The Influence of Oxygen on the Induction of Radiation Damage in DNA in Mammalian Cells after Sensitization by Intracellular Glutathione Depletion

Summary

Treatment of mammalian cells with buthionine sulphoximine (BSO) or diethyl maleate (DEM) results in a decrease in the intracellular GSH (glutathione) and non-protein-bound SH (NPSH) levels. The effect of depletion of GSH and NPSH on radiosensitivity was studied in relation to the concentration of oxygen during irradiation. Single- and double-strand breaks (ssb and dsb) and cell killing were used as criteria for radiation damage. Under aerobic conditions, BSO and DEM treatment gave a small sensitization of 10–20 per cent for the three types of radiation damage. Also under severely hypoxic conditions (0·01 μm oxygen in the medium) the sensitizing effect of both compounds on the induction of ssb and dsb and on cell killing was small (0–30 per cent). At somewhat higher concentrations of oxygen (0·5–10 μm) however, the sensitization amounted to about 90 per cent for the induction of ssb and dsb and about 50 per cent for cell killing. These results strengthen the widely accepted idea that intracellular SH-compounds compete with oxygen and other electron-affinic radiosensitizers with respect to reaction with radiation-induced damage, thus preventing the fixation of DNA damages by oxygen. These results imply that the extent to which SH-compounds affect the radiosensitivity of cells in vivo depends strongly on the local concentration of oxygen.     

Heat-induced morphological alterations in non-tolerant and thermotolerant cells.

CHO cells were heated at 43 degrees C or 45 degrees C for various durations up to 300 min. Survival values varied from 5 x 10(-1) to 10(-7). Unheated, non-tolerant control cells were compared with cells made thermotolerant (TT) by incubating at 37 degrees C for 6 or 12 h after treatment with either sodium arsenite (100 microM-As) or 45.5 degrees C for 10 min, respectively. Groups also were included in which heat-induced TT cells were heated at 43 degrees C for 5 h immediately before they were challenged at 45 degrees C; in these groups, cycloheximide was sometimes added to inhibit protein synthesis before and/or during heating at 43 degrees C. Morphological alterations were quantified immediately and at various times after heating by using phase-contrast microscopy to determine the percentage of cells that were severely blebbed and rounded. About 800 cells were analysed per datum point. When effects of heat on thermotolerant cells were compared with effects of heat on non-tolerant cells, heat-induced thermotolerance (HTT) was observed by an increase in survival, and by a reduction in the percentage of cells with morphological alteration observed immediately after the challenging heat. After the As treatment, very little thermotolerance was observed for morphological alterations immediately after the challenging heat, although thermotolerance was observed for survival. However, as the cells were incubated for 12 or 24 h at 37 degrees C after the challenging heat treatment, recovery from morphological alterations was observed in the As-TT cells. Possible mechanisms for the difference between HTT and As-TT are discussed.     

Heat-induced Morphological Alterations in Non-tolerant and Thermotolerant Cells

Summary

CHO cells were heated at 43°C or 45°C for various durations up to 300 min. Survival values varied from 5 × 10^?1 to 10^?7. Unheated, non-tolerant control cells were compared with cells made thermotolerant (TT) by incubating at 37°C for 6 or 12 h after treatment with either sodium arsenite (100 µm-As) or 45·5°C for 10 min, respectively. Groups also were included in which heat-induced TT cells were heated at 43°C for 5 h immediately before they were challenged at 45°C; in these groups, cycloheximide was sometimes added to inhibit protein synthesis before and/or during heating at 43°C. Morphological alterations were quantified immediately and at various times after heating by using phase-contrast microscopy to determine the percentage of cells that were severely blebbed and rounded. About 800 cells were analysed per datum point. When effects of heat on thermotolerant cells were compared with effects of heat on non-tolerant cells, heat-induced thermotolerance (HTT) was observed by an increase in survival, and by a reduction in the percentage of cells with morphological alteration observed immediately after the challenging heat. After the As treatment, very little thermotolerance was observed for morphological alterations immediately after the challenging heat, although thermotolerance was observed for survival. However, as the cells were incubated for 12 or 24 h at 37°C after the challenging heat treatment, recovery from morphological alterations was observed in the As-TT cells. Possible mechanisms for the difference between HTT and As-TT are discussed.     

Differences in Heat-induced Cell Killing as Determined in Three Mammalian Cell Lines Do Not Correspond with the Extent of Heat Radiosensitization

Summary

Three different cell lines, Ehrlich ascites tumour (EAT) cells, HeLa S₃ cells and LM mouse fibroblasts, were used to investigate whether or not the extent of heat killing (44°C) and heat radio-sensitization (44°C before 0–6 Gy X-irradiation) are related. Although HeLa cells were the most heat-resistant cell line and showed the least heat radiosensitization, we found that the most heat-sensitive EAT cells (D₀, EAT = 8·0 min; D₀, LM = 10·0 min; D₀ HeLa = 12·5 min) showed less radiosensitization than the more heat-resistant LM fibroblasts (TER_HeLa < TER_EAT < TER_LM). Therefore, it is concluded that the routes leading to heat-induced cell death are not identical to those determining heat radiosensitization. Furthermore the inactivation of DNA polymerase α and β activities by heat seemed not to correlate with heat survival alone but showed a positive relationship to heat radiosensitization. The possibility of these enzymes being a determinant in heat radiosensitization is discussed.     

Effects of Amiloride on Hyperthermic Cell Killing of Normal and Thermotolerant Mouse Fibroblast LM Cells

Summary

The effect of amiloride on hyperthermic cell killing of normal and thermotolerant mouse fibroblast LM cells was investigated under normal (pH 7·4) and acidic (pH 6·8) conditions. Amiloride is known to inhibit the Na⁺/H⁺ exchanger in the plasma membrane, the main pH regulating mechanism in mammalian cells. The effects of low pH and amiloride on the mouse fibroblasts were qualitatively similar. For normal cells, mainly a reduction of the shoulder of the survival curve was observed, while an increase of the slope of the exponential part of the survival curve was found in thermotolerant cells. When a combination of 3 mmol dm^?3 amiloride and low pH was used the effect on the hyperthermic sensitivity of normal and thermotolerant cells was not additive. This may be explained by a similarity in the mechanism of action of the two treatments, viz. inhibition of the Na⁺/H⁺ exchange, which is probably complete when 3 mmol dm^?3 amiloride is used. The amiloride sensitivity of normal and thermotolerant fibroblasts is dose dependent in the range of 0·1 to 3 mmol dm^?3. Because the D₀ of control cells is almost independent of the amount of amiloride, a concentration-dependent reduction of the thermotolerance ratio is found, especially at higher concentrations of amiloride.     

Influence of glutathione on the induction of chromosome aberrations, delay in cell cycle kinetics and cell cycle regulator proteins in irradiated mouse bone marrow cells

PURPOSE: Reduced glutathione (GSH) is an endogenous thiol and has long been thought to affect the sensitivity of cells to radiation. The aim was to see the influence of GSH on: (i) the production of all types of radiation-induced chromosome aberrations (CA), and (ii) the radiation-induced delay in cell cycle and the levels of cell cycle regulator proteins. MATERIALS AND METHODS: Cell cycle kinetics were determined by scoring the mitotic index (MI). CA and MI were scored in gamma-irradiated buthionine sulfoximine (BSO) (10 h) or GSH (1 h) pretreated and untreated mouse bone marrow cells (BMC). The expression of p53 and p21 proteins after 2 and 6 h of irradiation and for the B-cell lymphoma 2 (Bcl-2) associated X-protein (Bax) after 24 h of irradiation with or without BSO or GSH treatment was analyzed by immunoblotting. RESULTS: Radiation delays mouse BMC in their passage through the cell cycle and induces CA. Exogenous addition of GSH protected CA uniformly at lower doses of radiation but differentially at higher doses, whereas GSH-depletion by BSO increased the frequency of radiation-induced CA. Both GSH and BSO-pretreated cells reduced the delay in cell kinetics after irradiation. Levels of both p53 and p21 were enhanced after irradiation to BSO-pretreated cells. However, in GSH-pretreated cells the level of these proteins was reduced. CONCLUSION: Data indicate that the induction of CA and delay in cell kinetics by radiation may not always be interlinked and that the level of endogenous GSH exerts its influence on these parameters. Both GSH and BSO pretreatment reduce delays in cell kinetics of irradiated cells which may die apoptotically, since they have either a higher frequency of exchange aberrations or CA, respectively.     

Spatial Characterization of Glutathione Depletion in the KHT Sarcoma Using Flow Cytometry

Summary

Intravenous administration of the fluorescent DNA stain Hoechst 33342 to tumour-bearing mice was used to label cells proportionally to their proximity from the vasculature. Flow cytometry was used to sort cells from the tumour into populations based on their Hoechst 33342-derived fluorescence. The cell populations were then assayed for their glutathione (GSH) content and their radiosensitivity. Tumours from mice pretreated with buthionine sulphoximine (BSO) were compared with untreated animals. The major findings of this study suggest that the cellular GSH concentration within tumours decreases with distance from the vasculature, and that the GSH concentration within cells from all locations in the tumour can be depleted by enzymatically inhibiting its synthesis using BSO. This depletion of GSH resulted in a small degree of hypoxic radiosensitization of cells both distal and proximal to the vasculature.     

Plasma-membrane fluidity studies of murine neuroblastoma and malignant melanoma cells under irradiation]

Murine neuroblastoma (C-1300 NMB) and malignant melanoma (B16) cells were radiated in presence of radiopharmaceutics. Sensibilization was carried out with BSO and protection with TMX. Changes in fluidity of the plasma membrane, in cellular GSH contents and cell cycle were observed. After radiation fluidity of the plasma membrane is increased, whereas intracellular GSH decreased. These changes were intensified by BSO and reduced by TMX. Fluidity of the plasma membrane correlates with intracellular GSH and also with cell cycle. It is suggested that changes in plasma membrane fluidity can be used as an additional parameter for the determination of sensitivity towards radiation.     

丁胱亚磺酰亚胺对胶质瘤细胞谷光甘肽及放射增敏作用的实验研究

研究巯基修饰剂ＢＳＯ对胶质瘤细胞ＧＳＨ含量的修饰作用和放射增敏作用。方法利用Ｔｉｅｔｚｅ还原酶法观察ＢＳＯ对体外培养的胶质瘤细胞株及裸小鼠移植瘤模型胶质瘤细胞ＧＳＨ的作用。利用ＭＴＴ法观察ＢＳＯ对胶质瘤细胞放射敏感性的影响。结果经ＢＳＯ作用后胶质瘤细胞的ＧＳＨ含量显著下降，放射敏感性增加。结论无论是离体还是整体用药，ＢＳＯ均能降低胶质瘤细胞的ＧＳＨ含量。ＢＳＯ对胶质瘤细胞有放射增敏作用。     

Modulation of the clastogenic activity of gamma-irradiation in buthionine sulphoximine-mediated glutathione depleted mammalian cells

Purpose: Chromosome aberrations (CA) were used as an endpoint to investigate the effect of buthionine sulphoximine (BSO), a potent glutathione-depleting agent, on the radiosensitivity of mammalian cells. The aim was to obtain information about the role of glutathione (GSH) in physicochemical and biochemical processes in irradiated cells. Materials and methods: CA were scored from first cycle metaphases in irradiated BSO-pretreated and untreated samples. BSO exposure was for 10h in mouse bone marrow cells in vivo and 5h for human lymphocytes in vitro. In further experiments fresh blood was irradiated on ice and immediately after irradiation GSH/GSH-ester was added. Results: In both the systems BSO-treated samples showed higher sensitivity to radiation than BSO untreated samples. The frequency of all types of CA increased except exchange aberrations. GSH/GSH-ester treatment given after irradiating the cells at 4 C reduced the frequency of deletions and increased the frequency of exchange aberrations. Conclusions: Data indicate that BSO-mediated GSH depletion increased radiation-induced chromosome aberrations, apart from exchange aberrations. This could be due to reduction in the free-radical scavenging effect of GSH, a failure in rejoining of DNA double-strand breaks, or induction of apoptosis.     

Modulation of the clastogenic activity of gamma-irradiation in buthionine sulphoximine-mediated glutathione depleted mammalian cells.

PURPOSE: Chromosome aberrations (CA) were used as an end-point to investigate the effect of buthionine sulphoximine (BSO), a potent glutathione-depleting agent, on the radiosensitivity of mammalian cells. The aim was to obtain information about the role of glutathione (GSH) in physicochemical and biochemical processes in irradiated cells. MATERIALS AND METHODS: CA were scored from first cycle metaphases in irradiated BSO-pretreated and untreated samples. BSO exposure was for 10h in mouse bone marrow cells in vivo and 5 h for human lymphocytes in vitro. In further experiments fresh blood was irradiated on ice and immediately after irradiation GSH/GSH-ester was added. RESULTS: In both the systems BSO-treated samples showed higher sensitivity to radiation than BSO untreated samples. The frequency of all types of CA increased except exchange aberrations. GSH/GSH-ester treatment given after irradiating the cells at 4 degrees C reduced the frequency of deletions and increased the frequency of exchange aberrations. CONCLUSIONS: Data indicate that BSO-mediated GSH depletion increased radiation-induced chromosome aberrations, apart from exchange aberrations. This could be due to reduction in the free-radical scavenging effect of GSH, a failure in rejoining of DNA double-strand breaks, or induction of apoptosis.     

Spatial characterization of glutathione depletion in the KHT sarcoma using flow cytometry.

Intravenous administration of the fluorescent DNA stain Hoechst 33342 to tumour-bearing mice was used to label cells proportionally to their proximity from the vasculature. Flow cytometry was used to sort cells from the tumour into populations based on their Hoechst 33342-derived fluorescence. The cell populations were then assayed for their glutathione (GSH) content and their radiosensitivity. Tumours from mice pretreated with buthionine sulphoximine (BSO) were compared with untreated animals. The major findings of this study suggest that the cellular GSH concentration within tumours decreases with distance from the vasculature, and that the GSH concentration within cells from all locations in the tumour can be depleted by enzymatically inhibiting its synthesis using BSO. This depletion of GSH resulted in a small degree of hypoxic radiosensitization of cells both distal and proximal to the vasculature.     

Enhanced Glycosylation of a 50 kD Protein during Development of Thermotolerance in CHO Cells

Summary

During the development of thermotolerance, Chinese hamster ovary cells not only synthesized classical heat shock proteins, but also incorporated [³H]d-glucose or mannose into a glycoprotein with a M_r of approximately 50 kD. The glycosylation of the 50 kD protein correlated with the expression of thermotolerance under conditions when tolerance was induced either by acute or chronic heat conditioning. A phosphoprotein with the same molecular weight as the 50 kD glycoprotein was dephosphorylated immediately after heat conditioning. Both phosphate and glucose label in the ion front were enhanced immediately after heating, and may represent elevated levels of sugar phosphates. However, the composition of the ion front material remains to be determined. The data are consistent with a hypothesis that attributes increased heat resistance of thermotolerant cells to the glycosylation of specific heat-sensitive cellular sites.     

Membrane Radiosensitivity of Fatty Acid Supplemented Fibroblasts as Assayed by the Loss of Intracellular Potassium

Summary

Leakage of potassium from mouse fibroblast LM cells, X-irradiated at 0°C with doses up to 400 Gy is shown to be related to plasma membrane lipid composition. Fatty acid supplemented cells, containing about 40 per cent polyunsaturated fatty acids (PUFA) in their membranes were much more sensitive to radiation, as measured by increased permeability, than normal cells, which contained 7 per cent PUFA. The damage observed after irradiation at 0°C was partially repaired during a post-irradiation incubation at 22°C. The o.e.r. for potassium leakage was about 4 for normal fibroblasts and 8 for the PUFA-supplemented cells. No oxygen-dependent radiation damage could be observed in cells treated with high amounts of vitamin E. Depletion of glutathione in PUFA cells sensitized oxic cells to radiation damage, resulting in an increase of the o.e.r. from 8 to 17. No lipid peroxidation (malondialdehyde production and disappearance of fatty acyl chains) could be demonstrated. While PUFA, normal and vitamin E grown cells showed a differential sensitivity in radiation-induced potassium leakage and trypan blue uptake (high doses, interphase death), no difference in radiation-induced clonogenic ability (reproductive death) could be observed after the different cell treatments. The experiments reported are supportive of a role of membranes in the mechanism of radiation-induced interphase death and show that increased damage may be expected when high amounts of polyunsaturated membrane lipids are present under conditions of low amounts of appropriate antioxidants.     

Enhancement of SR 2508 (etanidazole) radiosensitization by buthionine sulphoximine at low-dose-rate irradiation.

SR 2508 (etanidazole) (1 mM) or buthionine sulphoximine (BSO, 50 microM) or both drugs together did not radiosensitize oxic V79 Chinese hamster cells irradiated at either an acute dose rate (2.35 Gy/min) or at a low dose rate (0.117 Gy/min). BSO pretreatment (15 h at 37 degrees C) depleted cellular glutathione (GSH) to less than or equal to 1% of control level and radiosensitized hypoxic cells at both dose rates with an enhancement ratio (ER) of 1.2. SR 2508 alone radiosensitized hypoxic cells equally at both dose rates with an ER of 1.5. However, ER values of 2.2 and 2.5 were obtained with 1 mM SR 2508 in GSH-depleted cells at acute and low dose rate, respectively, with no significant difference between the two, i.e. there is no dose rate dependence for this potentiation. Since BSO increases SR 2508 radiosensitization and the combined BSO + SR 2508 treatment is extremely cytotoxic to hypoxic cells, our results suggest that combining BSO with SR 2508 will be useful in brachytherapy as well as external-beam therapy if the toxicity from both drugs in vivo is less than the gain in radiosensitization achieved.     

Rapid-mixing Studies of Radiosensitivity with Thiol-depleted Mammalian Cells

Summary

A moderate reduction in the non-protein thiol content of V79 379A Chinese hamster cells, obtained by pretreatment with buthionine sulphoximine (BSO), diethyl maleate (DEM) or N-ethyl maleimide (NEM), increases both the absolute radiosensitivity of the cells in hypoxia and the radiosensitizing effect of adding oxygen 7 ms after irradiation. Combined pretreatment of cells with BSO and NEM removes most of the non-protein thiol and some of the protein thiol; such treatment further increases the radiosensitivity of hypoxic cells but there is no further effect of adding oxygen 7 ms after irradiation. Addition of 2-mercaptoethanol to cells 7 ms after irradiation gives protection factors that increase with increasing severity of thiol depletion. Substantial radioprotection can still be observed when 2-mercaptoethanol is added 70 ms after irradiation of cells pretreated with BSO and NEM; there is no effect of adding 2-mercaptoethanol to such cells 50 s after irradiation. These observations support the repair–fixation model of radiation damage and suggest that, in addition to the established role of non-protein thiol in chemical repair of radiation damage, other endogenous reducing agents such as protein thiol may be important in determining cellular radiosensitivity. A relatively long-lived thiol-modifiable component of radiation damage has been observed within hypoxic thiol-depleted cells.     

Effect of thermotolerance and step-down heating on thermal radiosensitization in CHO cells.

Chinese hamster ovary cells were exposed to single or fractionated heat treatments followed by irradiation on ice with graded doses of X-rays. The dose-response curves obtained were fitted by the linear-quadratic equation -ln(S/S0) = alpha D + beta D2 and analysed in terms of TER10%, alpha and beta. Thermal enhancement ratio, TER10%, was reduced when heat sensitivity was lowered either by chronic (pretreatment 40 degrees C, 16 h) or acute (43 degrees C, 45 min-37 degrees C, 10 h) thermotolerance, but was enhanced after step-down heating (43-40 degrees C or 45-40 degrees C). It could be shown that thermal radiosensitization, as expressed by TER10%, is modified by thermotolerance or step-down heating only to the extent to which cellular survival is modified by the corresponding pretreatments. However, the relative change of alpha and beta was found to be different for thermotolerance and step-down heating. For thermotolerant cells the values for alpha and beta were reduced by about the same factor, whereas step-down heating caused an increase in both parameters, which was greater for alpha than for beta. Data analysis showed that the modification of thermal radiosensitization by thermotolerance can be interpreted as if the cells were heated at the given temperature for a shorter time, whereas after step-down heating the cells responded as if they were exposed to a higher temperature prior to irradiation.     

The Effect of Artificially Induced Hyperglycemia on the Radiation Response of the Lewis Lung and EMT6 Tumor Models

Summary

The effect of hyperglycemia, induced by administration of glucose, on the radiation response of the Lewis lung and EMT6 tumor models has been evaluated. Neither acute (single i.p. injection of 8 mg/glucose) nor chronic (multiple i.p. injections of 6 mg/g glucose plus glucose in the water bottles) administrations of glucose increased the radiation response of either tumor. A combination of a single i.p. injection of glucose and a reduction of the O₂ content of the inspired gas to 10 per cent did by itself reduce cell survival by 55–75 per cent in the EMT6 and 80–90 per cent in the Lewis lung carcinoma. However, this treatment had little effect on the shape of the radiation dose–response curve, and simply gave rise to a parallel shift of the survival curve, indicating that this treatment had little or no specificity for hypoxic cells.     

Reduction of Intracellular Glutathione Content and Radiosensitivity

Summary

The intracellular glutathione (GSH) content of HeLa, CHO and V79 cells was reduced by incubating the cells in growth medium containing buthionine sulphoximine or diethyl maleate (DEM). Clonogenicity, single-strand DNA breaks (ssb) and double-strand DNA breaks (dsb) were used as criteria for radiation-induced damage after X- or γ-irradiation. In survival experiments, DEM gave a slightly larger sensitization although it gave a smaller reduction of the intracellular GSH. In general, sensitization was larger for dsb than for ssb, also the reduction of the o.e.r. was generally larger for dsb than for ssb. This may be due to the higher dose rate in case of dsb experiments resulting in a higher rate of radiochemical oxygen consumption. In general, no effect was found on post-irradiation repair of ssb and dsb.     

Inhibition of protein kinase C activity promotes heat-induced apoptosis in RIF-1 but not in TR-4 cells.

PURPOSE: Inhibition of protein kinase C (PKC) activity has been demonstrated to reduce thermotolerance (TT), presumably by decreasing heat shock protein (HSP) production. Therefore, the interest was in evaluating this relationship further in two isogenic murine tumour cell lines: RIF-1 and its thermoresistant TR-4 selectant. MATERIALS AND METHODS: TT was induced in RIF-1 and TR-4 cells (45 degrees C for 15 min, then 37 degrees C for 6 h) with or without Ro31-8220, a specific inhibitor of PKC. PKC activity was assayed by determining the catalytic transfer of ATP to a specific substrate peptide. Survival was determined using the clonogenic assay. Apoptosis was quantitated by counting the number of cells demonstrating apoptosis after staining with acridine-orange/ ethidium bromide. Production of the inducible form of HSP70 was assessed using Western blot. RESULTS: At 2 microM Ro31-8220, >80% of PKC activity was inhibited in both cell lines, which was associated with no cytotoxicity at 37 degrees C. Basal HSP70 level was approximately 10-fold higher in the TR-4 compared with the RIF-1 cells. Upon TT induction, HSP70 level increased significantly in both cell lines, which was suppressed in the presence of Ro31-8220, but the relative amount of HSP70 remained high in the TR-4 cells. At 24 h, heat-induced apoptosis increased from 4 to 38% in RIF-1 cells in the presence of Ro31-8220, which was associated with a 26% reduction in clonogenic survival after thermotolerant heating. In contrast, <1% of TR-4 cells demonstrated apoptosis even with the highest dose of Ro31-8220, and no effect on survival was observed. CONCLUSION: Inhibition of PKC activity reduces HSP70 induction, which in turn is associated with promotion of heat-induced apoptosis in RIF-1 cells. However, the survival signals in the TR-4 cells are so strong, that even 80% inhibition of PKC activity has minimal impact on heat-induced apoptosis and survival in this thermoresistant cell line.