The effect of hyperthermia on radiation-induced carcinogenesis

Ten groups of mice were exposed to either a single (30 Gy) or multiple (six fractions of 6 Gy) X-ray doses to the leg. Eight of these groups had the irradiated leg made hyperthermic for 45 min immediately following the X irradiation to temperatures of 37 to 43 degrees C. Eight control groups had their legs made hyperthermic with a single exposure or six exposures to heat as the only treatment. In mice exposed to radiation only, the postexposure subcutaneous temperature was 36.0 +/- 1.1 degrees C. Hyperthermia alone was not carcinogenic. At none of the hyperthermic temperatures was the incidence of tumors in the treated leg different from that induced by X rays alone. The incidence of tumors developing in anatomic sites other than the treated leg was decreased in mice where the leg was exposed to hyperthermia compared to mice where the leg was irradiated. A systemic effect of local hyperthermia is suggested to account for this observation. In mice given single X-ray doses and hyperthermia, temperatures of 37, 39, or 41 degrees C did not influence radiation damage as measured by the acute skin reactions. A hyperthermic temperature of 43 degrees C potentiated the acute radiation reaction (thermal enhancement factor 1.1). In the group subjected to hyperthermic temperatures of 37 or 39 degrees C and X rays given in six fractions, the skin reaction was no different from that of the group receiving X rays alone. Hyperthermic temperatures of 41 and 43 degrees C resulted in a thermal enhancement of 1.16 and 1.36 for the acute skin reactions. From Day 50 to Day 600 after treatment, the skin reactions showed regular fluctuations with a 150-day periodicity. Following a fractionated schedule of combined hyperthermia and X rays, late damage to the leg was less than that following X irradiation alone. Mice subjected to X rays and hyperthermic temperatures of 41 and 43 degrees C had a lower median survival time than the mice treated with hyperthermia alone. This effect was not associated with tumor incidence.     

The effects of hyperthermia and ionizing radiation in normal and ataxia telangiectasia human fibroblast lines

The effects of 45 degrees C hyperthermia and gamma radiation have been studied in three normal human fibroblast lines (GM38, GM730, WI38) and compared to the effects in two lines derived from patients with the hereditary disease ataxia telangiectasia (AT3BI, AT5BI). All lines, both normal and gamma-sensitive AT, showed a similar resistance to killing by heat alone, suggesting that the defect responsible for the increased radiation sensitivity in AT lines does not confer increased heat sensitivity. Shouldered survival curves were obtained in each case indicating the ability to accumulate sublethal heat damage. All normal and AT cell lines exhibited increased resistance to the lethal effects of heat in response to a thermal stress, indicating that the defect that causes radiosensitivity in AT cell lines does not prevent the induction of thermotolerance. Heat (45 degrees C, 30 min) was shown to increase the sensitivity of the normal cell lines to killing by gamma radiation. The thermal enhancement ratios obtained ranged from about 2.5 to 3.0. The same heat treatment, however, produced very little increase in the radiation sensitivity of the AT cells. Thermal enhancement ratios of about 1.2 were obtained in these lines. We hypothesize that, in normal cells, this heat treatment inactivates the process which is already defective in AT lines, and that this process may be required for the proper rejoining of double-strand breaks produced during the repair of other radiation-induced lesions.     

Hyperthermic potentiation of unrejoined DNA strand breaks following irradiation

Previous reports have suggested that the potentiation of cellular radiation sensitivity by hyperthermia may be due to its inhibition of the repair of single-strand breaks in DNA. Such inhibition could result in increased numbers of unrejoined breaks at long times following irradiation, lesions that are presumed to be lethal to the cell. As a test of this hypothesis, the amounts of residual strand-break damage in cells following combined hyperthermia and ionizing radiation were measured. The results show that hyperthermia does significantly enhance the relative number of unrejoined strand breaks as measured by the technique of alkaline elution and that the degree of enhancement is dependent on both the temperature and duration of the hyperthermia treatment. For example, compared to unheated cells, the proportion of unrejoined breaks measured 8 hr after irradiation was increased by a factor of 1.5 in cells that were treated for 30 min at 43 degrees C, by a factor of 6 for cells treated for 30 min at 45 degrees C, and by a factor of 4 for cells treated at 43 degrees C for 2 hr. In experiments in which the sequence of heat and irradiation were varied, a high degree of correlation was observed between the resulting level of cell killing and the relative numbers of unrejoined strand breaks. The greatest effects on both of these parameters were observed in those protocols in which the irradiation was delivered either during, just before, or just after the heat treatment.     

Inhibitors of poly(ADP-ribose) synthetase enhance the cytotoxicity of 42 degrees C and 45 degrees C hyperthermia in cultured Chinese hamster cells

Two inhibitors of poly(ADP-ribose) synthetase, 5-methylnicotinamide and m-methoxybenzamide, enhanced the cytotoxicity of 42 degrees C and 45 degrees C hyperthermia in cultured Chinese hamster V79 cells. The inhibitors showed minimal toxicity for cells treated at 37 degrees C, and did not appreciably alter cellular ATP levels under any of the experimental conditions used. Enhanced cell killing occurred when the inhibitors were added after an acute (5-10 min) 45 degrees C heat shock, and after 50 and 100 min exposures to 42 degrees C. When present during heating at 42 degrees C, the inhibitors reduced the shoulder of the 42 degrees C survival curves but did not appreciably affect the slopes. The results suggest a possible role for poly(ADP-ribose) synthetase in the survival response of V79 cells to hyperthermia.     

Thermal enhancement of the effectiveness of gamma radiation for induction of reproductive death in cultured mammalian cells.

The induction by gamma radiation of reproductive death in cultured cells derived from a rat ureter carcinoma (RUC-2) and from Chinese-hamster lung tissue (CH-V79) was shown to be enhanced by hyperthermic treatments at 41, 43 and 45 degrees C. The degree of enhancement was found to depend on the line of cells studied, the temperature employed and the level of damage considered. The influence of accumulating sublethal damage was decreased by hyperthermia, and the final slope of the radiation survival curve was increased. The degree of enhancement of lethal damage was found to depend on the time interval between the heat treatment and irradiation, especially at 41 degrees C.     

Effect of thermotolerance on thermal radiosensitization in hepatoma cells

The interaction between hyperthermia and X irradiation was determined in cultured Reuber H35 hepatoma cells with different states of thermosensitivity. Incubation at 41 degrees C followed by 4-Gy X rays resulted after 2 hr in a stabilization of cell survival for heat or plus X rays, with a maximum synergism factor of 1.6. Thermotolerance did not develop during incubation at 41.7 or 42.5 degrees C. When heat treatment of cells was followed by irradiation, the synergism factor for thermal radiosensitization increased with both the amount of thermal cell killing and the amount of X-ray cell killing; the influence of thermal exposure on the synergism factor was greater than that of the X-ray dose. Cells were made thermotolerant either by incubation at 42.5 degrees C for 30 or 60 min followed by an interval at 37 degrees C, or by continuous incubation at 41 degrees C. In both cases thermotolerance was measured by incubation at 42.5 degrees C. No difference was observed between the maximum thermotolerance achieved with both methods. When cells were irradiated in addition to the second heat treatment, thermal radiosensitization was strongly reduced concomitant with the decreased sensitivity to killing by heat.     

Hyperthermic killing and hyperthermic radiosensitization in Chinese hamster ovary cells: effects of pH and thermal tolerance

To quantitatively relate heat killing and heat radiosensitization, asynchronous or G1 Chinese hamster ovary (CHO) cells at pH 7.1 or 6.75 were heated and/or X-irradiated 10 min later. Since no progression of G1 cells into S phase occurred during the heat and radiation treatments, cell cycle artifacts were minimized. However, results obtained for asynchronous and G1 cells were similar. Hyperthermic radiosensitization was expressed as the thermal enhancement factor (TEF), defined as the ratio of the D0 of the radiation survival curve to that of the D0 of the radiation survival curve for heat plus radiation. The TEF increased continuously with increased heat killing at 45.5 degrees C, and for a given amount of heat killing, the amount of heat radiosensitization was the same for both pH's. When cells were heated chronically at 42.4 degrees C at pH 7.4, the TEF increased initially to 2.0-2.5 and then returned to near 1.0 during continued heating as thermal tolerance developed for both heat killing and heat radiosensitization. However, the shoulder (Dq) of the radiation survival curve for heat plus radiation did not manifest thermal tolerance; i.e., it decreased continuously with increased heat killing, independent of temperature, pH, or the development of thermotolerance. These results suggest that heat killing and heat radiosensitization have a target(s) in common (TEF results), along with either a different target(s) or a difference in the manifestation of heat damage (Dq results). For clinical considerations, the interaction between heat and radiation was expressed as (1) the thermal enhancement ratio (TER), which is the dose of X rays alone divided by the dose of X rays combined with heat to obtain an isosurvival, e.g., 10(-4), and (2) the thermal gain factor (TGF), the ratio of the TER at pH 6.75 to the TER at pH 7.4. Since low pH reduced the rate of development of thermal tolerance during heating at low temperatures, low pH enhanced heat killing more at 42-42.5 degrees C than at 45.5 degrees C where thermal tolerance did not develop. Therefore, the increase in the TGF after chronic heating at 42-42.5 degrees C was greater than after acute heating at 45.5 degrees C, due primarily to the increase in heat killing causing an even greater increase in heat radiosensitization. These findings agree with animal experiments suggesting that in the clinic, a therapeutic gain for tumor cells at low pH may be greater for temperatures of 42-42.5 degrees C than of 45.5 degrees C.     

Effects of gamma radiation and hyperthermia on DNA repair synthesis and the level of NAD+ in cultured human mononuclear leukocytes

DNA repair has been investigated, estimated by unscheduled DNA synthesis (UDS) and the cellular NAD+ pool, after exposing human mononuclear leukocytes to hyperthermia and gamma radiation separately and in combination. It was found that gamma radiation induced a decline in UDS with increasing temperature through the temperature region studied (37-45 degrees C). At 42.5 degrees C the gamma-ray-induced UDS was reduced to about 70% of that at 37 degrees C. Following gamma-ray damage the NAD+ pool dropped to about 20% of control values. Without hyperthermic treatment the cells completely recovered to the original level within 5 hr. Moderate hyperthermia (42.5 degrees C for 45 min) followed by gamma-ray damage altered the kinetics so that even after 8 hr the NAD+ pool had recovered to only 70% of the original level. After heat treatment at 44 degrees C for 45 min prior to gamma radiation the cells did not recover at all, presumably because of the cytotoxic effects from the combined treatment.     

Enhancement of cell killing by induction of apoptosis after treatment with mild hyperthermia at 42 degrees C and cisplatin.

Ohtsubo, T., Igawa, H., Saito, T., Matsumoto, H., Park, H. J., Song, C. W., Kano, E. and Saito, H. Enhancement of Cell Killing by Induction of Apoptosis after Treatment with Mild Hyperthermia at 42 degrees C and Cisplatin. Radiat. Res. 156, 103-109 (2001).We examined the interactive effects of cisplatin (1.0 microg/ml) combined with hyperthermia on cell killing and on the induction of apoptosis in IMC-3 human maxillary carcinoma cells. The cytotoxic effects of hyperthermia on IMC-3 cells at 44 degrees C were greater than at 42 degrees C, as has been reported for many other cells. The induction of apoptosis, DNA fragmentation and poly(ADP-ribose) polymerase cleavage were greater after hyperthermia at 44 degrees C for 30 min compared with treatment at 42 degrees C for 105 min, even though both of these heat doses were isoeffective in reducing cell survival to 50%. Treatment with cisplatin at 37 degrees C for up to 120 min did not result in cytotoxicity or the induction of apoptosis. The enhancement ratio for treatment with cisplatin at 42 degrees C was greater than that at 44 degrees C. More apoptosis was induced after the treatment with cisplatin at 42 degrees C compared to treatment with cisplatin at 44 degrees C. Taking these findings together, the combination of cisplatin and hyperthermia at 42 degrees C appeared to be more effective than cisplatin with hyperthermia at 44 degrees C for the induction of apoptosis in IMC-3 cells.     

Heterogeneity in radiosensitization by heat of cloned cell lines derived from a single human melanoma xenograft

Heterogeneity in radiosensitization by heat was studied using one uncloned and five cloned cell lines isolated from a single tumour of a human melanoma xenograft. Cells from passages 7-12 in vitro were given heat treatments of 42.5 degrees C (45 min), 43.5 degrees C (45 min) or 44.5 degrees C (45 min) immediately after exposure to graded doses of radiation. The survival curves after irradiation alone had similar D0 values but differed in the size of the shoulder. The heterogeneity in heat radiosensitization was reflected in differences in decrease of the D0 values. The thermal enhancement ratios, calculated from the D0 values, were in the ranges 1.2 +/- 0.2-1.7 +/- 0.2 (42.5 degrees C), 1.4 +/- 0.3-2.4 +/- 0.4 (43.5 degrees C) and 2.3 +/- 0.4-3.4 +/- 0.4 (44.5 degrees C). Moreover, at 43.5 degrees C the heterogeneity was also reflected in different modifications of the shape of the survival curves. Two lines showed survival curves with a significant shoulder and a relatively low D0 value whereas two other lines had lost the shoulder almost completely but showed relatively high D0 values. All lines showed survival curves with a broad shoulder after heating at 42.5 degrees C, whereas none of the lines showed survival curves with a significant shoulder after heating at 44.5 degrees C.     

Ultrastructural study of mitochondrial damage in CHO cells exposed to hyperthermia

A unique direct-view stereo electron microscope technique was used to visualize the structure and three-dimensional distributions of mitochondria in CHO cells in situ following hyperthermic treatments. Aberrations induced by various heating regimens were recorded. The protocol included a trypsin digestion that may have enhanced the expression of the initial heat damage. The developed damage was observed as increasing levels of mitochondrial distortion, swelling, and dissociation. Minimal damage was induced at 42 degrees C for exposures of up to 4 h, while significant damage was induced at 43 degrees C for exposures of more than 30 min and at 45 degrees C for exposures of more than 10 min. For moderate exposures, a partial recovery of mitochondrial integrity was observed when the heat treatment was followed by incubation at 37 degrees C for 24 h. Mitochondrial damage was related to the heat dose in that increasing treatment temperature resulted in greater damage, but when compared to cell survival the damage did not parallel cell killing under all time-temperature conditions.     

DNA lesions in hyperthermic cell killing: effects of thermotolerance, procaine, and erythritol

When HeLa S3 cells were subjected to 45 degrees C hyperthermia, DNA lesions were detected by the use of the alkaline unwinding/hydroxylapatite method. The number of lesions formed was not affected when the cells were made thermotolerant by either an acute (15 min 44 degrees C + 5 h 37 degrees C) or a chronic (5 h 42 degrees C) pretreatment before 45 degrees C hyperthermia. The presence of 10 mM procaine (heat sensitizer) or 0.5 M erythritol (heat protector) during hyperthermia also had no effect on the rate of formation of heat-induced alkali labile DNA lesions. These observations do not support a concept where DNA lesions are considered to be the ultimate cause of hyperthermic cell killing. Both drugs, however, influenced the rate of repair of radiation-induced strand breaks when present during preirradiation heat treatment. We conclude that the initial number of heat-induced alkali labile DNA lesions is not directly related to cell survival. It cannot be excluded, however, that differences in posthyperthermic repair of these lesions may lead to a positive correlation between residual DNA damage and survival after the different experimental conditions.     

Effect of hypothermia on cell kinetics and response to hyperthermia and X rays

Hyperthermia is a potent radio enhancer. Studies using hypothermia in combination with irradiation have given confusing results due to lack of uniformity in experimental design. This report shows that hypothermia might have potential significance in the treatment of malignant cells with both thermo- and radiotherapy. Reuber H35 hepatoma cells, clone KRC-7 were used to study the effect of hypothermia on cell kinetics and subsequent response to hyperthermia and/or X rays. Cells were incubated at 8.5 degrees C or between 25 and 37 degrees C for 24 hr prior to hyperthermia or irradiation. Hypothermia caused sensitization to both hyperthermia and X rays. Maximum sensitization was observed between 25 and 30 degrees C and no sensitization was found at 8.5 degrees C. At 25 degrees C maximum sensitization was achieved in approximately 24 hr, cell proliferation was almost completely blocked, and cells gradually accumulated in the G2 phase of the cell cycle. In contrast to the effect of hypothermia on either hyperthermia or X rays alone, thermal radiosensitization was decreased in hypothermically pretreated cells (24 hr at 25 degrees C) compared to control cells (37 degrees C). The expression of thermotolerance and the rate of development at 37 degrees C after an initial heating at 42.5 degrees C were not influenced after preincubation at 25 degrees C for 24 hr. The expression of thermotolerance for heat or heat plus X rays during incubation at 41 degrees C occurred in a significantly smaller number of cells after 24 hr preincubation at 25 degrees C. The enhanced thermo- and radiosensitivity in hypothermically treated cells disappeared in approximately 6 hr after return to 37 degrees C.     

Influence of hyperthermia and gamma radiation on ADP-ribosyl transferase, NAD+, and ATP pools in human mononuclear leukocytes

Effects of hyperthermia (42.5 degrees C) and gamma radiation (30 Gy) on ADP-ribosyl transferase, NAD+, and ATP pools in human mononuclear leukocytes have been investigated. It was found that the gamma-ray activation level of the enzyme was not influenced by this hyperthermia for 45 min. Following deprivation of ATP synthesis by 2,4-dinitrophenol, an uncoupler of the oxidative phosphorylation, and omitting glucose from the culture medium, the NAD+ pool was reduced to about 60% of control value. The potentiation of ATP production by exogenously supplied adenosine was reduced after a combined treatment of the cells with hyperthermia and gamma radiation. Mitochondrial and endoplasmic changes within the mononuclear leukocytes were also observed. Based on these findings a model for the hyperthermia effect is proposed.     

Effects of cycloheximide on thermotolerance expression, heat shock protein synthesis, and heat shock protein mRNA accumulation in rat fibroblasts.

A single hyperthermic exposure can render cells transiently resistant to subsequent high temperature stresses. Treatment of rat embryonic fibroblasts with cycloheximide for 6 h after a 20-min interval at 45 degrees C inhibits protein synthesis, including heat shock protein (hsp) synthesis, and results in an accumulation of hsp 70 mRNA, but has no effect on subsequent survival responses to 45 degrees C hyperthermia. hsp 70 mRNA levels decreased within 1 h after removal of cycloheximide but then appeared to stabilize during the next 2 h (3 h after drug removal and 9 h after heat shock). hsp 70 mRNA accumulation could be further increased by a second heat shock at 45 degrees C for 20 min 6 h after the first hyperthermic exposure in cycloheximide-treated cells. Both normal protein and hsp synthesis appeared increased during the 6-h interval after hyperthermia in cultures which received two exposures to 45 degrees C for 20 min compared with those which received only one treatment. No increased hsp synthesis was observed in cultures treated with cycloheximide, even though hsp 70 mRNA levels appeared elevated. These data indicate that, although heat shock induces the accumulation of hsp 70 mRNA in both normal and thermotolerant cells, neither general protein synthesis nor hsp synthesis is required during the interval between two hyperthermic stresses for Rat-1 cells to express either thermotolerance (survival resistance) or resistance to heat shock-induced inhibition of protein synthesis.     

Effects of m-aminobenzamide on the response of Chinese hamster cells to hyperthermia and/or radiation

The modifying effects of m-aminobenzamide (m-ABA), an inhibitor of poly(ADP-ribose) synthesis, on 42 degrees C hyperthermia- and/or radiation-induced cell killing were examined in Chinese hamster V-79 cells. When cells were exposed to 42 degrees C hyperthermia in combination with m-ABA (10 mM), cell survival decreased compared with that for 42 degrees C hyperthermia alone. Thermosensitizing effects of m-ABA changed with treatments in a decreasing order of during and after heating greater than during heating greater than after heating. Treatments with m-ABA during and/or after X irradiation enhanced radiation-induced cell killing. When cells were exposed to combined treatment with X irradiation, 42 degrees C hyperthermia (60 min), and m-ABA (24 hr), cell survival decreased markedly compared with that for X irradiation alone. However, with both X----42 degrees C and X----42 degrees C----m-ABA, the enhancement ratios (ER), designated as D0 ratio, were similar. These results suggest that the mechanisms of radiosensitization by m-ABA may be similar to those of 42 degrees C hyperthermia.     

Hyperthermic inactivation of diploid yeast and the interaction of damage caused by hyperthermia and ionizing radiation.

Inactivation of diploid yeast by hyperthermia has been studied. DO and Dq decrease with temperature for euoxic and anoxic conditions. The Arrhenius plot shows a break at 52 degrees C; the inactivation energies above and below this temperature are 153 and 94kcal/mol respectively. Hyperthermia (20 min at 51 degrees C) also potentiates the lethal action of gamma rays in diploid yeast cells under both euoxic and anoxic conditions. The interaction between hyperthermic and radiation damage appears to be largely at the sublethal level. The euoxic cells, the hyperthermic potentiation decreases with increasing time between the application of hyperthermia and radiation, being completely lost after 24 hours. However, in the anoxic cells there was no decrease in the hyperthermic potentiation with increasing time interval. These results suggest that yeast cells are capable of repairing hyperthermic sublethal damage, but require oxygen to do so. Thus there is a similarity in the process of repair of sublethal damage caused by ionizing radiation on the one hand and hyperthermia on the other.     

Hyperthermia-induced modulation of killing and mutation by UV and N-methyl-N'-nitro-N-nitrosoguanidine in V79 cells

Hyperthermic exposures of V79 cells did not affect the killing by UV light, whereas it enhanced MNNG-induced killing. Such hyperthermic exposure increased the mutation induction (resistance to 6-thioguanine) by both UV and MNNG. The timing of heat exposure, before or after the treatments, had no effect on the result in cases of cytotoxicity and mutagenesis.     

Relative biological effectiveness of the alpha-particle emitter (211)At for double-strand break induction in human fibroblasts

The purpose of this study was to quantify and to determine the distribution of DNA double-strand breaks (DSBs) in human cells irradiated in vitro and to evaluate the relative biological effectiveness (RBE) of the alpha-particle emitter (211)At for DSB induction. The influence of the irradiation temperature on the induction of DSBs was also investigated. Human fibroblasts were irradiated as intact cells with alpha particles from (211)At, (60)Co gamma rays and X rays. The numbers and distributions of DSBs were determined by pulsed-field gel electrophoresis with fragment analysis for separation of DNA fragments in sizes 10 kbp-5.7 Mbp. A non-random distribution was found for DSB induction after irradiation with alpha particles from (211)At, while irradiation with low-LET radiation led to more random distributions. The RBEs for DSB induction were 2.1 and 3.1 for (60)Co gamma rays and X rays as the reference radiation, respectively. In the experiments studying temperature effects, nuclear monolayers were irradiated with (211)At alpha particles or (60)Co gamma rays at 2 degrees C or 37 degrees C and intact cells were irradiated with (211)At alpha particles at the same temperatures. The dose-modifying factor (DMF(temp)) for irradiation of nuclear monolayers at 37 degrees C compared with 2 degrees C was 1.7 for (211)At alpha particles and 1.6 for (60)Co gamma rays. No temperature effect was observed for intact cells irradiated with (211)At. In conclusion, irradiation with alpha particles from (211)At induced two to three times more DSB than gamma rays and X rays.     

Growth factors and hyperthermia. I. The relationship between hyperthermic cell killing and the mitogenic response to serum and growth factors

Chinese hamster ovary HA-1 cells were tested for their ability to respond to mitogenic stimulation after hyperthermia at 45 degrees C. Cells were arrested by 24 h incubation in serum-free Eagle's MEM. Heating of arrested cells in serum-free medium did not alter heat sensitivity compared to exponentially growing cells heated in serum-containing medium. After hyperthermia cells exhibited a delay in the ability to undergo mitogenesis. Recovery of the capacity for mitogenesis occurred during the 24 h following heating and was able to take place in the absence of serum. After recovery in serum-free medium, cells were simultaneously assayed for survival and mitogenesis as measured by [3H]Thy uptake. With increasing heating time, surviving fraction and mitogenesis decreased. The reduction in survival was similar to the reduction in [3H]Thy incorporation. The relationship between mitogenesis and cell death was studied in more detail with flow cytometry. At a relatively mild heat dose of 30 min at 45 degrees C (survival = 30%), a small population of cells (9%) was found to be clonogenically dead yet capable of being stimulated to progress from G1 to G2-M. At a more severe heat dose of 40 min at 45 degrees C (survival = 3%), stimulation of dead cells could not be detected. Therefore, hyperthermia impairs mitogenic ability, but at low heat doses, a subpopulation of killed cells can still be stimulated to progress through the cell cycle.