The response of mouse skin to hyperthermia combined with fast neutrons or X-rays

The effects of hyperthermia combined with fast neutrons (mean energy approximately 7.5 MeV) or X-rays (250 kVp) were studied in the skin of the mouse ear and foot. Hyperthermia was achieved by immersion in water at temperatures of 41.5-43.0 degrees C for 1 hour. The heat treatments used caused no observable tissue injury other than transient erythema but they enhanced the response to both neutrons and X-rays. The enhancement of neutron damage increased as the heating temperature was increased, as is well known for X-rays. When heat was given after irradiation the thermal enhancement ratio (t.e.r.) for neutrons was similar to that for X-rays. When heat was given before irradiation the neutron t.e.r. was less than that for X-rays. Consequently, the relative biological effectiveness of fast neutrons compared with X-rays was not altered by giving heat after irradiation but it was reduced by giving heat before irradiation.     

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.     

Thermal enhancement of the radiation damage in the mouse foot at different heat and radiation dose: influence of thermotolerance

We studied the reaction of the mouse foot after combined X-irradiation and heat treatment. Acute reactions after heat differ from those after irradiation, however, after healing of the lesions, the same symptoms of deformity of the mouse foot remain. Prior heat treatment, 30 min at 43 degrees C, of the foot led to thermotolerance and this thermotolerance resulted in resistance to combined irradiation-heat treatments and hence to a decreased thermal enhancement of radiation effects. Resistance could be observed up to 168 h after prior heat treatment. The development of resistance to combined treatment at higher irradiation dose (15 or 20 Gy) and less severe heating was slower than at lower irradiation dose (10 Gy) and more severe heating. Thermal enhancement was confirmed to be dependent on the sequence of, and the interval between irradiation and heat treatment. When the mouse foot was made thermotolerant by prior heat treatment, thermal enhancement was always reduced, regardless of the sequence, when the combined heat and radiation treatments were given with an interval of less than 12 h. Thermotolerance led to an apparent decrease in the effective temperature employed in a combined treatment equivalent to approximately 1.0 degrees C, at temperatures above 43 degrees C in a 1 h heat treatment.     

Influence of hyperthermia on gamma-ray-induced mutation in V79 cells

Asynchronously growing V79 cells were assayed for mutation induction following exposure to hyperthermia either immediately before or after being irradiated with 60Co gamma rays. Hyperthermia exposures consisted of either 43.5 degrees C for 30 min or 45 degrees C for 10 min. Each of these heat treatments resulted in a survival level of 42%. For all sequences of combined treatment with hyperthermia and radiation, cell killing by gamma rays was enhanced. Mutation induction by gamma rays was enhanced when heat preceded gamma irradiation, but no increase was observed when heat was given after gamma exposures. Treatment at 45 degrees C for 10 min gave a higher yield in mutants at all gamma doses studied compared to treatment at 43.5 degrees C for 30 min. When heat-treated cells were incubated for different periods before being exposed to gamma rays, thermal enhancement of radiation killing was lost after 24 h. In contrast, only 5-6 h incubation was needed for loss of mutation induction enhancement.     

The effect of combined heat and ultrasound on multicellular tumour spheroids

The effect of combined ultrasound and heat treatments on Chinese hamster multicellular spheroids of varying size was investigated using growth rate, single cell survival and ultrastructural damage as endpoints. Ultrasonic irradiation at 37 degrees C had no effect on the growth rate of 200-730 microns spheroids. Similarly there was no effect on the growth rate of 350 microns spheroids when irradiated during a 60 min exposure to 41.5 degrees C. However, spheroids of 200-700 mm diameter showed growth delay when held at 43 degrees C for 1 h. The effect was enhanced with concomitant ultrasound irradiation but was not dependent on spheroid size. When 200 and 400 microns spheroids held at 43 degrees C for 60 min were irradiated with different ultrasonic intensities a dose-dependent decrease in surviving fraction and a dose-dependent increase in growth delay was obtained. When surviving fraction was plotted as a function of growth delay a good correlation was obtained, suggesting that the combination of heat and ultrasound irradiation does not produce cytostasis in the surviving cells of either 200 or 400 microns spheroids. At the ultrastructural level increased cytoplasmic vacuolation was the only result of ultrasonic irradiation at 37 degrees C. Exposure to 43 degrees C for 60 min was required to elicit thermal damage. This took the form of membrane evagination at the spheroid surface, vacuolation of the cytoplasm, grouping of organelles around the periphery of the nucleus, and fragmentation of the nucleolus. These effects were enhanced with concomitant ultrasonic irradiation but other features were also noted, viz. disaggregation of polyribosomes, dilation of the rough endoplasmic reticulum and blebbing of the nuclear membrane. Damage was independent of spheroid size. These results are in agreement with previous data obtained from single-cell studies. Indicating that there is a non-thermal, non-cavitational component to the cell killing in multicellular spheroids resulting from combined heat and ultrasound treatment.     

Radiosensitivity and thermosensitization of thermotolerant Chinese hamster cells and RIF-1 tumors

CHO cells subline HA-1 were made thermotolerant by a priming heat treatment (43 degrees C, 30 min). Later, 4, 16, or 24 hr, they were either irradiated or heated (43 degrees C, 30 min) and irradiated. Thermotolerance had no effect on the radiation sensitivity of the cells as measured by the D0 value of the clonogenic survival curve. However, the N value of the curve (width of shoulder) showed a significant increase at 24 hr, indicating an increased capacity to accumulate sublethal damage. This indicates that the fractionation schedule 43 degrees C, 30 min + 37 degrees C, 24 hr + 43 degrees C, 30 min + X ray required approximately 100 rad more radiation than 43 degrees C, 30 min + X ray to reduce survival to the same level. The same priming treatment was given to RIF-1 tumors growing in C3H mice. Later, 24 hr, when the tumors were either irradiated or heated (43 degrees C, 30 min) and irradiated, it was found that thermotolerance had no effect on the radiosensitivity of the cells as measured by in vitro assay. However, thermal radiosensitization was not apparent 24 hr after the priming treatment.     

Hyperthermia in a differentiating murine erythroleukemia cell line: cell killing by heat and radiation

The survival response of Friend erythroleukemia cells (a differentiating cell system) to heat and radiation has been examined. The Friend erythroleukemia cells (FELC) were more heat and radiation sensitive than V79 cells, and the heat and radiation survival curves possessed shoulders, showing the ability of the cells to accumulate sublethal damage. Thermal tolerance was expressed after prolonged heating at 41.0-42.0 degrees C. Thermal radiosensitization by heating at 42.0 or 45.0 degrees C was greatest for simultaneous heat and radiation treatments, and recovery occurred when the cells were incubated at 37 degrees C between the heat and radiation or radiation and heat treatments. Arrhenius analysis of the FELC heat survival data showed that the curve for thermal inactivation possessed a break at about 43.0 degrees C and that the thermal inactivation energies above and below the break point were comparable to those for V79 cells and other cell lines reported in the literature.     

Influence of prior heat treatment on the effects of heat alone or combined with X-rays on mouse stromal tissue

The tumour bed effect assay was used to study the sensitivity of mouse stromal tissue to heat applied alone or combined with irradiation. Prior heat treatment, 30 min at 43 degrees C, of the tumour bed led to thermotolerance. After priming, thermotolerance developed fully within 24 h and it had disappeared completely after about 10 days. The kinetics of development and decay of thermotolerance in this slowly dividing tissue is similar to that which we had observed previously in skin. When decay rates of several normal tissues with different proliferation characteristics are compared, it is obvious that there is not a clear relationship between proliferation rate of the presumed target cells in the tissue and thermotolerance decay rate.     

Thermotolerance induced by heat, sodium arsenite, or puromycin: its inhibition and differences between 43 degrees C and 45 degrees C

When CHO cells were treated either for 10 min at 45-45.5 degrees C or for 1 hr with 100 microM sodium arsenite (ARS) or for 2 hr with 20 micrograms/ml puromycin (PUR-20), they became thermotolerant to a heat treatment at 45-45.5 degrees C administered 4-14 hr later, with thermotolerance ratios at 10(-3) isosurvival of 4-6, 2-3.2, and 1.7, respectively. These treatments caused an increase in synthesis of HSP families (70, 87, and 110 kDa) relative to total protein synthesis. However, for a given amount of thermotolerance, the ARS and PUR-20 treatments induced 4 times more synthesis than the heat treatment. This decreased effectiveness of the ARS treatment may occur because ARS has been reported to stimulate minimal redistribution of HSP-70 to the nucleus and nucleolus. Inhibiting protein synthesis with cycloheximide (CHM, 10 micrograms/ml) or PUR (100 micrograms/ml) after the initial treatments greatly inhibited thermotolerance to 45-45.5 degrees C in all cases. However, for a challenge at 43 degrees C, thermotolerance was inhibited only for the ARS and PUR-20 treatments. CHM did not suppress heat-induced thermotolerance to 43 degrees C, which was the same as heat protection observed when CHM was added before and during heating at 43 degrees C without the initial heat treatment. These differences between the initial treatments and between 43 and 45 degrees C may possibly be explained by reports that show that heat causes more redistribution of HSP-70 to the nucleus and nucleolus than ARS and that redistribution of HSP-70 can occur during heating at 42 degrees C with or without the presence of CHM. Heating cells at 43 degrees C for 5 hr after thermotolerance had developed induced additional thermotolerance, as measured with a challenge at 45 degrees C immediately after heating at 43 degrees C. Compared to the nonthermotolerant cells, thermotolerance ratios were 10 for the ARS treatment and 8.5 for the initial heat treatment. Adding CHM (10 micrograms/ml) or PUR (100 micrograms/ml) to inhibit protein synthesis during heating at 43 degrees C did not greatly reduce this additional thermotolerance. If, however, protein synthesis was inhibited between the initial heat treatment and heating at 43 degrees C, protein synthesis was required during 43 degrees C for the development of additional thermotolerance to 45 degrees C.(ABSTRACT TRUNCATED AT 400 WORDS)     

Inhibition of repair of X-ray-induced DNA damage by heat: the role of hyperthermic inhibition of DNA polymerase alpha activity

HeLa S3 cells growing in suspension have been used to investigate possible mechanisms underlying the inhibitory action of hyperthermia (44 degrees C) on the repair of DNA strand breaks as caused by a 6-Gy X-irradiation treatment. The role of hyperthermic inactivation of DNA polymerase alpha was investigated using the specific DNA polymerase alpha inhibitor, aphidicolin. It was found that both heat and aphidicolin (greater than or equal to 2 micrograms ml-1) could decrease DNA repair rates in a dose-dependent way. When the applications of heat and aphidicolin were combined, each at nonmaximal doses, no full additivity in effects was observed on DNA repair rates. When the heat and radiation treatment were separated in time by postheat incubation at 37 degrees C, restoration to normal repair kinetics was observed within 8 h after hyperthermia. When heat was combined with aphidicolin addition, restoration of the aphidicolin effect to control level was also observed about 8 h after hyperthermia. It is suggested that although DNA polymerase alpha seems to be involved in the repair of X-ray-induced DNA damage, and although this enzyme is partially inactivated by heat, other forms of heat damage have to be taken into account to explain the observed repair inhibition.     

Thermal sensitivity and radiosensitization in V79 cells after BrdUrd or IdUrd incorporation

Chinese hamster V79 cells were exposed to 10(-5) moles/liter bromodeoxyuridine (BrdUrd) or iododeoxyuridine (IdUrd) for 16 or 29 hr and then tested for thermal sensitivity, radiosensitivity, and sensitivity to the combined treatments of heat and radiation. BrdUrd and IdUrd treatment of cells resulted in enhanced radiosensitivity which increased with exposure time but had little or no effect on thermal sensitivity. For 42.0 degrees C heating, no effect was observed, while for 45.0 degrees C heating, a small decrease in thermal sensitivity occurred for both 16- and 29-hr exposure times, in the combined treatment of heat and radiation, the presence of BrdUrd or IdUrd resulted in about the same thermal enhancement in radiosensitivity. BrdUrd and IdUrd uptake into cellular DNA were measured using high-pressure liquid chromatography and, after a 29-hr exposure to 10(-5) moles/liter of BrdUrd or IdUrd, approximately 40% of the thymidine was substituted.     

Effects of hyperthermia and X-irradiation on mouse stromal tissue

The sensitivity of normal stroma to heat, irradiation and heat combined with irradiation has been studied using the tumour bed effect (TBE) assay. Irradiation before implantation led to a TBE. This TBE was dose dependent below 15 Gy, the TBE remaining relatively constant above 15 Gy. The interval (0-90 days) between irradiation and tumour implantation did not influence the magnitude of the TBE. Hyperthermia with large heat doses (45-60 min at 44 degrees C) before implantation may lead to a TBE. The interval between hyperthermia and tumour implantation proved to be very important. Our results show that the recovery from heat-induced stromal damage is very rapid. When the interval between hyperthermia and tumour implantation is 10 days or longer, no TBE could be observed. Irradiation combined with large heat doses (30-60 min at 44 degrees C) decreased the radiation-induced TBE. However, the combination of irradiation with mild heat treatments (15 min at 44 degrees C) could lead to a larger TBE than after irradiation alone. When hyperthermia was given prior to irradiation, the interval between heat and irradiation proved to be very important. With large intervals (21 days or longer) the TBE values were about the same as with irradiation alone. When heat was given after irradiation it always reduced the irradiation-induced TBE.     

Covalent DNA-protein crosslinking occurs after hyperthermia and radiation

Covalent DNA-protein crosslinks occur in exponentially growing mouse leukemia cells (L1210) after exposure to ionizing radiation. The amount of DNA-protein crosslinks as measured by a filter binding assay is dose dependent upon X irradiation. Although hyperthermia and radiation in combination are synergistic with respect to cell lethality, the combination does not result in an increase of DNA-protein crosslinks when assayed immediately following treatments. Hyperthermia (43 degrees C/15 min) given prior to radiation does not alter the radiation dose dependency of the amount of initial crosslinking. In addition, the amount of DNA-protein crosslinking produced by heat plus radiation is independent of the length of heating the cells at 43 degrees C. The DNA-protein crosslinks produced by 50-Gy X ray alone are removed after 2 hr at 37 degrees C. However, if hyperthermia (43 degrees C/15 min) is given prior to 100-Gy X ray, the removal of DNA-protein crosslinks is delayed until 4.0 hr after radiation. Phospho-serine and phospho-threonine bonds are not produced with either radiation or the combination of hyperthermia plus radiation as judged by the resistance of the bonds to guanidine hydrochloride. However, hyperthermia plus radiation causes an increase in phosphate to nitrogen type bonding. These results show that radiation alone causes covalent DNA-protein crosslinks. Hyperthermia in combination with radiation does not increase the total amount of the crosslinks but delays the removal of the crosslinks and alters the distribution of the types of chemical bonding. These data suggest that the synergistic action on hyperthermia with radiation is more related to the rate of removal and the type of chemical bonding involved in the covalent DNA-protein crosslinks rather than the amount of DNA-protein crosslinks.     

An unexpected effect of hyperthermia on the expression of X-ray damage in mouse skin

The interaction between hyperthermia and X irradiation in the expression of injury to skin was investigated in the tail of adult mice. The X-ray treatments when given alone resulted in skin reactions which ranged in severity from "no observable gross injury" to "moist desquamation over most of the tail," the peak reaction occurring at approximately 20 days. When hyperthermia was given alone, the maximal reaction observed was "foci of moist desquamation, accompanied by severe erythema and edema" which, in contrast to the radiation response, peaked 1 to 2 days after treatment. For the combined treatments, hyperthermia at a temperature between 43.0 and 44.5 degrees C for 30 min was given either 3, 6, 9, or 10 days after X irradiation. When the interval was 3 days, there appeared to be no interaction between the treatments. As the interval was lengthened, so that hyperthermia was given 6 or more days after irradiation, i.e., within 7 days of the time of appearance of gross radiation injury, the severity of the observed skin reaction was greater than the individual responses following either treatment given alone. Using a 9-day interval, it could be seen that both the thermal and radiation reactions were enhanced in a dose-dependent manner. The peak times for each reaction were not significantly altered by the additional treatment. The results are discussed with reference to possible modes of interaction between X irradiation and hyperthermia in an in vivo system.     

Thermal radiosensitization and thermotolerance in cultured cells from a murine mammary carcinoma

Cultured murine mammary carcinoma cells M8013 could be made thermotolerant by a priming heat treatment, 30 min at 43 degrees C, applied 5 h prior to subsequent heat treatment. The sensitivity of non-tolerant and thermotolerant cells to either radiation or heat combined with radiation was investigated. Analysis of survival curves with respect to D0 and N showed that thermotolerance had no influence on the radiation sensitivity of the cells. Thermal enhancement of radiation effects (in combined heat/irradiation treatments) was however reduced as a result of thermotolerance. When thermal enhancement ratios were (D0) plotted as a function of the cell killing effects of heat treatment alone, thermotolerance did not seem to have any influence. This latter observation suggests that thermotolerance modifies the effectiveness of the heat treatment for heat-induced cell lethality and radiosensitization equally. Comparison of our in vitro results with several in vivo data on normal tissues suggest that the reduction in 'effective' treatment temperature which has been observed in the in vivo studies as a result of thermotolerance may be explained by equal modification of the effects of heat by thermotolerance both for its direct effects and the radiosensitization.     

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.     

Thermotolerance and thermosensitization in CHO and R1H cells: a comparative study

In CHO and R1H cells thermotolerance was induced by a pre-incubation at 40 degrees C, by an acute heat shock at 43 degrees C followed by a time interval at 37 degrees C, and during continuous heating at 42 degrees C. Thermotolerance, which was tested at 43 degrees C, primarily causes an increase in D0 of the heat-response curve. The degree of maximum thermotolerance was found to be generally more pronounced in CHO than in R1H cells, but the time interval at 37 degrees C, as well as at 40 degrees C, to reach this maximum level was the same in both cell lines. CHO and R1H cells could be sensitized to 40 degrees C by a pre-treatment at 43 degrees C. When compared for the same survival rate after pre-treatment at 43 degrees C alone the degree of thermosensitization was about the same in both cell lines. In either cell line thermosensitization was found to be suppressed when cells were made thermotolerant by a previous incubation at 40 degrees C for 16 hours.     

Correlation between cellular survival and potassium loss in mouse fibroblasts after hyperthermia alone and after a combined treatment with X rays

Mouse fibroblast LM cells have been heated at 44 degrees C for different periods. Potassium content of the cells was measured at certain intervals during the postheating period at 37 degrees C for up to 24 hr. The level of K+ decreased gradually in time starting within some hours after the heat treatment. The rate of K+ loss as well as the ultimate level reached was heat-dose dependent. When the potassium content of the cell population was determined 16 hr after the heat treatment, a correlation was observed between the concentration of potassium and the level of cell survival. When X irradiation was applied immediately after hyperthermia, radiosensitization on the level of cell survival was obtained as expected, the extent being dependent on the severity of heat treatments. No added K+ loss was observed, however, when hyperthermia was combined with radiation. It is suggested that plasma membrane related functions are disturbed by the heat treatment. This points to membranes as possible candidates for primary targets in the case of cell inactivation by heat alone, and not with respect to the radiosensitization by hyperthermia.     

Cross-regulation of iNOS and COX-2 by its products in murine macrophages under stress conditions.

Exposure of macrophages to heat shock induces rapid synthesis of heat shock proteins (HSPs) which are important for cell homeostasis. Prostaglandins (PGs) and nitric oxide (NO) are important cell regulatory molecules. We have therefore investigated the interactions between these molecules in the LPS-induced expression of iNOS and COX-2 and in the mitochondrial activity of macrophages. Cultures of the murine macrophage cell line, J774, were exposed to heat shock (43 degrees C, 30 min) and stimulated with LPS (1 microg/ml), concomitantly or after 8h of cell recovery. NO production was measured by Griess reaction; PGE(2) by ELISA; HSP70, iNOS and COX-2 by immunobloting; mitochondrial activity by MTT assay. Heat shock induced HSP70, but not iNOS or COX-2 whereas LPS induced iNOS and COX-2 but not HSP70. When heat shock and LPS were given concomitantly, iNOS but not COX-2 expression was reduced. When a period of 8h was given between heat shock and LPS stimulation, iNOS, COX-2, PGE(2) and NO levels were significantly increased. Under these conditions, the expression of COX-2 was reduced by L-NAME (NO-synthesis inhibitor) and of iNOS by nimesulide (PGs-synthesis inhibitor). Such cross-regulation was not observed in cells at 37 degrees C. These treatments significantly reduced MTT levels in cells at 37 degrees C but not in cells submitted to heat shock. These results suggest that HSPs and cross-regulation of iNOS and COX-2 by their products might be of relevance in the control of cell homeostasis during stress conditions.     

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.