Induction Of Thermotolerance Research Articles

Induction of thermotolerance and mitotic recombination by heat-shock in Ustilago maydiss

Wild-type cells of Ustilago maydis which have been briefly heated at 42 °C subsequently acquire resistance to the lethal effects of a higher temperature (48 °C). This induced thermotolerance develops over a 2 h period after treatment at 42 °C and is dependent on protein synthesis. It is distinct from the cellular response to DNA-damaging agents, since mutants deficient in this and other repair processes show the same induced thermotolerance as wild-type cells. However, UV-light treatment does induce some resistance to subsequent heat treatment. A single 48 °C heat treatment increases recombination in heteroallelic diploids, but the same treatment of thermotolerant cells does not stimulate recombination. This suggests that heat treatment can damage DNA, but thermotolerant cells are protected from such damage.

The Effects of Hyperthermia and Ionizing Radiation in Normal and Ataxia Telangiectasia Human Fibroblast Lines

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

Is DNA Damage the Signal for Induction of Thermal Resistance? Induction by Radiation in Yeast

Yeast, as well as higher eukaryotes, are induced to increase thermal resistance (thermotolerance) by prior exposure to a heat stress. Prior exposure to an acute dose of either 60Co gamma or 254-nm ultraviolet radiation, at sublethal or fractionally lethal doses, is shown to cause a marked increase in the resistance of Saccharomyces cerevisiae to killing by heat. Following a radiation exposure, thermal resistance increased with time during incubation in nutrient medium, and the degree of resistance reached was proportional to the dose received. Partial induction by radiation followed by maximum induction by heat did not produce an additive response when compared to a maximum induction by heat alone, suggesting that the same process was induced by both heat and radiation. Irradiation with 254-nm uv light followed by an immediate, partial photoreversal of the pyrimidine dimers with long-wavelength uv light resulted in a reduced level of resistance compared to cells not exposed to the photoreversal light, indicating that the cells specifically recognized pyrimidine dimers as a signal to increase their thermal resistance. Exposure to 254-nm uv or ionizing radiation induced thermal resistance in mutants defective in either excision repair (rad3, uv-sensitive) or recombinational repair (rad52, gamma-sensitive), suggesting that recognition and repair of DNA damage by these systems are not a part of the signal which initiates an increase in resistance to heat. The amount of induction, per unit dose, was greater in the DNA repair-deficient mutants than in the wild-type cells, suggesting that an increase in the length of time during which damage remains in the DNA results in an increase in the effectiveness of the induction. These data indicate that types of DNA damage as diverse as those produced by ionizing radiation and by ultraviolet light are recognized as a signal by the yeast cell to increase its thermal resistance. It is therefore suggested that heat-induced alterations in DNA or in DNA-dependent chromosomal organization may be the signal for heat induction of thermotolerance in this and other eukaryotes.

The induction of thermotolerance in the ear of the mouse by fractionated hyperthermia

The development of thermotolerance in ears of mice was investigated after fractionated hyperthermia. Ears were heated at 43.5°C by immersion in a water bath and the response was measured in terms of the heating time required to cause thermal necrosis in 50% of the ears (NT 50). Three types of treatment were given: (1) single treatments, for which the NT 50 was 42 minutes; (2) priming treatments, which caused little visible effect but induced thermotolerance. These treatments were given as 1–10 daily fractions, the total heating time ranging from 20–630 minutes; (3) test treatments which were given at various times after priming and were varied to estimate the NT 50. Thermotolerance was defined as an increase in the test NT 50 for preheated ears relative to the single treatment NT 50. It has been suggested that thermotolerance induced by a single priming treatment may be increased by giving additional heat treatments which would not be tolerated by normal cells. In the mouse ear, the maximum thermotolerance induced by a single priming treatment of 20 min at 43.5°C was seen after 24 hr when the test NT 50 was about 2.5 times the single NT 50. The effect of giving up to nine additional daily treatments of 70 min, each of which would cause necrosis in ears that had not received prior hyperthermia, was measured. The maximum thermotolerance observed was equal to that after a single 20 minute priming treatment but thermotolerance decreased as the number of 70 min treatments was increased from four to nine. The effects of repeating a treatment (20 min or 5 min) which was tolerated by normal ears and induced maximal or less than maximal resistance were compared. The interval between each fraction (24 hr or 12 hr respectively) was equal to the time at which maximal thermotolerance was observed after one treatment. For each regimen, the degree of resistance seen after 2 to 10 exposures was similar to that after the appropriate single treatment. This resistance was maintained throughout the course of priming treatment and decayed after the last fraction. Thus for this regimen, thermotolerance depended on the duration of each treatment rather than on the number of treatments given.

Cellular ATP Content of Heated Chinese Hamster Ovary Cells

Hyperthermia at either 41.5 or 45 degrees C with variable heating times to reduce cell survival up to three orders of magnitude did not decrease significantly cellular ATP content when measured either immediately or up to 7 hr after a heat treatment. Similarly, cellular ATP content was not significantly reduced with step-down heating, precooling prior to hyperthermia, or thermotolerance induction. The data suggest that heat-induced depletion of intracellular ATP content is not a critical factor in the thermal death of cells heated under normal culture conditions.

Yeast thermotolerance does not require protein synthesis

Heat shock at 37 degrees C induces synthesis of stress (heat shock) proteins in Saccharomyces cerevisiae and also induces thermotolerance. Amino acid analogs that are powerful inducers of stress protein synthesis failed to induce thermotolerance, suggesting that the stress proteins do not play a causal role in acquired thermotolerance at 37 degrees C. This suggestion was confirmed by the observation that protein synthesis was not required for the induction of thermotolerance at 37 degrees C.

Thiols, Thiol Depletion, and Thermosensitivity

Hyperthermia sensitization or tolerance is subject to cellular events that may occur at membrane, nuclear, and cytoplasmic sites. We have studied the effects of elevated temperatures on the oxidative-reductive state of the cell by measuring and altering glutathione (GSH) concentrations. GSH plays a pivotal role in maintaining the overall cellular redox state and detoxification of peroxides. Continuous heating at 42.5 degrees C or acute exposure at 43 degrees C or 45.5 degrees C resulted in rapid elevations of cellular GSH to 120-200% of control values. Qualitatively, the more severe the heat exposure, the quicker the maximal GSH levels are attained. Ethanol, a compound that also induces thermal tolerance, likewise increases intracellular GSH concentrations. GSH depletion by two different modalities, diethylmaleate (DEM) and buthionine sulfoximine (BSO), results in thermal sensitization. It was demonstrated that once thermotolerance has been induced, depletion of GSH has minimal effects on subsequent heating and thermotolerance. Heat shock protein (HSP) synthesis is lessened by treatment with buthionine sulfoximine; the extent of the decrease in HSP production correlates with the decrease in thermotolerance. The exogenous thiol cysteine combined with heat treatment results in thermosensitization. Exogenous cysteine is found to oxidize to cysteine and to enhance oxygen consumption. The use of N-acetylcysteine resulted in less oxygen consumption and less thermosensitization. A proposed mechanism of peroxide-induced cell damage is suggested by exogenous thiols, as well as an involvement of GSH in the initial aspects of thermotolerance induction.

Induction of thermotolerance and enhanced heat shock protein synthesis in chinese hamster fibroblasts by sodium arsenite and by ethanol

Synthesis of a family of proteins called "heat shock" proteins is enhanced in cells in response to a wide variety of environmental stresses. This suggests that these proteins may have functions essential to cell survival under stressful conditions. A causative relationship between heat shock protein synthesis and development of thermotolerance would imply that agents known to induce heat shock protein synthesis, such as sodium arsenite, also induce thermotolerance. Conversely, agents known to induce thermotolerance, such as ethanol, would also enhance heat shock protein synthesis. To test this hypothesis, I have examined the effect of sodium arsenite or ethanol treatment on protein synthesis and cell survival in Chinese hamster ovary HA-1 cells. After either sodium arsenite or ethanol treatment, the synthesis of heat shock proteins was greatly enhanced over that of untreated cells. In parallel, cell survival was increased as much as 10(4)-fold when cells exposed to either agent were challenged by a subsequent heat treatment. The synthesis of heat shock proteins correlated well with the development of thermotolerance. A qualitative analysis of individual proteins suggests that the synthesis of 70,000 and 87,000 molecular weight proteins most closely mirrored the development of thermotolerance. The results, therefore, strongly reinforce the hypothesis that a causal relationship exists between the enhanced synthesis of heat shock protein and cell survival under specific stresses.

The Cell Cycle Dependence of Thermotolerance: I. CHO Cells Heated at 42°C

This report extends our investigations of the cell cycle dependence of the expression of thermotolerance to include tolerance expressed by Chinese hamster ovary (CHO) cells exposed to 45.0 degrees C hyperthermia. We examined the response of asynchronous cells following exposure at 45.0 degrees C. A maximum in thermotolerance under these conditions was reached approximately 12 hr after a 15-min exposure to 45.0 degrees C hyperthermia and progressively decreased thereafter. Cells were delayed in S and G2 phase for 24 hr, after which time cell growth resumed. We then characterized the response of CHO cell populations synchronized in G1 or early or late S phase. We observed that the expression of tolerance depended on the position of cells in the cell cycle and was modulated by changes in the sensitivity of cells as they progressed through the cell cycle subsequent to the tolerance induction dose. We measured the variation in the sensitivity of these cells to 45.0 degrees C hyperthermia throughout the cell cycle and found substantial changes as cells progressed through S phase. Cells in early S phase were the most sensitive to heat at this temperature, and as these cells progressed through S phase, they became progressively more resistant. In addition, G1 cells were delayed for approximately 15 to 18 hr by a 15-min, 45.0 degrees C heat pulse, whereas S-phase cells were delayed to a lesser extent. The data presented in this report suggest that the induction of thermotolerance is relatively non-cell-cycle specific, but the magnitude of expression of tolerance depends on the position of cells in the cell cycle at the time of the subsequent challenge heat dose.

Modification of the Thermal Response by D 2 O: II. Thermotolerance and the Specific Inhibition of Development

In a companion paper it was shown that D/sub 2/O protects cells against the lethal effects of heat and that D/sub 2/O interfers with the induction of thermotolerance. In this paper we investigate the latter phenomenon with particular reference to an operational model of thermotolerance. Plateau-phase Chinese hamster ovary cells (HA-1) were treated at elevated temperatures permissive for the development of thermotolerance (40-42.5/sup 0/C) in media prepared with H/sub 2/O or D/sub 2/O. Cells were given a more lethal dose of heat (45/sup 0/C/45 min with surviving fraction approx. =10/sup -4/) to assess the degree of thermotolerance. Cells pretreated in H/sub 2/O media expressed a two to three log increase in survival, while thermotolerance was effectively suppressed in cells pretreated in D/sub 2/O. However, cells treated in D/sub 2/O media for 4 hr at 42/sup 0/C, then permitted a 37/sup 0/C incubation period in H/sub 2/O media, rapidly recovered their thermotolerance. Interpreting results within the framework of a three-component model of thermotolerance (trigger, development, decay), we suggest the following: (1) D/sub 2/O inhibits the development component of tolerance; (2) the effect of D/sub 2/O on the trigger component is such that the cells behave as if they were treated atmore » a temperature lower by 2/sup 0/C in H/sub 2/O media; (3) the protection afforded by D/sub 2/O is mechanistically different from that of thermotolerance, at least in the sense that they augment each other.« less

Importance of preheating temperature and time for the induction of thermotolerance in a solid tumour in vivo.

The importance of the priming heat treatment temperature and heating time for the degree and kinetics of thermotolerance was investigated in a C3H mammary carcinoma inoculated into the feet of CDF1 mice. A single heat treatment in the range 41.5-44.5 degrees C resulted in a linear relationship between heating time and tumour growth time (i.e. the time for tumours to reach a volume five times that of the first treatment day). An Arrhenius plot showed an inflection point at 42.5 degrees C with activation energies of 635 and 1508 kJ/mol, respectively, above and below 42.5 degrees C. The degree and kinetics of thermotolerance were independent of the preheating temperature, if the heating time was adjusted to give the same level of heat damage. A pretreatment at these temperatures with a tumour growth time of approximately 10 days, equivalent to 30 min at 43.5 degrees C, resulted in maximal thermotolerance at a 16-h interval with a thermotolerance ratio (TTRmax) of approximately 5.2. Preheating of the tumours at 43.5 degrees C for 3.5, 7.5, 15, 30, or 45 min, showed that if the preheating time was increased, both the TTRmax and the time interval necessary to develop TTRmax increased, both being linear functions of the duration of the preheating time. Maximal thermotolerance was obtained at intervals of 2, 4, 8, 16, and 28 h with TTRmax of 1.6, 2.2, 3.7, 5.2, and 7.7, respectively.

Induction of heat shock proteins and thermotolerance by ethanol in [formula omitted

The temperature sensitivity of Saccharomyces cerevisiae and the conditions of moderate heat pretreatment required to induce thermotolerance are established. Ethanol is identified as an inducer of heat shock proteins and an inducer of thermotolerance.

Thermal Sensitivity and Thermotolerance in Normal Porcine Tissues

We have previously presented a histopathological grading scheme for thermal damage in normal porcine adipose and skeletal muscle tissues. Here we have used this scheme to assess the heat sensitivity of these tissues, and evaluate the protective benefit of thermotolerance as induced by a prior thermal exposure. Tissues were exposed to temperatures ranging from 40-50 degrees for 30 min. Half of all sites also received a thermal exposure of 41.0-43.0 degrees 4 hr earlier. Biopsies for histological evaluation were obtained at 18 to 24 hr (acute) and at 28 to 31 days following treatment. Only mild acute injury was seen in the early samples, following either single or double heat exposures, at all temperature levels. Minimal chronic damage was also seen in the late samples following single exposures of 45 degrees or less. Higher single exposures caused important chronic lesions, the severity of which was dose dependent. Regions that had received the earlier conditioning thermal exposure showed a significant protection against the subsequent thermal exposure. In such regions, mean (chronic) pathology scores were reduced by 76 to 86% over the temperature range 45-48 degrees. The degree of acute damage failed to predict the degree of chronic damage. Overall, induction of thermotolerance provided an advantage of 2 degrees or more in normal tissue protection.

Induction of Thermotolerance and Evidence for a Well-Defined, Thermotropic Cooperative Process

Heat-induced thermal resistance is not only an interesting biological phenomenon, but may also present an important consideration in the application of hyperthermia in cancer therapy. Characterization of the temperature dependence and kinetics of the induction of thermotolerance serves two purposes. First, such data are essential to the rational design of clinical heat treatment protocols, and second, knowledge of the kinetics and temperature dependence may yield some insight into the mechanism of thermotolerance and thermal inactivation. Split-dose experiments were performed using plateau-phase Chinese hamster HA-1 cells. The effects of varying the temperature (41-470C) and the duration of the first heat treatment on the subsequent expression of thermotolerance were studied. Our results indicate that cells initially treated at 41 and 420C are essentially at their most resistant state at the end of the first treatment, while an initial exposure to 430C or higher requires approximately 8 hr of incubation at 370C before maximal thermotolerance is expressed. Based on our three-component model of thermotolerance (trigger-development-decay), we found that neither the rate of development nor the rate of decay was significantly affected by the duration of the first treatment. By plotting the survival at maximal thermotolerance as a function of the duration of the initial heat treatment (at temperatures of 40-470C), we were able to display the kinetics of the trigger. From these data we constructed an Arrhenius plot which yielded two linear, parallel segments with an unusually sharp discontinuity near 43?C. Over both temperature ranges, 43-47 and 40-42.50C, the activation energy is approximately 120 kcal/mole, consistent with the involvement of protein structure alterations in the heat-actuated event. Furthermore, the discontinuity in the Arrhenius plot implies the existence of a sharp thermotropic cooperative process occurring at approximately 430C.

The effect of reduced pH on the induction of thermotolerance.

The effect of low pH on thermotolerance induction was studied in Chinese hamster ovary cells. The thermotolerance ratio (TTR), defined as the ratio of the Do of a subsequent heat survival curve following heat challenge to the Do of the single-dose heat survival curve, was found at pH 7.2 to be maximal at 3.6 seven hours following a 10-min 45 degrees initial heat dose. When heating and induction times were at pH 6.6, the TTR ws reduced to 3.1. When the pH was reduced to 6.6 only during the induction period, the TTR was even lower at 2.5. Heat sensitization at low pH was distinguishable from inhibition of thermotolerance induction. Both the heat sensitization and reduced thermotolerance induction at low pH may enhance the thermal response of tumors relative to normal tissues.

The Effect of Low pH on Thermotolerance Induction Using Fractionated 45°C Hyperthermia

The Effect of Low pH on Thermotolerance Induction Using Fractionated 45°C Hyperthermia

Induction of thermotolerance and sensitization in CHO cells by combined hyperthermic treatments at 40 and 43°C

Chinese hamster ovary cells exposed to combined hyperthermic treatments at 40 and 43°C showed an increase or decrease in cellular sensitivity depending on the temporal order of the application of the two temperatures. Treatments at 40°C prior to hyperthermia at 43°C induced pronounced thermoresistance; the D o of the subsequent 43°C survival curves increased by factors of 1.1, 1.9 or 3.5 and the D q by factors of 2.2, 2.4 or 2.5 for a 1, 3 or 5-hr pre-treatment, respectively. The magnitude of thermotolerance is fully developed by the end of pre-treatment and is not further enhanced by an incubation at 37°C following the 40°C pre-treatment. When the two thermal doses are separated by incubation at 37°C, the magnitude of thermotolerance remained constant for about 2 hr followed by a slow decline. After 24 hr at 37°C, survival is still considerably higher than expected without induced thermotolerance. If the order of the two hyperthermic treatments is reversed, CHO cells are rendered sensitive to 40°C, which is virtually non-lethal when it is not preceded by high hyperthermia. After hyperthermic treatments at 43°C for 30 or 60 min, inactivation at 40°C occurs exponentially, the D o amounting to 1.88 ± 0.03 hr and 1.49 ± 0.08 hr, respectively. This sensitization completely disappears in the course of 7 hr when the cells are incubated at 37°C after conditioning with 43°C hyperthermia.

Interaction of Hyperthermia and Radiation in CHO Cells: Recovery Kinetics

The sensitivity to hyperthermic damage (450C + 0.050) and the interaction of hyperthermic damage with radiation damage was determined in Chinese hamster ovary (CHO) fibroblasts in monolayer culture. The survival curve for asynchronous cells heated at 450C was exponential with a shoulder (Do = 3.6 min, D, = 7.5 min, n = 7.3). Fractionation of two equal heat treatments each of 17.5 min indicated that cell survival was maximally enhanced at a fractionation interval of 12 hr to a survival ratio of approximately 400. Of this, a factor of 60 was due to induction of thermotolerance in terms of an increased Do. Recovery of the capacity to accumulate sublethal damage (return of extrapolation number to 7.3) was complete and occurred within the first 4 hr. By 72 hr the Do returned to normal although the D, persisted at 20 min at 45?C. Heating (17.5 min at 450C) followed immediately by radiation (450 rad) or radiation followed immediately by heating did not produce significantly different survival values. Hyperthermia reduced the Do of the radiation-survival curve by a factor of 1.9 and increased the extrapolation number by a factor of 3. The D, remained unchanged. For intervals up to 48 hr, prior hyperthermia increased the extrapolation number and D, of the radiation-survival curve to approximately 1000 and 650 rad, respectively. However, the Do remained unchanged at approximately 83 rad over the entire interval of 0-72 hr. For the sequence of radiation followed by hyperthermia, the D, of the hyperthermiasurvival curve was reduced to 2.9 min at 450C by simultaneous irradiation but returned to near-normal values after a fractionation interval of 2 hr. The Do of the hyperthermiasurvival curve was unaffected by prior irradiation. Hyperthermia decreased the slope and widened the shoulder of the radiation-survival curve while prior irradiation only affected the shoulder of the subsequent hyperthermiasurvival curve. The recovery kinetics and extensive resistance of heated cells to a second heat dose are difficult to explain solely by the rate process theory of protein denaturation. However, denaturation of membrane or chromatin proteins associated with intracellular recovery mechanisms may account for the effects of hyperthermia alone on cell survival and its modification of the radiation response.