On the Importance of the Level of Glutathione and the Activity of the Pentose Phosphate Pathway in Heat Sensitivity and Thermotolerance

Summary

Heating of Ehrlich ascites tumour (EAT) cells and mouse fibroblast LM cells to 43 or 44°C respectively, results in an increased level of reduced gluthathione (GSH). The maximum elevation in GSH was to 140 per cent for LM cells and to 120 per cent for EAT cells. No increase of GSH in EAT cells was observed after heating at 44°C. LM cells were treated with diethylmaleate (DEM) and the EAT cells with buthionine-sulphoximine (BSO) at non-toxic doses to deplete the levels of GSH. No effect on thermosensitivity or on the development of thermotolerance was observed when the DEM and BSO treatments were chosen such that the lowering of GSH was just down to the level of detection (about 5 per cent of control). When higher concentrations of DEM were used, thermal sensitization was observed. The activity of the pentose phosphate pathway (PPP) was also investigated because of its importance in supplying NADPH for the regeneration of GSH from GSSG and for the endogenous production of polyols. Hyperthermia was found to enhance markedly the flux of glucose through the PPP. While the DEM treatment inhibited glucose oxidation through the PPP, BSO addition to the cells resulted in a slightly increased activity of the PPP. The PPP activity of thermotolerant cells was lower (fibroblasts) or hardly affected (EAT cells) compared to control cells. The extent of PPP activation by hyperthermia was comparable for thermotolerant and control cells. For the two cell lines studied neither a high level of GSH nor an active PPP is a prerequisite for the development of thermotolerance.     

Thermal Radiosensitization in CHO Cells by Prior Heating at 41–46°C

Summary

CHO cells were exposed to heat at temperatures ranging from 41°C to 46°C followed by irradiation on ice with graded doses of X-rays. The dose–response curves obtained were analysed in terms of D_10%, D₀ and Dq and thermal enhancement was expressed by the corresponding values TER_10%, TEF and TEQ, respectively. TER_10% and TEF were shown to increase linearly with heating time, the increase being steeper at higher temperatures. The dose–response curves were also analysed using the equation ? ln(S/S₀) = αD + βD²; the values of α and β obtained from curve-fitting were found to increase with heating time. For temperatures below 43°C the relative increase in α was greater than that in β; the Arrhenius activation energies were E_a = 890 kJ mol^?1 for α and E_a = 1830 kJ mol^?1 for β. At temperatures exceeding 43°C the relative increase of α and β was similar and the corresponding activation energies were about the same (E_a ≈ 700 kJ mol^?1). The increase in the α-term was attributed to a depressed repair of double-strand breaks, whereas the increase of β was assumed to be a consequence of an insufficient repair of base damage.     

Inhibition of Repair of Radiation-induced DNA Damage by Thermal Shock in Chinese Hamster Ovary Cells

Summary

The effect of exposure to elevated temperatures (41–45°C) on the repair of radiation-induced DNA strand breaks was measured in monolayer cultured Chinese hamster ovary (CHO) cells. Prior exposure of cells to temperatures between 43 and 45°C resulted in significant decreases in the rate of repair of DNA damage. Exposure to 45°C for 15 min slowed the rate of DNA repair to 0·17 of the control repair rate. The T_o for inactivation of DNA repair was observed to be 34, 13 and 6 min at 43, 44 and 45°C, respectively. Stepdown-heating (45°C for 15 min followed by repair at 41°C) resulted in greater inhibition of DNA repair (0·11 of the control rate) than was observed after acute heating alone. Repair at 41°C was observed to proceed in unheated cells at a faster rate than at 37°C. An Arrhenius analysis of the inactivation kinetics of DNA repair between 43 and 45°C indicated an activation energy of 140 kcal mol^?1 of protein for the inhibition of DNA repair. In general, the results were inconsistent with either a retardation of the DNA repair rate or an increase in unrepaired DNA lesions being responsible for heat-induced radiosensitization.     

Induction and Repair of DNA Strand Breaks in X-irradiated Proliferating and Quiescent CHO Cells

Summary

Induction and repair of DNA strand breaks was studied in X-irradiated proliferating and quiescent CHO cells using the alkaline unwinding technique. The results showed that induction of strand breaks is identical for both states of proliferation, whereas repair is different. The decrease in the number of DNA strand breaks with incubation time at 37°C is best described by a sum of three exponential components I, II and III. The half-times of component I were similar (τ_Ip = 1·73 min versus τ_Iq = 1·66 min) whereas strand breaks comprising component II were repaired slightly faster (τ_IIp = 17·0 min versus τ_IIq = 14·2 min) and those comprising component III were repaired significantly faster (τ_IIIp = 218 min versus τ_IIIq = 113 min) in quiescent as compared with proliferating cells. In contrast, the initial fractions, f, of the three components were closely similar for both states of growth (proliferating cells: f_I = 0·69, f_II = 0·25, f_III = 0·06; quiescent cells: f_I = 0·65, f_II = 0·29, f_III = 0·06). Radiosensitivity as assayed by colony formation was found to be lower for quiescent cells than for proliferating cells. By fitting the survival data to the linear-quadratic equation, ?ln(S/S₀) = αD + βD², the ratios α_p/α_q = 1·7 and β_p/β_q = 1·2 were obtained, which are similar to the ratios of the half-times of components III and II (τ_IIIp/τ_IIIq = 1·9; τ_IIp/τ_IIq = 1·2). This result indicates that the DNA damage represented by components II and III might be involved in cell killing.     

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.     

High Radiosensitivity of the MOLT-4 Leukaemic Cell Line

Summary

Radiation survival of MOLT-4, a leukaemic T-lymphocyte cell line, was measured by counting colonies formed in 0·8 per cent methyl cellulose. The survival curve was a simple exponential and showed the cells to be radiation sensitive, with D₀ = 0·49 ± 0·02 Gy and extrapolation number n = 0·92 ± 0·09. No increase in survival as measured by colony-forming ability or trypan blue dye exclusion was seen when the dose was split into two fractions, separated by a 5 h incubation period. Electron microscopy and trypan blue dye exclusion showed that 5 h after exposure to high doses, MOLT-4 cells began to die and displayed condensed, marginated chromatin and cellular vesticulation.     

Enhancement of Hyperthermic Cell Killing by Non-thermal Effect of Ultrasound

Summary

The present study was performed to elucidate the mechanism of enhanced hyperthermic cell killing by a non-thermal effect (cavitation and direct effect) of ultrasound under various gas conditions. Cavitation, as indicated by formation of DNA double-strand breaks and liberation of potassium iodide, was completely inhibited under N₂O-saturated conditions, while it was promoted under O₂-, Ar-, and N₂-saturated conditions. Mouse L cells were treated with ultrasound (1 MHz continuous wave, spatial peak temporal average intensity; 3·7 W/cm²) and/or 44°C hyperthermia in medium saturated with O₂, Ar, N₂ (with cavitation) or N₂O (with direct effect). The synergism on cell killing by ultrasound and 44°C hyperthermia was observed under N₂O-saturated conditions (enhancement ratio = 1·39). On the other hand, additive enhancement was observed under O₂-, Ar-, or N₂-saturated conditions. In addition, when cells were treated with 44°C hyperthermia before or after sonication under N₂O-saturated conditions, synergistic cell killing was not observed. These results suggested that the direct effect of ultrasound alone did not influence cell killing, but enhanced the hyperthermic cell killing synergistically, when both agents simultaneously acted on the cells.     

Response of Human Bone Marrow Progenitor Cells to X-rays in Vitro

Summary

In 25 out of 33 cases the survival response of myeloid progenitor cells from fresh human bone marrows, X-irradiated in vitro in phosphate-buffered saline at 23°C, was characterized by a two-term exponential relationship, with D₀ values of 0·42 ± 0·19 Gy and 1·38 ± 0·37 Gy, respectively. In the remaining eight cases for which the colony-forming efficiency was 10 times lower, survival followed a single exponential function with D₀ = 1·18 ± 0·15 Gy. The biphasic response at 23°C became a single exponential response when the temperature at irradiation was 30 or 37°C.     

Cell Inactivation and DNA Single- and Double-strand Breaks in Cultured Mammalian Cells Irradiated by a Thermal Neutron Beam

Summary

The effects on the cellular viability and induction and repair kinetics of DNA strand breaks in HeLa cells were examined after exposure to a thermal neutron beam and compared with those after γ-irradiation. The thermal neutron survival curve had no initial shoulder. The relative biological effectiveness (r.b.e.) value of the neutron beam was determined to be 2·2 for cell killing (ratio of D₀ values), 1·8 and 0·89 for single strand breakage (ssb) by alkaline sedimentation and alkaline elution respectively, and for double strand breakage (dsb) 2·6 by neutral elution. No difference was observed between thermal neutrons and γ-rays in the repair kinetics of ssb and dsb. It is suggested that the effect induced by the intracellular nuclear reaction, ¹⁴N(n,p)¹⁴C is mainly responsible for the high r.b.e. values observed.     

Survival of HeLa Cells Cultured in Vitro and Exposed to Protracted Gamma-irradiation

Summary

Experiments are described in which S₃ HeLa cells were exposed to gamma-radiation at dose-rates of 9·5 and 19 rad/hour (chronic irradiation). The cell-survival, in terms of reproductive integrity, was compared with the same cell-line irradiated at 44·9 rad/min (acute irradiation). It was found that while a ‘type C’ multi-hit curve was necessary to describe the survival of cells irradiated at 44·9 rad/min, a simple exponential function of dose was sufficient to describe the survival at 9·5 and 19 rad/min. The data were compared with the results of Berry and Cohen (1962).     

A Comparison of the Intracellular Uptake and Radiosensitization Efficiency in Different Media of Uncharged 2-nitroimidazoles of Varying Lipophilicity

Summary

The effect of varying octanol: water partition coefficients, P, (range 0·026–260) on the uptake of uncharged 2-nitroimidazoles into Chinese hamster V79 379A cells has been studied. Average intracellular concentrations were measured by high performance liquid chromatography after centrifuging cells through oil or an aqueous medium. The ratio of intracellular concentration of radiosensitizer to extracellular concentration (C_i/C_e) for misonidazole (P = 0·43) was 0·85 for the oil method and 0·68 for the aqueous method. For values of P less than about 0·05 uptake was initially very slow and C_i was always less than C_e. When P ≧ 0·1 uptake was rapid and then remained unchanged for times up to 3 h; for P ≧ 10, C_i/C_e increased rapidly as P increased. Ro 31-1405 (P = 260) concentrated by a factor of 7 inside the cell. Although uptake was identical for cells suspended in full growth medium and PBS, radiosensitization was greater for cells in PBS: 1 mmol dm^?3 misonidazole produced an enhancement ratio of 1·6 in full growth medium and 1·9 in PBS. This increase in radiosensitization could not be accounted for by protein binding. However, measurements on cellular non-protein sulphydryl (NPSH) demonstrated the levels to be reduced to about 60 per cent for cells in PBS. Similar reductions in NPSH levels have previously been shown not to increase the radiosensitivity of control cells but to increase greatly the effectiveness of nitroimidazole radiosensitizers.     

Radiation Sensitivities Are Not Related to the Sizes of DNA Supercoiled Domains in L5178Y-R and L5178Y-S Cells

Summary

Survival of murine lymphoblasts L5178Y-R and L5178Y-S irradiated with ⁶⁰Co gamma radiation was determined. The parameters of the survival curves were D₀ = 1·18 Gy, n = 1·56 and D₀ = 0·55 Gy, n = 1·00 for L5178Y-R and L5178Y-S cells respectively. The sizes of DNA supercoiled domains were estimated using sedimentation of nucleoids from cells irradiated with doses from 1 to 7 Gy. These sizes were 2·44 × 10⁹ and 5·13 × 10⁸ Da for L5178Y-R cells and 1·30 × 10⁹ and 4·07 × 10⁸ Da for L5178Y-S cells. Hence, higher radiosensitivity of L5178Y-S cells was not compatible with the larger size of the DNA supercoiled domains, as suggested by Filippovich et al. (1982). We have not found any simple relation between the sizes of DNA supercoiled domains and the susceptibility of L5178Y sublines to ionizing radiation.     

Heat Resistance and Thermotolerance in a Radiation-resistant Cell Line

Exponentially growing TN-368 lepidopteran insect cells have a normal growth temperature of 28°C. These cells were heated in water baths at various temperatures between 33 and 44°C under conditions of constant or fractionated heating. Determinations of cell survival using colony formation as well as measurements of DNA and protein synthesis were performed to assess relative heat resistance and development of thermotolerance. The results demonstrate a marked heat resistance over previously reported findings from the same laboratory for dipteran Drosophila cells in culture. The degree of heat resistance is remarkable, especially when compared to the heat resistance of mammalian cells, i.e. TN-368 cell survival at 41·5 and 44°C was somewhat similar to mammalian cell survival, even though these temperatures are 13·5 and 16°C above the normal growth temperature for TN-368 cells and 4·5 and 7°C above the growth temperature of mammalian cells. Furthermore, the lepidopteran cells maintain the ability to develop a notable amount of thermotolerance in addition to this heat resistance. Thermotolerance development alone is capable of enhancing survival by an additional 10000-fold. Thermotolerance could also be detected at the level of protein synthesis as a more rapid recovery following heat treatment. In contrast, DNA synthesis inhibition was prolonged even further in cells receiving a prior heat treatment to induce thermotolerance. In summary, it appears that, in addition to their pronounced radiation resistance, the TN-368 cells are also quite resistant to heat. It remains to be seen whether a single mechanism could be responsible for resistance to these agents which act very differently.     

Effect of heat on dsb repair in G1- and S-phase studied in the human HeLa S3 cell line

Purpose : The aim of this study was to investigate the relation between double-strand breaks and thermal radiosensitization in dependence on cell-cycle position. Materials and methods : The experiments were performed with the human tumour cell line HeLa S3. Cells synchronized in G1- and S-phase were exposed to X-rays alone or in combination with prior heating at 44° C for 20 min. Cell kill was determined by means of colony forming assay, double-strand breaks (dsb) using constant-field gel electrophoresis and apoptotic cell death was scored using the fraction of detached cells. Results : In both cell-cycle phases heating at 44° C for 20min prior to irradiation resulted in an increased cellular radiosensitivity, whereby the thermal enhancement ratio (TER) was significantly higher in S- than in G1-phase cells with TER=2.1 and 1.2, respectively. Prior heating at 44° C did not affect the number of radiation-induced dsb but was found to modify their repair as measured for a X-ray dose of 40Gy. In both cell cycle phases dsb repair kinetics measured after irradiation alone could be described by a fast and a slow component with the majority of dsb being repaired with fast kinetics. Prior heating at 44° C was found to have only a minor effect on these half-times but mainly to affect the number of slowly rejoined dsb. In G1-phase cells the number of slowly rejoined dsb measured 300min after irradiation was enhanced by a factor of 1.8 and in S-phase cells even by a factor of 3.2. Fraction of apoptotically dying cells was low after X-irradiation alone but was clearly enhanced after combined treatment, which was especially pronounced for S-phase cells. Conclusions : The pronounced thermal radiosensitization found for S-phase cells was attributed to the heat-mediated increase in the number of slowly rejoined dsb and partly also to the enhanced fraction of apoptotically dying cells when compared to G1-phase cells.     

Differential Response to Gamma Radiation of Human Stomach Cancer Cells in Vitro

Summary

In vitro effects of radiation were studied in two permanent cell lines (AGS and SII) from two patients with adenocarcinoma of the stomach and three permanent sublines from each cell line. Radiation survival parameters for AGS and SII parent cell lines and sublines were determined after in vitro irradiation of their cells with 0·5 to 10 Gy of ⁶⁰Co gamma rays. The AGS and SII cell lines had different growth properties, DNA contents and radiation survival curves. Surviving fractions of SII parent cells (76 chromosomes) after 2·0 and 10 Gy were 1·22 and 17·8 times greater, respectively, than values for AGS parent cells (47 chromosomes). Sensitivities (D₀) were 1·08 and 1·45 Gy for AGS and SII parent lines, respectively. The D₀ values for AGS parent cells and sublines were similar (1·01 to 1·08 Gy), but SII parent cells and sublines had D₀ values of 1·45, 1·36, 1·37 and 1·12 Gy (for SII-A). Also, the SII parent cells had survival fractions after 2·0 and 10 Gy that were 1·3 and 11·3 times greater, respectively, than values for the SII-A cells. These data show differences in radiation responses among stomach cancer cell lines and sublines that may relate to DNA content, but there was no consistent correlation between radiation response and a particular cell characteristic.     

Radiation-induced DNA Double-strand Break Frequencies in Human Squamous Cell Carcinoma Cell Lines of Different Radiation Sensitivities

Summary

DNA neutral (pH 9·6) filter elution was used to measure radiation-induced DNA double-strand break (dsb) frequencies in eight human squamous cell carcinoma cell lines with radiosensitivities (D₀) ranging from 1·07 to 2·66 Gy and D¯ values ranging from 1·46 to 4·08 Gy. The elution profiles of unirradiated samples from more radiosensitive cell lines were all steeper in slope than the profiles from resistant cells. The shapes of the dsb induction curves were curvilinear and there was some variability from cell line to cell line in the dose-response for the induction of DNA dsb after exposures to 5–100 Gy ⁶⁰Co γ-rays. There was no relation between the shapes of the survival curves and the shapes of the dose-responses for the induction of DNA dsb. At low doses (5–25 Gy), three out of four of the more sensitive cell lines (D¯ < 2·5 Gy) had larger initial break frequencies than the more resistant lines (D¯ > 3·0 Gy). Although the low-dose (5–25 Gy) elution results were variable, they do suggest that DNA neutral elution will detect differences between sensitive and resistant tumour cells in initial DNA dsb frequencies.     

Effect of Dose Rate on Cell Killing and DNA Strand Break Repair in CHO Cells Exposed to Internal β-rays from Incorporated [3H]thymidine

Summary

Survival as well as repair of DNA strand breaks were studied in CHO cells after exposure to internal β-rays from incorporated [³H]thymidine at 4°C (equivalent to an exposure at ‘infinitely high’ dose rate) and at 37°C (low dose rate). DNA strand breaks were determined by the alkaline unwinding technique. In cells exposed at 4°C cell killing was five times higher (D₀ = 250 decays per cell) than in cells exposed at 37°C (D₀ = 1280 decays per cell). Strand breaks induced by ³H decay at 37°C were repaired with the same kinetics as those generated at 4°C. Therefore the different degrees of cell killing at 4°C and 37°C cannot be attributed to a difference in the repair kinetics for DNA strand breaks.     

Murine Haemopoietic Stem Cells with Long-term Engraftment and Marrow Repopulating Ability are More Resistant to Gamma-radiation than are Spleen Colony Forming Cells

The radiation sensitivity of various subsets in the haemopoietic stem cell hierarchy was defined using a limiting dilution type long-term bone marrow culture technique that was previously shown to allow quantification of cells with spleen colony-forming potential (day-12 CFU-S) and in vivo marrow repopulating ability (MRA). Primitive stem cells that generate new in vitro clonable colony-forming cells (CFU-C) in the irradiated marrow (MRA) and have long-term repopulation ability (LTRA) in vitro (cobblestone area forming cell, CAFC day-28) had D₀ values of 1·25 and 1·38 Gy, respectively. A lower D₀ was found for the less primitive CFU-S day-12, CAFC day-12 and cells with erythroid repopulating ability (0·91, 1·08 and 0·97 Gy, respectively). CFU-S day-7 were the most radiosensitive (D₀ equalling 0·79 Gy), while CFU-C and CAFC day-5 were relatively resistant to irradiation (D₀ 1·33 and 1·77 Gy). Split-dose irradiation with a 6 h interval gave dose sparing for stem cells with MRA and even more with in vitro LTRA, less for CFU-S day-12 and CAFC day-10 and none for CFU-S day-7. The cell survival data of the specified stem cell populations were compared with the ability of a fixed number of B6-Gpi-1^a donor bone marrow cells to provide for short- and long-term engraftment in single- and split-dose irradiated cognenic B6-Gpi-1^b mice. Serial blood glucose phosphate isomerase (Gpi) phenotyping showed less chimerism in the split as compared to the single radiation dose groups beyond 4 weeks after transplant. Radiation dose-response curves corresponding to stable chimerism at 12 weeks for single and fractionated doses revealed appreciable split-dose recovery (D₂–D₁) in the order of 2 Gy. This was comparable to D₂–D₁ estimates for MRA and late-developing CAFC (1·27 and 1·43 Gy, respectively), but differed from the poor dose recovery in cells corresponding to the committed CFU-S day-7/12 and CAFC day-10 population (0·14–0·33 Gy). These data are together consistent with differential radiosensitivity and repair in the haemopoietic stem cell hierarchy, and provide a cellular basis for explaining the dose-sparing effect of fractionated total-body irradiation conditioning on long-term host marrow repopulation.     

The efficacy of weekly and bi-weekly heat training to maintain the physiological benefits of heat acclimation

ObjectivesTo examine the efficacy of weekly and bi-weekly heat training to maintain heat acclimatization (HAz) and heat acclimation (HA) for 8 weeks in aerobically trained athletes.DesignRandomized, between-group.MethodsTwenty-four males (mean [m ± standard deviation [sd]; (age, 34 ± 12 y; body mass, 72.6 ± 8.8 kg, VO_2peak, 57.7 ± 6.8 mL·kg^?1·min^?1) completed five trials (baseline, following HAz, following HA (HAz + HA), four weeks into heat training [HT_WK4], and eight weeks into HT [HT_WK8] that involved 60 min of steady-state exercise (59.1 ± 1.8% vVO_2peak) in an environmental laboratory (wet bulb globe temperature [WBGT], 29.6 ± 1.4 °C) on a motorized treadmill. Throughout exercise, heart rate (HR) and rectal temperature (T_rec) were recorded. Following HAz + HA, participants were assigned to three groups: control group (HT₀), once per week heat training (HT₁), and twice per week heat training (HT₂). HT involved heated exercise (WBGT, 33.3 ± 1.3 °C) to achieve hyperthermia (38.5–39.75 °C) for 60 min. Repeated measures ANOVAs were used to determine differences.ResultsHAz + HA resulted in significant improvements in HR (p < 0.001) and T_rec (p < 0.001). At HT_WK8, HR was significantly higher in HT₀ (174 ± 22 beats?min^?1) compared to HT₂ (151 ± 17 beats?min^?1, p < 0.023), but was not different than HT₁ (159 ± 17 beats?min^?1, p = 0.112). There was no difference in % change of T_rec from post-HAz + HA to HT_WK4 (0.6 ± 1.3%; p = 0.218), however, HT_WK8 (1.8 ± 1.4%) was significantly greater than post-HAz + HA in HT₀ (p = 0.009).ConclusionsBi-weekly HT provided clear evidence for the ability to maintain physiological adaptions for 8 weeks following HA.     

Endogenous Superoxide Dismutase and Catalase Activities and Radiation Resistance in Mouse Cell Lines

Summary

The relationship between the endogenous cytoplasmic levels of the enzymes superoxide dismutase and catalase and the inhibition of cell proliferation by radiation has been studied in 11 mouse cell lines. The resistance of these mouse cell lines to radiation was found to vary by over 25-fold. No correlation was found between the cytoplasmic level of CuZn-superoxide dismutase or catalase and the resistance to radiation as measured by extrapolation number (EN), quasi-threshold dose (D_q), or D₀. None of the cell lines had detectable cytoplasmic Mn-superoxide dismutase. The apparent K_i of potassium cyanide for mouse CuZn-superoxide dismutase was determined (K_i = 6·5 µmol dm^?3).