Winner of the Lund Science Award 1992. Thermosensitization induced by step-down heating. A review on heat-induced sensitization to hyperthermia alone or hyperthermia combined with radiation.

A few minute's exposure to a high temperature (sensitizing treatment, ST) may substantially increase the cytotoxic and the radiosensitizing effect of a subsequent heating at a lower temperature (test treatment, TT). This phenomenon, which is known as step-down heating (SDH) or thermosensitization, has been observed both in cultured cells in vitro and in tumours and normal tissues in vivo. The effect of SDH increases with a lowering of TT temperature, but it is rapidly lost at temperatures very close to 37 degrees C. SDH-induced thermosensitization decays within a few hours, when an interval is inserted between ST and TT. In vitro results suggest an exponential decay of the SDH effect with half times ranging from 1.5- to 3.1 h. The effect of SDH increases with increasing ST time or temperature. For single heating, the Arrhenius plot is biphasic with activation energies of 500-800 and 1200-1700 kJ/mol above and below a break point temperature in the region 42.5-43.0 degrees C, respectively. For SDH, the Arrhenius plot gradually becomes monophasic with increasing severity of ST and it approaches asymptotically to an activation energy of about 400 kJ/mol. The reduction of the activation energy depends on cell survival after the priming ST and not on the specific ST heating time or temperature. SDH strongly enhances hyperthermic radiosensitization with a 5-6-fold reduction of the radiation dose required to achieve tumour control. The thermosensitizing and the radiosensitizing effects of SDH have several features in common. Both effects become more prominent when the TT temperature is decreased and when the ST heating time or temperature increases. In addition, the decay kinetics for both effects are comparable. For heat alone, the effect of SDH in tumour and normal tissue seems to be quantitatively similar. However, the therapeutic ratio may be increased by combining SDH with radiation. Biologically, the critical subcellular targets involved in the SDH effect have not been revealed. However, the ability of SDH to inhibit the clearance of heat-induced aggregation of proteins in the nucleus is interesting. Blockage of the nuclear function by proteins is a central theory in the present molecular biological models for both cell kill by heat and heat radiosensitization. Clinically, SDH may be an advantage since even a short exposure to high temperature increases the effect of an otherwise inadequate heat treatment. The disadvantages are that SDH complicates thermal dose calculations, and may cause unacceptable damage to normal tissue.     

Step-down heating in a C3H mammary carcinoma in vivo: Effects of varying the time and temperature of the sensitizing treatment

The effect of step-down heating (SDH), consisting of an initial sensitizing treatment (ST) performed at either 44·5°C or 43·5°C followed by a lower temperature test treatment (TT), was investigated in a C3H mammary carcinoma in vivo. A linear relationship between heating time and tumour growth delay was observed for all temperature combinations applied. At a given TT temperature, SDH increased the slope of the dose-response curve compared to the curve for tumours, single-heated without an initial ST. The slope of the SDH curves increased asymptotically towards a plateau value as the ST time at 44–5°C was increased. The time-temperature relationship for single heating was described by a biphasic Arrhenius curve with activation energies of 1361±34 and 666±54 kJ/mol below and above an inflection point at 42·5°C, respectively. For SDH, the Arrhenius curve gradually became straight with increasing ST time, and the activation energy saturated at a value of 425 ±25 kJ/mol. The reduction of the activation energy at an ST temperature of 43·5°C was due rather to the extent of ST heat damage than to the ST time or temperature used. These results may be relevant for calculations of thermal doses, since even a short temperature peak (e.g. 44·5°C/5 min) significantly changed the time-temperature relationship.     

Time-temperature relationships for step-down heating in normal and thermotolerant cells

Normal and thermotolerant H35 cells were submitted to step-down heating (SDH). SDH can significantly reduce the induction and expression of thermotolerance. For SDH a sensitizing treatment (ST) at 44 · 6°C was followed by a test treatment (TT) at a lower hyperthermic temperature. The comparison between the thermotolerant and non-thermotolerant condition was based on isosurvival ST doses. For both conditions dose-effect relationships were obtained by plotting the ST-surviving fraction against the D₀ of a TT. The TT was at either 41 or 42–5°C, representing respectively, a permissive or a non-permisive condition for chronic induction of thermotolerance (CIT). The complex dose-effect relationships are partly exponential. In non-thermotolerant cells tested at 42–5°C the dose-effect relationship between ST and TT is relatively weak. At 41°C, however, the expression of CIT is strongly inhibited after a ST that kills < 20% of the cells. At higher ST doses the response is comparable with that at 42–5°C. In thermotolerant cells a high degree of thermosensitization is also observed for relatively low ST doses, but in contrast with non-thermotolerant cells a stronger dose-effect relationship remains at the higher ST doses. Ultimately this results in a comparatively higher degree of thermosensitization that can be achieved in non-thermotolerant cells. For example, at an isosurviving fraction of 0–15 the reduction of D₀ in non-thermotolerant cells at 42 · 5°C is less than five times, whereas in thermotolerant cells, the D₀ reduction is between 40 and 50 times. A similar reduction is found in non-thermotolerant cells tested at 41 °C. Subsequently, an isosurvival ST dose of about 40% was used in combination with a TT that was varied between 39 and 44°C. D0's were plotted in an Arrhenius diagram to obtain a time-temperature relationship for the effect of SDH on thermotolerant and non-thermotolerant cells. The four plots are all biphasic with a downward inflection. Thermotolerance causes an upward shift of the inflection point of 2°C relative to single-heated cells, whereas SDH causes a downward shift of 1 °C in single-heated cells and of 2°C in thermotolerant cells. For most of the temperature range, i.e. 39–43 · 5°C, SDH decreases the activation energies.     

Factors of importance for the development of the step-down heating effect in a C3H mammary carcinoma in vivo

The effect of step-down heating (SDH) was investigated in a C3H mammary carcinoma inoculated into the feet of CDF1 mice. The SDH effect was evaluated by comparing slopes of time versus growth delay curves of SDH-heated with the curve for single-heated controls. The effect was quantified by a ratio: 'step-down ratio' (SDR), defined as slope (SDH-heated)/slope (single-heated). Step-down heating resulted in thermosensitization in contrast to step-up heating which did not affect the heat sensitivity. The kinetics of the step-down heating effect was investigated by inserting an interval between a 44.5 degrees C/10 min sensitizing treatment (ST) and a 42.0 degrees C test treatment (TT). The effect of SDH was maximal with no interval between ST and TT (SDR = 2.3), decayed within 2 h and turned into thermotolerance. This thermotolerance was maximal after 12 h and decayed within 120 h. The effect of varying the TT temperature was investigated in the range 39.0-44.5 degrees C (ST = 44.5 degrees C/10 min). Below 42.5 degrees C the SDR value increased exponentially, and even a 39 degrees C TT produced a significant heat damage. An Arrhenius analysis was made showing a straight line in the whole temperature range with an activation energy of 526 kJ/mol and an increased activation entropy. These data show that thermosensitization can be induced by SDH in C3H mammary carcinomas in vivo. The effect seems to decay within 2 h, and by decreasing the heat activation energies the effect of low temperature heating is increased.     

Effect of step-down heating on hyperthermic radiosensitization in an experimental tumor and a normal tissue in vivo

The effect of step-down heating (SDH) on the radiosensitization induced by simultaneous hyperthermia and radiation was investigated in a C3H mammary carcinoma inoculated into the feet of CDF1 mice and the skin of normal CDF1 feet. SDH consisted of a sensitizing treatment (ST) of 44.5 degrees C/10 min followed by a test treatment (TT) of 41.5 degrees C for 30, 60 or 120 min. Simultaneous administration of radiation and hyperthermia was achieved by delivering radiation in the middle of the TT. The endpoint selected was the radiation dose needed to achieve either tumor control or moist desquamation in 50% of the animals. The results were evaluated by the thermal enhancement ratio (TER), defined as dose of radiation needed to achieve endpoint in relation to dose of combined radiation and hyperthermia needed to achieve the endpoint. SDH of tumors increased the TER significantly compared with step-up heating (SUH). The ratios between TCD50 values for corresponding SDH and SUH increased with TT heating time and at 120 min a 2.5-fold increase in the radiosensitizing effect was achieved. It has previously been shown that SDH alone causes thermosensitization in tumors by decreasing the activation energy. However, the effect was too small to explain the increased radiosensitization observed with SDH. In the normal tissue studies SDH combined with radiation treatment gave a lower TER compared to the SDH tumor results, suggesting a possible therapeutic gain.     

Step-down heating in a C3H mammary carcinoma in vivo: effects of varying the time and temperature of the sensitizing treatment

The effect of step-down heating (SDH), consisting of an initial sensitizing treatment (ST) performed at either 44.5 degrees C or 43.5 degrees C followed by a lower temperature test treatment (TT), was investigated in a C3H mammary carcinoma in vivo. A linear relationship between heating time and tumour growth delay was observed for all temperature combinations applied. At a given TT temperature, SDH increased the slope of the dose-response curve compared to the curve for tumours, single-heated without an initial ST. The slope of the SDH curves increased asymptotically towards a plateau value as the ST time at 44.5 degrees C was increased. The time-temperature relationship for single heating was described by a biphasic Arrhenius curve with activation energies of 1361 +/- 34 and 666 +/- 54 kJ/mol below and above an inflection point at 42.5 degrees C, respectively. For SDH, the Arrhenius curve gradually became straight with increasing ST time, and the activation energy saturated at a value of 425 +/- 25 kJ/mol. The reduction of the activation energy at an ST temperature of 43.5 degrees C was due rather to the extent of ST heat damage than to the ST time or temperature used. These results may be relevant for calculations of thermal doses, since even a short temperature peak (e.g. 44.5 degrees C/5 min) significantly changed the time-temperature relationship.     

Thermosensitization by step-down heating in mouse testis

Step-down heating (SDH) was investigated in mouse testis by giving an initial treatment of 3 min at 43.0°C followed immediately by a treatment in the temperature range 38.0–42.0°C. The dose-response curves for testis weight loss as a function of duration of hyperthermia were compared with those obtained using single-temperature treatments. In all cases the curves were linear, allowing the use of Arrhenius analysis. For single- temperature treatments the Arrhenius relationship showed an inflection at approximately 41°C with a small, but significant, increase in activation energy for hyperthermal temperatures below the transition. SDH increased the thermal sensitivity in this lower range, by approximately 1 °C, but the activation energy was not significantly altered. The results support the view that in vivo thermosensitization by SDH is not due solely to inhibition of development of thermotolerance.     

Arrhenius analysis of single-heated and step-down heated V79 and L cellsin vitro

The response to single heat treatment and Step-down heat (SDH) treatmentin vitro of V₇₉ and L cells was studied. Colony-forming ability was assayed in medium after treatmentin vitro. Time-response curves were established and subjected to Arrhenius analysis. The Arrhenius curves showed inflection points at 43° C for V₇₉ cells and at 42° C for L cells. The activation energies were 145 kcal/mole and 400 kcal/mole above and below 43° C (P<0.05), respectively, for V₇₉ cells, while 160 kcal/mole and 300 kcal/mole above and below 42° C (P<0.05), respectively, for L cells. Thermosensitivity of L cells are markedly higher than V₇₉ cells. Both V₇₉ and L cells were sensitized by SDH. The SDH effect was characterized by a reduction in shoulder (an addition effect to sublethal damage), an increase in slope (thermosensitization), and the delay and disappearance of thermotolerant “tail” for V₇₉ and L cells at 45° C to 40° C and 44° C to 42° C SDH treatment respectively. Particularly, 42° C to 39° C or 42° C to 40° C SDH for L cells resulted in thermosensitization effect up to a factor of 7.1 or 2.7, respectively. The effect was quantified by thermorsensitization ratio (TSR), defined as T₀ single heated/T₀SDH-heated. The relative ratio was much higher for V₇₉ than for L cells. Heat killing with SDH characterized by Arrhenius analysis showed that Step-down heating reduced the activation energy for heat killing more than single heating. The decrease of activation energy for L cells was markedly greater than for V₇₉. These data suggest that greater cellular sensitivity under step-down heating conditions may reflect a different mechanism for cell killing.     

The effect of step-down heating on mouse small intestinal mucosa

Step-down heating (SDH) was investigated in mouse small intestine by giving a primary (conditioning) treatment at or above 43 degrees C followed by a test treatment below 43 degrees C. Crypt dose-response curves following SDH were compared with those obtained using the test treatment alone; the SDH effect was characterized by a reduction in shoulder (an additive effect) and an increase in slope (thermosensitization). The thermosensitization ratio, defined as slope SDH-heated/slope single-heated, was independent of the conditioning temperature but increased to a maximum of approximately three as the duration of conditioning increased. Thermosensitization was eliminated when the conditioning treatment was itself sufficient to cause significant crypt loss and, also, when the interval between the two treatments was 0.5 h or longer. This period was less than that required for either recovery of the 'shoulder' on the crypt dose-response curve or the development of thermotolerance following the primary treatment. Thermotolerance which develops in intestine during prolonged hyperthermia (after approximately 100 min) was not affected by SDH and Arrhenius analysis indicated that the activation energy for temperatures below 43 degrees C was not significantly altered by SDH. In summary, the SDH effect on small intestine, assessed using the crypt loss endpoint, was similar to thermosensitization observed in vitro. However, the lower magnitude of the effect and its complex dependence on the primary heat treatment suggest either that crypt cells respond to SDH in a unique and characteristic manner or that the crypt assay in vivo and reproductive survival in vitro do not reflect the same endpoint.     

Housing temperature influences the pattern of heat shock protein induction in mice following mild whole body hyperthermia

Purpose: Researchers studying the murine response to stress generally use mice housed under standard, nationally mandated conditions as controls. Few investigators are concerned whether basic physical aspects of mouse housing could be an additional source of stress, capable of influencing the subsequent impact of an experimentally applied stressor. We have recently become aware of the potential for housing conditions to impact important physiological and immunological properties in mice.

Materials and methods: Here we sought to determine whether housing mice at standard temperature (ST; 22?°C) vs. thermoneutral temperature (TT; 30?°C) influences baseline expression of heat shock proteins (HSPs) and their typical induction following a whole body heating.

Results: There were no significant differences in baseline expression of HSPs at ST and TT. However, in several cases, the induction of Hsp70, Hsp110 and Hsp90 in tissues of mice maintained at ST was greater than at TT following 6?h of heating (which elevated core body temperature to 39.5?°C). This loss of HSP induction was also seen when mice housed at ST were treated with propranolol, a β-adrenergic receptor antagonist, used clinically to treat hypertension and stress.

Conclusions: Taken together, these data show that housing temperature significantly influences the expression of HSPs in mice after whole body heating and thus should be considered when stress responses are studied in mice.     

Thermosensitization by step-down heating in mouse testis

Step-down heating (SDH) was investigated in mouse testis by giving an initial treatment of 3 min at 43.0 degrees C followed immediately by a treatment in the temperature range 38.0-42.0 degrees C. The dose-response curves for testis weight loss as a function of duration of hyperthermia were compared with those obtained using single-temperature treatments. In all cases the curves were linear, allowing the use of Arrhenius analysis. For single-temperature treatments the Arrhenius relationship showed an inflection at approximately 41 degrees C with a small, but significant, increase in activation energy for hyperthermal temperatures below the transition. SDH increased the thermal sensitivity in this lower range, by approximately 1 degree C, but the activation energy was not significantly altered. The results support the view that in vivo thermosensitization by SDH is not due solely to inhibition of development of thermotolerance.     

Comparative study of thermoradiosensitization by misonidazole and metronidazole in vivo: antitumour effect and pharmacokinetics

Tumour control by local hyperthermia (43–5°C, 30 min) and radiation (20 Gy) given in combination with misonidazole (MISO) or metronidazole (METRO) was studied using FSa-II murine fibrosarcoma. When MISO or METRO (5 mmol/kg) was given 30 min before heat and subsequently treated with radiation, tumour regression was observed for both agents. Radiation dose-response curves for MISO and METRO with heating at 43·5°C for 30 min were identical. Mouse foot reaction was used to evaluate local toxicity following combined heat, a nitroimidazole and radiation treatment. MISO enhanced the magnitude of foot reaction and prolonged the recovery time compared with heat plus radiation controls. There were no observable differences of foot reaction between animals treated with heat plus radiation and those animals treated with heat, radiation and METRO. Pharmacokinetics of the nitroimidazoles heated at 43·5°C for 30 min in FSa-II tumours were investigated as a possible mechanism of thermal sensitization. Local hyperthermia did not alter the pharmacokinetics of METRO. Tumour concentration and tumour/plasma ratio of MISO were slightly decreased during heating. Since the hypoxic metabolism of the nitroimidazoles did not increase significantly during the heat treatment, the thermal enhancement of MISO or METRO radiosensitization cannot be explained by the increase in hypoxic cytotoxicity of the nitroimidazoles at elevated temperature alone. The two nitroimidazoles also were not accumulated in the tumour after heating. Therefore, alternation of pharmacokinetics is not the major mechanism for the thermal enhancement of nitroimidazole radiosensitization. The METRO radiosensitization effect became identical to that of MISO at elevated temperatures is of particular importance in clinical radiosensitization. The very low local and systemic toxicity together with the high efficacy of METRO at elevated temperatures will make it an attractive candidate as a future clinical radiosensitizer.     

Changes in electrical impedance of skeletal muscle measured during hyperthermia

The electrical impedance of rat skeletal muscle was measured from 100 Hz to 40 MHz during the application of typical hyperthermia heating regimens. Trials were performed employing freshly excised tissue heated to target temperatures from 39·5 to 50°C. Abrupt and rapid decreases in the low-frequency β-dispersion occurred very shortly after reaching hyperthermia temperatures. These rapid decreases continued at a rate and to an extent dependent upon the target temperature, and then, as heating continued, abruptly changed to a much slower rate which continued indefinitely. The initial rapid changes were associated with microscopically observed muscle fibre rounding and radial shrinkage, with accompanying increasing interstitial oedema. The subsequent slow changes were associated with a slow histolysis. The time-and temperature-dependence of the rapid resistivity changes evidenced similarities to typical hyperthermia endpoint responses. An Arrhenius analysis of the rate of the resistivity changes yielded a break at 43°C, with activation energies of 36·1 and 58·3 kcal/mol above and below this break. Preliminary in vivo impedance data displayed qualitative similarities to the excised tissue findings.     

Arrhenius relationships from the molecule and cell to the clinic

There are great differences in heat sensitivity between different cell types and tissues. However, for an isoeffefct induced in a specific cell type or tissue by heating for different durations at different temperatures varying from 43–44°C up to about 57°C, the duration of heating must be increased by a factor of about 2 (R value) when the temperature is decreased by 1°C. This same time-temperature relationship has been observed for heat inactivation of proteins, and changing only one amino acid out of 253 can shift the temperature for a given amount of protein denaturation from 46°C to either 43 or 49°C. For cytotoxic temperatures <43–44°C, R for mammalian cells and tissues is about 4–6. Many factors change the absolute heat sensitivity of mammalian cells by about 1°C, but these factors have little effect on Rs, although the transition in R at 43–44°C may be eliminated or shifted by about 1°C. R for heat radiosensitization are similar to those above for heat cytotoxicity, but Rs for heat chemosensitization are much smaller (usually about 1 · 1–1 · 2). In practically all of the clinical trials that have been conducted, heat and radiation have been separated by 30–60 min, for which the primary effect should be heat cytotoxicity and not heat radiosensitization. Data are presented showing the clinical application of the thermal isoeffect dose (TID) concept in which different heating protocols for different times at different temperatures are converted into equiv min at 43°C (EM₄₃). For several heat treatments in the clinic, the TIDs for each treatment can be added to give a cumulative equiv min at 43°C, viz., CEM₄₃. This TID concept was applied by Oleson et al. in a retrospective analysis of clinical data, with the intent of using this approach prospectively to guide future clinical studies. Considerations of laboratory data and the large variations in temperature distributions observed in human tumours indicate that thermal tolerance, which has been observed for mammalian cells for both heat killing and heat radiosensitization, probably is not very important in the clinic. However, if thermal tolerancve did occur in the clinical trials in which fractionation schemes were varied, it probably would not have been detected because with only the 2–3-fold change in treatment time that occurs when comparing one versus two fractions per week, or three versus six total fractions, little difference would be expected in the response of the tumours since both thermal doses were extremely low on the dose-response curve. Data are shown which indicate that in order to test for thermal tolerance in the clinic and to have a successful phase III trial, the thermal dose should be increased about five-fold compared with what has been achieved in previous clinical trials. This increase in thermal dose could be achieved by increasing the temperature about 1 · 5°C (from 39·5 to 41 ·0°C in 90% of the tumour) or by increasing the total treatment time about five-fold. The estimate is that 90% of the tumour should receive a cumulative thermal dose (CEM₄₃) of at least 25; this is abbreviated as a CEM₄₃T₉₀ of 25. This value of 25 compares with 5 observed by Oleson et al. in their soft tissue sarcoma study. Arguments also are presented that thermal doses much higher than the CEM₄₃T₉₀ induce the hyperthermic damage that causes the tumours to respond, and that the minimum CEM₄₃T₉₀ of 25 only predicts which tumours that receive a certain minimal thermal dose in <90% of the regions of the tumours will respond. For example, in addition to a minimal CEM₄₃T₉₀ of 25 a minimum CEM₄₃T₅₀ of about 400 also may be required for a response. Finally, continuous heating for ～2 days at about 41 °C during either interstitial low dose-rate irradiation or fractionated high dose-rate irradiation, which we estimate could give a CEM₄₃ of 75, should be considered in order to enhance heat radiosensitization of the tumour as well as heat cytotoxicity. In order to exploit the use of hyperthermia in the clinic, we need a better understanding of the biology and physiology of heat effects in tumours and various normal tissues. As an example of an approach for mechanistic studies, one specific study is described which demonstrates that damage to the centrosome of CHO cells heated during G₁ causes irregular divisions that result in multinucleated cells that do not continue dividing to form colonies. This may or may not be relevant for heat damage in vivo. However, since normal tissues vary in thermal sensitivity by a factor of 10, similar approaches are needed to describe the fundamental lethal events that occur in the cells comprising the different tissues.     

Effect of chronic thermotolerance on thermosensitization in Chinese hamster ovary cells studied at various temperatures

The effect of chronic thermotolerance on the thermal responses of Chinese hamster ovary (CHO) cells to single and step-down heating was studied. Thermotolerance was induced by pre-heating exponentially growing cells at 39°C for 9 h, followed by test treatments for variable times at temperatures ranging from 39 to 43 °C. In the temperature range studied, the heat sensitivity of thermotolerant CHO cells was characterized by an Arrhenius activation energy of E_a=1175 ± 40 kJ/mol. This value agreed well with E_a = 1180 ± 45 kJ/mol measured after single heating, indicating that the induction of chronic thermotolerance did not affect the activation energy for cell killing by heat. Thermosensitization was studied after a priming treatment at 43°C for 50 min followed by step-down heating at temperatures ranging from 39 to 43°C. The temperature dependence of the thermal response after step-down heating was characterized by an activation energy of E_a =490 ± 17 kJ/mol. When the cells were pre-treated for 1–16 h at 39°C prior to step-down heating (43°C, 50 min, followed by graded exposure to 39–43°C), the activation energy was gradually enhanced and approached E_a = 825 ± 42 kJ/mol for 39 z°C, 16 h. This change in E_a reflects the effect of thermotolerance on the priming treatment at 43°C for 50 min, whereas the effect on the final test treatment resulted in a parallel shift of the Arrhenius curve without changing the slope, indicating that the effect of thermotolerance on the priming and the test treatment is expressed in the Arrhenius diagram in different ways.     

Some peculiarities of the sequential action of heat and ionizing radiation on yeast cells

The dependence of the thermal enhancement ratio after a sequential action of heat and ionizing radiation on the dose and dose rate of ionizing radiation as well as on the temperature and duration of its application was studied for yeast cells. The combined effect of heat and ionizing radiation on cell killing depended on both the sequence of application (i.e. whether heat is applied prior to or following irradiation) and the temperature. The effectiveness of treatment with heat and ionizing radiation was greatly dependent on the duration of heat exposure. For an equal amount of cell killing from heat alone, long action of heat (50°C) was more effective for radiosensitization than a short acute action of high heat (58°C). For heating at 50°C, heating after irradiation produced more radiosensitization than heating before irradiation. However, high heating at 58°C before irradiation gave the same radiosensitization as heating after irradiation. These data confirm similar observations for mammalian cells. The results were interpreted by means of a mathematical model in which the synergistic effect of the sequential application of heat and ionizing radiation results from the additional lethal damage arising from the interaction of sublesions induced by both agents. These sublesions are not lethal after the action of these modalities, each taken alone. The model appears to be appropriate and the conclusions are valid.     

Protein synthesis, thermotolerance and step down heating

The heat sensitivity of mammalian cells is modified by the cells' previous thermal history. If CHO cells are exposed to 42 degrees C or lower, they show resistance to subsequent heating at higher temperatures ("thermotolerance," TT); if the sequence is reversed, then an increased sensitivity is seen ("step-down heating," SDH). There is considerable evidence that protein synthesis is required for the development of tolerance, but nothing is known about the molecular events leading to SDH. We now show that for HA1 cells, the rate of protein synthesis (rPS) is related to both TT and SDH. The rPS of TT cells is 30% higher than of unheated cells. There is only a transient reduction of rPS during the exposure of TT cells to temperatures up to 43 degrees C with recovery occurring during heating. At higher temperatures, the effect is more severe and no recovery is observed. No recovery is seen during heating at 43 degrees C in previously unheated cells. On the other hand, SDH sensitization occurs in unheated cells and only when there is a severe and prolonged inhibition of the rPS (less than 10% of the control value). TT cells do not show SDH and also only show transient rPS reductions. Our results indicate that proteins must be synthesized for the development of TT and that SDH develops primarily as a consequence of the inhibition of the development of TT.     

Evidence for an upper temperature limit for thermotolerance development in L1A2 tumour cells in vitro

The response of LI A2 cells in vitro to water bath hyperthermia was investigated. In previous studies in the temperature range 40–5–45 0°C., an Arrhenius plot for heat killing of L1A2 cells was linear, and thermotolerance did not develop during continuous heating. The present Arrhenius analysis was extended to the range 38–45°C., and the Arrhenius plot was biphasic with an inflection point at 40–5°C., below which the activation energy was significantly increased from 724 to 923 kJ/mol (P< 0 001). In order to test for the development of thermotolerance during continuous heating, the cells were heated in the range 38–41°C for 10 h, followed immediately by a graded test treatment at 42°C. Thermotolerance developed below 40–5°C as shown by an increased D₀ of the 42°C survival curve, but not at 40 5 and 4FC. Preheating for 90min at 42°C followed by a 10 h incubation at 37°C resulted in maximal thermotolerance with a thermotolerance ratio of approximately 4–3, a ratio also obtained if the cells were incubated for 10 h at temperatures of 38–40°C. No thermotolerance was observed at incubation temperatures of 40 5°C and above. Thus, in the L1A2 cells 40°C is the upper temperature permissive for thermotolerance development, and the data support the assumption that the inflection point on the Arrhenius plot reflects the upper limit for thermotolerance development.     

Simultaneous and sequential hyperthermia and radiation treatment of an experimental tumor and its surrounding normal tissue in vivo

In order to optimize the therapeutic effect of a combined hyperthermia-radiation treatment, the influence of sequence and interval between the two modalities on local tumor control and normal tissue damage was studied during variation of heating time and temperature. A C3H mouse mammary carcinoma transplanted into the feet of C3D2F1 mice was used as a model. Local hyperthermia was given to unanesthetized mice by immersion of the tumor-bearing foot into a water bath. Radiation was given either before, during or after heating. After simultaneous treatment an increasing thermal enhancement ratio (TER) was observed with increasing temperatures and/or increasing heating time with TER values ranging from 1.2 at 41° C to about 5 at 43.5° c after a one hour heating. In all simultaneous treatment schedules, the TER values were almost similar in tumor and normal tissue; no improvement in therapeutic gain was observed. Different results were obtained by using an interval between the two modalities. Hyperthermic treatment (42.5° C/60 min) given with intervals up to 24 hours before radiation showed no definitive improvement in therapeutic ratio. However, if radiation was given before the heating, the normal tissue completely recovered from thermal sensitization within 4 hours, whereas a marked thermal enhancement was persistent in the tumor for more than 24 hours. Thus, an increased therapeutic ratio could be obtained if radiation was given before heat and an interval of 4 hours or more was allowed. This improved therapeutic ratio was dependent on the temperature and ranged from about 1.1 at 41.5° C to 2.1 at 43.5° C given for one hour. These data indicate that if both tumor and normal tissue are heated, the optimal tumor effect may not be a hyperthermic radiosensitization, but rather a direct heat killing of radioresistant tumor cells. This special heat sensitivity of radioresistant tumor cells may be explained by the characteristic environmental conditions (e.g. chronic hypoxia and acidity) influencing such cells.     

Thermotolerance in human glioma cells

Human glioma cells held in plateau phase were tested for the development of chronic and acute thermotolerance. Long duration, mild hyperthermia at 39–42°C for up to 48 h showed no development of chronic thermotolerance. Heating at 45°C immediately after mild hyperthermia showed that acute thermotolerance did develop for 40–42°C heating. This thermotolerance developed at about the same rate for the three inducing temperatures (40–42°C) but the decay characteristics were temperature dependent. In fact, for 42°C heating thermosensitization to subsequent 45°C heating was achieved after 48 h of heating. These data show that chronic and acute thermotolerance may be different in human glioma cells.