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.     

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.     

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.     

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.     

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.     

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

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.     

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.     

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.     

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.     

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°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°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 of human melanoma xenografts: effects of the tumour microenvironment.

Thermosensitisation by step-down heating (SDH) has previously been demonstrated in experimental rodent tumours. The purpose of the study reported here was to investigate whether the SDH effect in tumours in part may be attributed to heat-induced alterations in the capillary network and/or the microenvironment. Two human melanoma xenograft lines differing substantially in vascular parameters were selected for the study. A thermostatically regulated water bath was used for heat treatment. The conditioning treatment (44.5 degrees C or 45.5 degrees C for 15 min) was given in vivo, whereas the test treatment (42.0 degrees C for 45, 90, 135 or 180 min) was given either in vitro or in vivo. Treatment response was measured in vitro using a cell clonogenicity assay. Fraction of occluded vessels following heat treatment was assessed by examination of histological sections from tumours whose vascular network was filled with a contrast agent. Tumour bioenergetic status and tumour pH were measured by 31P magnetic resonance spectroscopy. The conditioning heat treatments caused significant vessel occlusion, decreased tumour bioenergetic status and decreased tumour pH in both tumour lines. The SDH effect measured when the test treatment was given in vivo was significantly increased relative to that measured when the test treatment was given in vitro. The magnitude of the increase showed a close relationship to fraction of occluded vessels, tumour bioenergetic status and tumour pH measured 90 min after treatment with 44.5 degrees C or 45.5 degrees C for 15 min. The increased SDH effect in vivo was probably attributable to tumour cells that were heat sensitive owing to the induction of low nutritional status and pH during the conditioning treatment. Consequently, the SDH effect in some tumours may in part be due to heat-induced alterations in the microenvironment. This suggests that SDH may be exploited clinically to achieve increased cell inactivation in tumours relative to the surrounding normal tissues.     

Cell killing and sensitization to heat shock by hypothermic incubation of asynchronous and synchronized mouse neuroblastoma cells

The effect of hypothermia on cell survival and on subsequent response to hyperthermia was studied in asynchronous and synchronized Neuro-2A cells. Cell cycle progression was blocked at temperatures below 27 degrees C. Immediately after shift to hypothermic temperatures, cells became more sensitive to hyperthermia. Development of thermosensitization was time and temperature dependent. Thermosensitization of cells by hypothermia was high at 0 degrees C and 15 degrees-30 degrees C and less at 5 degrees-10 degrees C. Sensitization started to occur before hypothermic cell death became manifest and developed gradually. Hypothermic cell death was observed when the cells were incubated for more than 1 day at temperatures of 0 degrees-24 degrees C with a minimal cell death during incubation at 6 degrees C. Thermosensitization of cells by hypothermia depended on the position of the cell in the cell cycle at the time of shift to hypothermic temperatures. Cells in late G1 and early S phase became more thermosensitive than did cells in G1 or late S-G2 phase. Furthermore G1-S cells were more sensitive to prolonged hypothermia alone than were G1 or late S-G2 cells. In contrast, late S-G2 cells were most sensitive to hyperthermia alone. It is concluded that the temperature- and cell cycle-dependent way of hypothermic induced cell death was similar to the thermosensitization of cells by hypothermia. But thermosensitization became manifest prior to the actual cell death, following hypothermic treatment.     

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

A comparison between the effect of step-down heating (SDH) obtained in a C3H mammary carcinoma grown in the feet of CDFl mice and the skin of normal CDFl feet is presented. Water-bath heating was used, and SDH was obtained by giving a 44.7°C/10 min treatment followed by heating at 42.2°C for variable times. Single heating at 42.2°C and step-up heating (SUH), i.e. 42.2°C followed by 44.7°C/10 min, were used as controls. The endpoint was the heating time at 42.2°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°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.     

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 degrees 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 degrees 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 degrees 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 degrees C and for step-down heating in the range 41.7-44.7 degrees 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 degrees C/10 min treatment and a test treatment performed at 42.2 degrees 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 chronically induced thermotolerance on thermosensitization in CHO cells

Chronic thermotolerance was induced in Chinese hamster ovary (CHO) cells by pretreatment at 40°C for various times ranging from 15 min to 16 h. The thermotolerant cells were either exposed to single heat treatments at 43 °C or subjected to step-down heating consisting of a priming treatment at 43 °C for 90 min immediately followed by a graded test treatment at 40°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 °C and by reducing the response to the test treatment at 40°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°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°C and 40°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.     

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.     

Depletion of cellular glutathione by exogenous spermine in V79 cells: implications for spermine-induced hyperthermic sensitization

The relationship between spermine-induced thermosensitization and modulation in the cellular redox state as measured by glutathione levels was studied using Chinese hamster V79 cells. Marked cellular glutathione depletion was observed for cells treated with exogenous 1 mM spermine at 37 degrees C or 43 degrees C. Glutathione depletion and thermal sensitization by spermine were found to be cell density dependent with maximum depletion and sensitization observed at low cell densities. These findings are discussed in the context that treatment of cells with exogenous polyamines such as spermine can result in cellular oxidative stress which may in part contribute to spermine-induced thermal sensitization.     

Intrinsic thermal response, thermotolerance development and stepdown heating in murine bone marrow progenitor cells.

Thermal response, thermotolerance development and stepdown heating (SDH) in the murine bone marrow granulocyte-macrophage (CFU-GM) progenitors were determined in vitro. Marrow was removed from femora and tibia, heated in McCoy's 5A medium plus 15% FBS and cultured in soft agar in the presence of three different sources of colony stimulating factor. D0's (+/- SE) for survival curves of CFU-GM heated in vitro were 147 +/- 13, 71 +/- 9, 37 +/- 2, 19 +/- 0.7, 11 +/- 1, and 4.3 +/- 0.3 min, for temperatures of 41.8, 42, 42.3, 42.5, 43 and 44 degrees C, respectively. Arrhenius analysis showed inactivation enthalpies of 812 +/- 9 KJoules/mole (193 +/- 2 Kcal/mole) above, and 2142 +/- 157 KJoules/mole (509 +/- 37 Kcal/mole) below, an inflection at 42.5 degrees C. Thermotolerance development was evident during prolonged hyperthermia exposure at temperatures below 42.5 degrees C (chronic hyperthermia) as a change in the slope of the survival curves after approximately 110 min of heating. Thermotolerance development at 37 degrees C after exposure to temperatures of 43 degrees C or greater (acute hyperthermia) was assessed by fractionated heat treatments consisting of an initial heat treatment (15 min at 44 degrees C) followed by incubation at 37 degrees C and challenge with 15 min or 25 min at 44 degrees C. Maximum thermotolerance occurred after 210 and 330 min at 37 degrees C, respectively. The half-time for maximum thermotolerance development was 36 min. Depending on the amount of heat damage and the maximum amount of thermotolerance development, the decay of thermotolerance was complete after approximately 48-72 h at 37 degrees C. An exposure of 10 min at 44 degrees C before incubation at 40 or 41 degrees C (stepdown heating) reduced the slope of the 40 or 41 degrees C survival curves by inhibiting thermotolerance development that would have otherwise occurred. D0's were 100 +/- 19 and 45 +/- 5 min for 40 and 41 degrees C incubation preceded by 10 min at 44 degrees C, respectively. These studies indicate that whole-body or regional hyperthermia protocols designed either to treat solid tumours or to purge leukemic stem cells from marrow ex vivo should avoid inadvertent temperature elevations to large volumes of marrow. Although, marrow progenitors are capable of thermotolerance development during exposure to temperatures up to 42.3 degrees C, results suggest that conditions of stepdown heating may prevent thermotolerance development.     

Effects of hyperthermia on the colony-stimulating factor production by the lung.

Hyperthermic treatment of the murine lung in the range of 40-46 degrees C inhibited the production of colony-stimulating factors by the lung in vitro. This inhibition was dose dependent. Thermodynamic analysis was used to determine the activation energies. The Arrhenius plot contained a transition at 43 degrees C. At temperatures below and above the transition temperature, the activation energies were 40.49 and 197 kcal/mole, respectively. Below the transition temperature, the effect of hyperthermia was characterized by a delayed response represented by the broad initial shoulder of the hyperthermic dose-response curves. To investigate the mechanism of hyperthermia-induced reduction of the colony-stimulating factor production, the effect of hyperthermia on the protein synthesis by the lung was also studied. The results indicated an immediate response to hyperthermia, characterized by the absence of the initial shoulder and the high slope of the hyperthermic dose response curves. The corresponding Arrhenius plot did not have any transition point. The single activation energy calculated was 97.25 kcal/mole. It is concluded that the hyperthermic depression of the colony-stimulating factor production by the lung cannot be explained solely on the basis of the effects of hyperthermia on the protein synthesis.