulder.Supported in part by NIH, NCI grants P01 CA 19298 and P30 CA 14520Supported in part by an American Cancer Society Clinical Fellowship     

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.     

Effect of confluent holding on potentially lethal damage repair, cell cycle progression, and chromosomal aberrations in human normal and ataxia-telangiectasia fibroblasts

The effects of confluent holding recovery on survival, chromosomal aberrations, and progression through the life cycle after subculture of human diploid fibroblasts X-irradiated during density inhibition of growth have been examined. The responses of three normal strains were determined and compared with those of four ataxia-telangiectasia (AT), an AT heterozygote, and two hereditary retinoblastoma strains. The capacity for potentially lethal damage repair (PLDR) was slightly reduced in retinoblastoma cells and almost absent in AT cells, but normal in an AT heterozygote. The decline in chromosomal aberrations seen in normal cells during confluent holding was absent in AT cells, consistent with the lack of PLDR. Following subculture, all irradiated AT fibroblasts progressed through the cell cycle to the first mitosis with no delay. AT heterozygotic and retinoblastoma cells showed both an enhanced delay in the initiation of DNA synthesis and a large fraction of cells irreversibly blocked in G1 as compared with normal cells. Both the delayed entry into S and the G1 block were reduced by confluent holding. These results indicate that AT homozygotic and heterozygotic cells respond quite differently to X irradiation.     

Abnormal sensitivity of some Cockayne's syndrome cell strains to UV- and gamma-rays. Association with a reduced ability to repair potentially lethal damage

Cockayne's syndrome (CS) is a rare autosomal recessive genetic disease characterized by mental and physical retardation, microcephaly, dwarfism, retinitis pigmentosa and a hypersensitivity to sunlight. Cells originating from patients also exhibit, in vitro, a hypersensitivity to UV radiation. Using a colony assay in vitro, we studied the sensitivity of 5 CS cell strains (GM739, BOR, CS697, CS698 and KA) and two normal ones (HF19 and GP) to UV- and gamma-irradiation. The 5 CS strains appear to be UV-hypersensitive but the sensitivity varies widely from one strain to another. Hypersensitivity to gamma-rays has been reported for 4 out of the 5 CS cell strains investigated. However, these CS cell strains are less sensitive to gamma-rays than are ataxia telangiectasia cells. The KA cell strain exhibits a normal response to gamma-irradiation. Repair of potentially lethal damage (PLD) after UV- and gamma-irradiation was investigated by using unfed plateau-cell cultures. Under these conditions, control cells show a great capacity to repair PLD (10- to 30-fold survival increase at 1% survival level). The two CS strains (GM739 and BOR), which are hypersensitive to both UV- and gamma-irradiation, exhibit no or only little PLD repair after treatment. In contrast, the normal response of KA cells to gamma-rays is associated with a normal PLD repair capability. This latter cell strain exhibits an intermediate sensitivity to UV and shows an intermediate PLD repair capacity. The response of CS cell strains after gamma-irradiation suggests a genetic heterogeneity. Three complementation groups are described in CS cells when dealing with UV radiosensitivity. However, variations in gamma-ray sensitivity are reported for cells within the same UV complementation group.     

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.