Step-down Heating Research Articles

Effects on the expression of heat shock proteins by step-down heating and hypothermia in rat hepatoma cells with a different degree of heat sensitivity.

Thermosensitization induced by pretreatment at supra- and subnormal temperatures, rate of protein synthesis and expression of the major heat shock proteins under such conditions was investigated in relation to intrinsic heat sensitivity of rat hepatoma cells, i.e. Reuber H35 and HTC. The high degree of heat susceptibility of H35 cells was reflected by a high degree of thermosensitization after pretreatment by heat (step-down heating) at temperatures of 42-44 degrees C for 30 min or cold for 16 h at temperatures ranging from 0 to 25 degrees C. Sensitization under step-down heating conditions was found to be paralleled by a delayed recovery of protein synthesis. Despite an increased relative rate, enhancement of the absolute rate of synthesis of the major heat shock proteins, HSP28, HSP60, HSP68, HSP70, HSP84 and HSP100, was less pronounced during step-down exposure. Comparable results were obtained during recovery of sensitized H35 cells at 37 degrees C after exposure to heat following pretreatment at 0 degrees C. Furthermore, clear differences in the regulation of the specific HSP synthesis, depending on the particular treatment protocol, were observed.

Use of nitroprusside to increase tissue temperature during local hyperthermia in normal and tumor-bearing dogs

The present study investigates the effects of nitroprusside, a potent vasodilating agent, on tissue temperature during local hyperthermia in five normal and five tumor-bearing dogs. Caudal thigh muscles were heated in normal dogs and muscle temperatures were recorded during hyperthermia. Tumor-bearing dogs received two hyperthermia treatments during a course of radiation therapy. Temperatures were recorded in tumor and surrounding normal tissues. Mean arterial pressure was decreased by approximately 40–45% during nitroprusside infusion and was associated with a compensatory increase in heart rate and increases in tissue temperature. In normal dogs, muscle temperatures increased an average of 1.7°C with nitroprusside administration. When nitroprusside was administered at the beginning of local hyperthermia to induce step-down heating, approximately 48% of the measured positions in caudal thigh muscle achieved a temperature ≥ 43°C, sufficient to induce step-down heating, during the hyperthermia episode. In tumor-bearing dogs, there was a significant increase in tumor and normal tissue temperatures during nitroprusside administration. Estimated T 90 and T 50 descriptors increased by 0.9°C and 1.6°C, respectively, for tumor tissue and by 0.4°C and 1.2°C, respectively, for normal tissue. Despite the increase in normal tissue temperatures no toxicity was observed in these dogs. Nitroprusside may be a useful agent for manipulation of tumor temperatures during the entire hyperthermia treatment or for a short time period at the initiation of treatment to induce step-down heating.

Significance of additive heat effect in the therapeutic gain factor in combined hyperthermia and radiotherapy: Murine tumor response and foot reaction

The thermal enhancement ratio (TER) and therapeutic gain factor (TGF) were evaluated for combined hyperthermia and radiation treatments of a murine fibrosarcoma, FSa-II. The TER is the ratio of the radiation dose that induces a given reaction without hyperthermia to that with hyperthermia. The TGF is defined as the ratio of TER for tumor response to TER for normal tissue response. Tumors in the subcutaneous tissue of the right foot were irradiated with graded radiation doses when they reached an average diameter of 6 mm (110 mm). Hyperthermia was given by immersing animal feet in a constant temperature water bath 10 min before or after irradiation. The tumor growth time to reach 500 mm 3 was obtained for each tumor and the median tumor growth time was calculated for each treatment group. For the normal tissue study, the non-tumor bearing murine foot was treated, as was the tumor, and the foot reaction was scored after treatment, according to our numerical score system for radiation damage, until the 35th post-treatment day and averaged. Using the fraction of animals showing a given average foot reaction score in a treatment group, the RD 50, or the radiation dose to induce the given foot reaction or greater, was calculated. A single heating at 45.5°C for 10 min and a step-down heating (first heat at 45.5°C for 10 min immediately followed by the second heat at 41.5°C for 60 min) prolonged the tumor growth time, indicating that hyperthermia per se resulted in some cell killing. The prolongation was greater following step-down heating than following single heating. These heat treatments alone induced no noticeable heat damage on the foot, but decreased the threshold dose observed on the radiation dose response curves for the foot reaction. Accordingly, TER and TGF were evaluated with or without normalizing this thermal effect. TER's for both tumor and foot responses without normalization were greater than the TER's after normalization and decreased with increasing radiation dose (between 1.9 and 7.1 or greater for tumor and between 1.3 and 4.3 or greater for foot reaction), whereas the normalized TER's were relatively constant (between 1.6 and 1.7 for tumor and between 0.7 and 1.5 for foot reaction). TGF's without normalization were greater than those obtained after normalization. The former was large at small doses and decreased with increasing radiation dose (between 1.5 and 4.0 or greater), whereas the latter was within 0.8 and 1.3 and relatively independent of radiation dose. TGF's following step-down heating were greater than the TGF following single heating (> 3.0 and between 1.5 and 2.0 for SDH and single heating given before radiation). This greater non-normalized TGF is likely due to the additive effect of hyperthermia, that is, due to the greater thermal effect on the tumor than the normal foot. Similarly, greater TGF's following step-down heating may be due to greater damage, induced by this heating, to the tumor. TGF following hyperthermia given before or after radiation appeared to depend on the heat dose applied.

Inhibition of heat shock protein synthesis and protein glycosylation by stepdown heating

Mammalian cells exhibit increased sensitivity to hyperthermic temperatures of 38–43 °C after an acute high-temperature heat shock; this phenomenon is known as the stepdown heating (SDH) effect. We characterized the SDH effect on (1) the synthesis of major heat shock proteins, HSP110, 90, 72/70, 60 ( 35S-amino acids label), (2) on heat-induced protein glycosylation ( 3H- d-mannose label), and (3) on thermotolerance expression, using cell survival as an endpoint. Partitioning of label between soluble and insoluble cell fractions was separately examined. Synthesis of high molecular weight HSPs (HSP110, 90, and 72/70) was increased both by acute (10 min, 45 °C) and chronic (1–6 h, 41.5 °C) hyperthermia, primarily in the soluble cytosol fraction. SDH (10 min, 45 °C + 1 to 6 h, 41.5 °C) completely inhibited labeling of HSP110, partially inhibited HSP90 labeling, and had virtually no effect on HSP72/70 synthesis, when compared with chronic hyperthermia alone. At the cell survival level, SDH increased sevenfold the rate of cell killing at 41.5 °C, but reduced the expression of thermotolerance by only a factor of two. This suggests that SDH sensitization did not result from changes in HSP72/70 synthesis, nor solely from inhibition of thermotolerance. 35S-labeled HSP60 and HSP50 were found primarily in the cellular pellet fraction after both acute and chronic hyperthermia. SDH completely inhibited 35S-labeling of both HSP60 and HSP50. Labeling of GP50 with 3H- d-mannose was also completely inhibited by SDH. Moreover, SDH progressively reduced N-acetylgalactosaminyl-transferase activity. The data demonstrate that heat sensitization by SDH is accompanied by complex and selectively inhibitory patterns of HSP synthesis and protein glycosylation. Profound inhibition of HSP110, HSP60, and HSP50/GP50 labeling suggests that these may be associated with mechanisms of SDH sensitization.

A Generalized Concept for Cell Killing by Heat: Effect of Chronically Induced Thermotolerance

Chronic thermotolerance was induced in Chinese hamster ovary (CHO) cells by pretreatment at 40 degrees C for various times ranging from 15 min to 16 h. The thermotolerant cells were either exposed to single heat treatments at 43 degrees C or subjected to step-down heating consisting of a priming treatment at 43 degrees C for 90 min followed immediately by a graded test treatment at 40 degrees C. Data evaluation using a mathematical model published previously (H. Jung, Radiat. Res. 106, 56-72, 1986) showed that the rate constants p for the production of nonlethal lesions at 43 and 40 degrees C decreased by a factor of 20 with the duration of the thermotolerance-inducing pretreatment at 40 degrees C and approached exponentially (half-time 1.24 +/- 0.11 h) a minimum value. By contrast, the rate constants c for the conversion of nonlethal lesions into lethal events were not altered by the induction of thermotolerance; this applied to both c (43 degrees C) and c (40 degrees C). After transferring thermotolerant cells (pretreatment at 40 degrees C for 3 h) to 37 degrees C, the rate constants p increased exponentially with time (doubling time 25 +/- 5 h). Similar kinetics for the development and decay of chronic thermotolerance was shown to be applicable also to extended heating at 40 degrees C and to various data from the literature.

Sensitization to hyperthermia induced in a normal tissue by step-down heating

The effect of step-down heating was investigated in the skin of the CDF1 mouse foot. Step-down heating was induced with a 44.7°C 10 min pretreatment followed by a test treatment at a lower temperature for variable time. Step-up heating, that is, a test treatment followed by a 44.7°C 10 min treatment, and single heating were used as controls. The normal tissue reaction was scored at five levels of damage (from slight redness and oedema to loss of a toe or greater reaction), and the heating time to induce each level in 50% of the animals, RD50, was used as the endpoint. The effect of step-down heating was quantified by the step-down ratio, calculated as the ratio of test heating times to obtain the endpoint. A significant reduction of the RD50 was seen at all score levels when the 44.7°C 10 min was given in a step-down heating schedule, and the effect increased with decreasing test treatment temperature. In contrast, the heat sensitivity was only marginally influenced by step-up heating. An analysis of the time-temperature relationship demonstrated a log-linear relationship between temperature and RD50 for single heating in the range 42.2–44.7°C and for step-down heating in the range 41.7–44.7°C. The curve for step-down heating showed a lesser slope indicating a decrease of the activation energy. The kinetics of the SDH effect were investigated by inserting an interval between a primary 44.7°C 10 min treatment and a test treatment performed at 42.2°C. The effect of step-down heating was maximal with no interval between the priming treatment and the test treatment. As the interval was increased to 1.5 hr the step-down sensitization disappeared, and with even longer intervals thermotolerance developed. From a clinical point of view, the present data indicate that step-down heating may increase the extent of both reversible and irreversible heat damage in the normal tissue.

Effect of Step-down Heating on the Interaction between Heat and Radiation in a C3H Mammary Carcinomain Vivo

The effect of step-down heating (SDH) on the interaction between heat and radiation was investigated in a C3H mammary carcinoma in vivo. SDH consisted of an initial sensitizing treatment (ST) performed at 44.5 degrees C or 43.5 degrees C followed by a lower temperature test treatment (TT) in the range 41.0-43.0 degrees C. Step-up heating (SUH), i.e. TT followed by ST, and single heating were used as controls. The end-point was the radiation dose needed to control 50% of the tumours (TCD50). The results were evaluated by calculating the thermal enhancement ratio (TER) defined as TER = TCD50 (radiation alone)/TCD50 (radiation and heat). For a simultaneous application of TT and radiation a significant enhancement of direct heat radiosensitization was observed with increasing ST time or ST temperature using SDH. In contrast, only a minor increase was seen with SUH. A comparison between TCD50 values for the corresponding SUH and SDH schedules revealed that the SDH effect was largest at 41.0-42.0 degrees C and decreased with increasing TT temperature. The radiosensitizing effect of SDH also decreased if an interval was allowed between ST and TT or between TT and radiation. However, as a result of an increased cytotoxicity towards hypoxic tumour cells, the TCD50 value for SDH remained significantly smaller than for SUH, even with a sequential combination of radiation and heat.

A comparison between the effect of step-down heating in a tumour and a normal tissue invivo

A comparison between the effect of step-down heating (SDH) obtained in a C3H mammary carcinoma grown in the feet of CDF1 mice and the skin of normal CDF1 feet is presented. Water-bath heating was used, and SDH was obtained by giving a 44.7 degrees C/10 min treatment followed by heating at 42.2 degrees C for variable times. Single heating at 42.2 degrees C and step-up heating (SUH), i.e. 42.2 degrees C followed by 44.7 degrees C/10 min, were used as controls. The endpoint was the heating time at 42.2 degrees C to obtain either a definite tumour growth time (TGT50) or a specific skin score level (RD50) in 50% of the animals. The effect of SDH and SUH was quantified by the step-down ratio (SDR), calculated as the ratio of the heating times at 42.2 degrees C to obtain the specific endpoint. In both assays the effect of SDH was seen as a significant left shift of the SDH dose-response curve compared to the curve for single heating and SUH. For the comparison of the tumour and the normal tissue response, damage levels with comparable heating times for single heating were used. The therapeutic effect was then investigated by calculating the therapeutic gain factor (TGF), where TGF = SDR(tumour)/SDR(normal tissue). Neither SUH nor SDH gave a TGF significantly different from 1. The results suggest that SDH may be used clinically to shorten the heating time without decreasing the therapeutic effect.

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 degrees C for 9 h, followed by test treatments for variable times at temperatures ranging from 39 to 43 degrees C. In the temperature range studied, the heat sensitivity of thermotolerant CHO cells was characterized by an Arrhenius activation energy of Ea = 1175 +/- 40 kJ/mol. This value agreed well with Ea = 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 degrees C for 50 min followed by step-down heating at temperatures ranging from 39 to 43 degrees C. The temperature dependence of the thermal response after step-down heating was characterized by an activation energy of Ea = 490 +/- 17 kJ/mol. When the cells were pre-treated for 1-16 h at 39 degrees C prior to step-down heating (43 degrees C, 50 min, followed by graded exposure to 39-43 degrees C), the activation energy was gradually enhanced and approached Ea = 825 +/- 42 kJ/mol for 39 degrees C, 16 h. This change in Ea reflects the effect of thermotolerance on the priming treatment at 43 degrees 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.

Effect of chronically induced thermotolerance on thermosensitization in CHO cells

Chronic thermotolerance was induced in Chinese hamster ovary (CHO) cells by pretreatment at 40 degrees C for various times ranging from 15 min to 16 h. The thermotolerant cells were either exposed to single heat treatments at 43 degrees C or subjected to step-down heating consisting of a priming treatment at 43 degrees C for 90 min immediately followed by a graded test treatment at 40 degrees C. The results showed that chronic thermotolerance affected the thermal sensitivity of step-down-heated CHO cells in two ways: by lowering the effectiveness of the priming treatment at 43 degrees C and by reducing the response to the test treatment at 40 degrees C. The effect on the priming treatment corresponds to a reduction in the effective heating time, i.e. the thermotolerant cells respond as if they were exposed to 43 degrees C for times shorter than 90 min. It was further shown that, for a given conditioning treatment, the effectiveness of both the priming and the test treatment was reduced by the same factor; the thermotolerance ratios determined for 43 degrees C and 40 degrees C showed an identical dependence on the duration of the thermotolerance-inducing conditioning treatment. Since thermotolerance development did not reverse heat sensitization by step-down heating, it is concluded that thermotolerance and thermosensitization are distinct phenomena which act independently.

Effect of Step-Down Heating on Brachytherapy in a Murine Tumor System

The effects of step-down heating combined with low-dose-rate irradiation (brachytherapy) were studied using a murine mammary adenocarcinoma (MTG-B) grown in the flanks of C3H mice. Treatment was initiated when tumors reached 0.9 to 1.1 cm in diameter. Step-down heating consisted of 7.5 min at 45 degrees C immediately followed by 7.5 min at 42 degrees C. Step-up heating consisted of 7.5 min at 42 degrees C immediately followed by 7.5 min at 45 degrees C. Step-down heating and step-up heating were compared to a single 45 degrees C, 15-min hyperthermia treatment. These hyperthermia protocols were combined before, in the middle of, or after brachytherapy. There were 4 untreated controls, 6 sham controls, and 11 treated animals in each of the brachytherapy-alone and combined treatment groups. The entire experiment was repeated at brachytherapy doses of 988, 1273, and 1603 cGy. In addition, the effects of step-down heating, step-up heating, and single-temperature hyperthermia were tested alone and in combination with sham treatment for each sequence. Based on daily measurements of tumor diameter, the growth delay to doubling volume was used as the biological end point. To compare the various treatment protocols, an isoeffect thermal enhancement ratio (TERiso) was calculated. Step-down heating after 988 cGy brachytherapy had a TERiso of 2.0 +/- 0.04, while step-up heating after 988 cGy brachytherapy had a TERiso of 1.7 +/- 0.05. Overall, the thermal enhancement ratios calculated from these growth delays indicate that step-down heating caused significantly greater hyperthermic radiosensitization than step-up heating when combined with brachytherapy.

Correlation between redistribution of a 26 kDa protein and development of chronic thermotolerance in various mammalian cell lines

Previous studies suggested that a 26 kDa protein might play an important role in protein synthesis-independent thermotolerance development in CHO cells. To determine if this phenomenon was universal, four mammalian cell lines, viz., CHO, HA-1, murine Swiss 3T3, and human HeLa, were studied. Cells were heated at 42 degrees C, and the level of 26 kDa protein in the nucleus was measured, together with clonogenic survival and protein synthesis. The results demonstrated that 1) the 26-kDa protein was present in the four different cell lines, and 2) the level of the 26 kDa protein in their nuclei was decreased by 30-70% after heating at 42 degrees C for 1 hr. However, restoration of this protein occurred along with development of chronic thermotolerance. The protein synthesis inhibitor cycloheximide (10 micrograms/ml) neither inhibited the development of chronic thermotolerance nor affected the restoration of the 26 kDa protein in the nucleus. In fact, this drug protected cells from hyperthermic killing and heat-induced reduction of 26 kDa protein in the nucleus. Heat sensitizers, quercetin (0.1 mM), 3,3'-dipentyloxacarbocyanine iodide (DiOC5[3]: 5 micrograms/ml), and stepdown heating (45 degrees C-10 min----42 degrees C), potentiated hyperthermic killing and inhibited or delayed the restoration of the 26 kDa protein to the nucleus. These results support a correlated, perhaps causal relationship between the restoration of the 26 kDa protein and chronic thermotolerance development in four different mammalian cell lines.

Time-Temperature Relationships for L1A2 Cells Step-Down Heated from 38 to 45°C in Vitro

The in vitro response of L1A2 cells to a single exposure to one temperature and to step-down heating was investigated. Single heating consisted of heating for a specified time at a constant temperature in the range 38.0-45.0 degrees C, whereas step-down heating involved a pretreatment of either 45.0 degrees C for 10 min or 42.0 degrees C for 90 min. The pretreatments were adjusted to give the same survival level. The survival curves for single heating had an initial shoulder followed by an exponential region, whereas for step-down heating they were strictly exponential and had no shoulder. The time-temperature relationship for cells exposed to single heating showed a biphasic Arrhenius curve with a downward inflection at 40.5 degrees C. Biphasic Arrhenius curves were also observed for step-down heating, but both the 45 degrees C/10 min and the 42 degrees C/90 min pretreatment showed an upward inflection that broke at 42.5 degrees C and 40.5 degrees C, respectively. The downward inflection on the Arrhenius curve for single heating has been attributed to thermotolerance development and the effect of step-down heating to a temporary inhibition of thermotolerance development. However, the present shape of the Arrhenius curves for step-down heating cannot be explained by inhibition of thermotolerance. It is therefore reasonable to assume that step-down heating is more than just the inhibition of thermotolerance, and that step-down heating and thermotolerance are distinct phenomena which act independently.

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.

Step-down heating in a C3H mammary carcinoma invivo: 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.

Step-down heating of CHO cells at 37.5–39° C

The thermosensitizing effect of step-down heating was studied using Chinese hamster ovary (CHO) cells. Exponentially growing cells were given priming heat treatments at 43 degrees C for 45 or 90 min, immediately followed by a second exposure to a temperature ranging from 37.5 to 39 degrees C. The measured rates of cell killing, 1/D0, increased exponentially with temperature; the slopes correspond to Arrhenius activation energies of Ea = 1200 +/- 150 kJ mol-1 and Ea = 1275 +/- 125 kJ mol-1 for cells preheated at 43 degrees C for 45 or 90 min, respectively. For the temperature range 39-43 degrees C an activation energy of Ea = 561 +/- 24 kJ mol-1 was obtained for step-down heated cells (43 degrees C, 45 min followed by T = 39-43 degrees C). These results indicate that there is a 'second inflection point' at 39 degrees C on the Arrhenius curve for step-down heating of CHO cells. Data evaluation using a mathematical model published previously (H. Jung, Radiation Research, 106, 56-72, 1986) showed that the rate constant c for the conversion of nonlethal lesions into lethal events increased with an activation energy of Ea = 1520 +/- 140 kJ mol-1 in the temperature range from 37.5-39 degrees C. For 39-45 degrees C the activation energy for c was Ea = 360 +/- 26 kJ mol-1, indicating that the temperature dependence of c shows a break at 39 degrees C similar to that observed on the 1/D0 Arrhenius plot.

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.

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 °C/10 min followed by a test treatment (TT) of 41.5 °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 TCD 50 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.

Stability of heating temperature on cytotoxicity

Chinese hamster V79 cells were used to test the cytotoxic effect of heat which alternates between high and low temperatures during the treatment period. This type of temperature fluctuation is often encountered in clinical hyperthermia. Two sets of heating protocols were used in the experiments: Temperature alternates (a) between 42 and 43° and (b) between 42 and 44° in cycle with equal total “thermal dose,” that is heating temperature × heating time. The effectiveness of these heating protocols expressed in average percentage of cell survival depend on the initial temperature attained and the length of the time of this temperature alternation. If the period of temperature change is short such as every 5 or 10 minutes, the cytotoxicity shows very little difference whether the initial treatment temperature is low or high. However, when the period is longer than 20 minutes, the difference in cell survival between the initial temperature at 42° and at higher temperatures is substantial. Cells obtained from logarithmic or plateau phases of growth yield the same result. This difference is likely resulted from a combination of thermotolerance and step-down heating mechanisms. In addition, the effects of heating temperature fluctuation on cytotoxicity is not altered by a 500 cGy of gamma-ray radiation applied either immediately before or after the heat treatment.

Effects of step-up and step-down heating combined with radiation on murine tumor and normal tissues.

Radiosensitizing effects of step-up heating (SUH) and step-down heating (SDH) on the tumor and skin were studied by using mammary adenocarcinoma transplanted to the foot of the C3H/He mouse. The tumor and skin responses were assessed by the tumor growth delay method and the skin reaction scoring method, respectively. Neither SDH (44 degrees, 10 min----42 degrees, 30 min) nor SUH (42 degrees, 30 min----44 degrees, 10 min) alone caused a substantial tumor or skin response. When the heat treatment was given immediately after irradiation, the thermal enhancement ratio (TER) was higher in SDH than in SUH for tumors as well as the skin. A therapeutic gain factor (TGF) of 1.2 was obtained in SUH, while no therapeutic benefit was found in SDH. SDH was applied at various times (0 to 3 hr) before or after irradiation. When SDH was given before irradiation, the TER was consistently high to almost the same degree for tumors and the skin regardless of the time interval, resulting in minimal or no therapeutic gain. With SDH after irradiation, the TER for the skin decreased with increase in the time interval, while the TER for the tumor was moderately enhanced. Therefore, the TGF increased with increase in the time interval and reached 2.2 when SDH was given 3 hr after radiation. SUH is slightly advantageous over SDH in terms of the TGF, and SDH should be given 3 hr after irradiation when selective tumor heating is not possible.