Reduction of DNA-polymerase beta activity of CHO cells by single and combined heat treatments

The effect of single and combined heat treatments on the activity of DNA polymerase beta was studied in CHO cells. The activity of polymerase beta was determined by measuring the amount of [3H]TTP incorporated into activated calf thymus DNA in the presence of aphidicolin, a specific inhibitor of DNA polymerase alpha. Biphasic response curves were obtained for all temperatures tested (40-46 degrees C) showing the sensitivity to decrease during heating. A constant activation energy of Ea = 120 +/- 10 kcal/mole was found for the initial heat sensitivity, whereas the Arrhenius plot for the final sensitivity is characterized by an inflection point at 43 degrees C with Ea = 360 +/- 40 kcal/mole or Ea = 130 +/- 20 kcal/mole for temperatures below or above 43 degrees C, respectively. The observed decrease of the polymerase activity is not due to a decrease in the number of active enzyme molecules but to a change in its affinity, since the inhibition is reversible when increasing concentrations of TTP are applied. When acute or chronic thermo-tolerance was induced by a priming heat treatment at 43 degrees C for 45 min followed by a time interval at 37 degrees C for 16 h or by a preincubation at 40 degrees C for 16 h, respectively, the thermal sensitivity of polymerase beta was lowered by a factor of up to 5. By contrast, pretreatment at a higher temperature followed by a lower temperature (step-down heating) did not alter the sensitivity of polymerase beta to the second treatment. The results indicate that heat-induced cell death cannot be the consequence of the reduction of the polymerase beta activity, confirming earlier studies on this subject.     

Exposure to pretreatment hypothermia as a determinant of heat killing

When Chinese hamster ovary (CHO) cells were exposed to 22 degrees C for 2 hr prior to 42.4 degrees C hyperthermia, neither the shoulder region of the survival curve nor the characteristic development of thermotolerance after 3-4 hr of heating were observed. Absolute cell survival after 4 hr at 42.4 degrees C was decreased by a factor of between 10 and 100 (depending on the rate of heating of nonprecooled controls). Conditioning at 30 degrees C for 2 hr, 26 degrees C for 2 hr, or 22 degrees C for 20 min followed by heating to 42.4 degrees C over 30 min did not result in sensitization. Prolonged (16 hr) conditioning at 30 degrees C, however, increased the cytotoxicity of immediate exposure to 41.4 or 45 degrees C with maximum sensitization to 45 degrees C occurring after 6 hr at 30 degrees C. Both 3- and 18-hr pretreatments at 30 degrees C similarly increased the cytotoxicity of 45-41.5 degrees C step-down heating (D0 = 28 min in precooled versus 40 min in nonprecooled cells).     

Thermosensitization,heat shock protein synthesis and development of thermotolerance in M-14 human tumor cells subjected to step-down heating

M-14 human tumor cells have been subjected to two regimens of step-down heating (SDH) consisting of a conditioning treatment at 42 degrees C for 1 h or at 44.5 degrees C for 20 min, immediately followed by heating at 40 degrees C. Both conditioning treatments thermosensitize the cells towards the subsequent heating at 40 degrees C; the thermosensitization ratio is 6.4 for cells conditioned at 42 degrees C for 1 h and 32.3 for cells conditioned at 44.5 degrees C for 20 min. The overall protein synthetic activity is reduced to 32.7% or 18.4% of control values following 1 h at 42 degrees C and 20 min at 44.5 degrees C, respectively; this inhibition is followed by a full recovery of the synthetic activity during the subsequent exposure at 40 degrees C. SDH-treated cells synthetize four heat shock proteins, with approximate molecular weights of 28, 64, 70 and 90 kDa. The pattern of HSPs induction observed in SDH-treated cells is similar to that found in cells subjected to single hyperthermic exposures. Cells subjected to the SDH sequence 42 degrees C/1 h-->40 degrees C/4 h develop thermotolerance, as indicated by a reduced sensitivity to further hyperthermic challenges.     

Heterogeneity in radiosensitization by heat of cloned cell lines derived from a single human melanoma xenograft

Heterogeneity in radiosensitization by heat was studied using one uncloned and five cloned cell lines isolated from a single tumour of a human melanoma xenograft. Cells from passages 7-12 in vitro were given heat treatments of 42.5 degrees C (45 min), 43.5 degrees C (45 min) or 44.5 degrees C (45 min) immediately after exposure to graded doses of radiation. The survival curves after irradiation alone had similar D0 values but differed in the size of the shoulder. The heterogeneity in heat radiosensitization was reflected in differences in decrease of the D0 values. The thermal enhancement ratios, calculated from the D0 values, were in the ranges 1.2 +/- 0.2-1.7 +/- 0.2 (42.5 degrees C), 1.4 +/- 0.3-2.4 +/- 0.4 (43.5 degrees C) and 2.3 +/- 0.4-3.4 +/- 0.4 (44.5 degrees C). Moreover, at 43.5 degrees C the heterogeneity was also reflected in different modifications of the shape of the survival curves. Two lines showed survival curves with a significant shoulder and a relatively low D0 value whereas two other lines had lost the shoulder almost completely but showed relatively high D0 values. All lines showed survival curves with a broad shoulder after heating at 42.5 degrees C, whereas none of the lines showed survival curves with a significant shoulder after heating at 44.5 degrees C.     

Amino acid pools in CHL V79 cells during induction of thermotolerance: reduction in free intracellular glutamine

The amino acid pools in Chinese hamster lung V79 cells were measured as a function of time during hyperthermic exposure at 40.5 degrees and 45.0 degrees C. Sixteen of the 20 protein amino acids were present in sufficient quantity to measure accurately. The total amino acid pool and all individual amino acids, except glutamine, remained relatively constant for at least 90 min at 40.5 degrees C and for 30 min at 45 degrees C. The glutamine pool decreased rapidly to 20% of its control value within 30 min at 40.5 degrees C with a T1/2 = 15 min. At 45 degrees C, the decrease was 36%. Thermotolerance developed at 40.5 degrees C with a T1/2 = 30 min; thus, glutamine depletion preceeds the development of thermotolerance. The depletion of glutamine is probably due to increased metabolism and oxidation of glutamine through the TCA cycle at hyperthermic temperatures. Glutamine, as is true for other amino acids, was shown to protect proteins from thermal inactivation and V79 cells from hyperthermic killing when added in excess (4-10 mM) to the medium during heat stress. However, the stability of the total amino acid pool during the development of thermotolerance indicates that resistance to heat does not result from the accumulation of amino acids which then protect against thermal damage. The effects of the large decrease in the glutamine pool are unknown, although glutamine depletion may act as a signal for part of the heat shock response.     

Isolation and characterization of heat-sensitive, thermotolerant defective mutants of CHO cells

A replica plating technique was utilized to isolate stable CHO cell mutants that are heat-sensitive and have altered capacities to develop thermotolerance. From a mutagen (EMS) treated population of CHO cells, two strains were isolated. One (HS-36) shows a greatly reduced ability to develop thermotolerance following an initial 45.0 degrees C heat shock. The other (HS-23) also shows a greatly reduced thermotolerance development following a short 45.0 degrees C induction dose, but a greater thermotolerance development following longer 45.0 degrees C induction doses. The dose-survival response following single-dose 45.0 degrees C heating of HS-23 cells suggests the presence of a resistant subpopulation which is not due to contamination from, or reversion to, wild-type cells. Both strains have unique morphological characteristics. Spheroids develop in the central portion of HS-36 colonies, though cells in monolayers are indistinguishable from wild-type parental cells. HS-23 cells grow in firmly attached monolayers, but more than 95% maintain a "rounded" morphology. The remainder show a "flattened" morphology typical of CHO cells. Both strains have parental CHO characteristics as determined by chromosome number, population doubling times, and survival responses to UV light and to gamma rays. Each has maintained its heat-sensitive and altered thermotolerance phenotype for a period of over 6 months in continuous log-phase culture.     

Temperature-dependent induction of thermotolerance by ethanol

Ethanol (1 M) cytotoxicity in asynchronous Chinese hamster ovary cells was strongly temperature dependent, yielding families of cell survival curves between 34 and 39 degrees C that were similar to those obtained at hyperthermic temperatures in medium without ethanol. Below 36 degrees C, survival curves were biphasic, indicating the development of thermotolerance during ethanol exposures. At room temperature (22 degrees C) ethanol was completely nontoxic with incubation periods up to 6 h. A comparison of survival curves with and without ethanol showed that the major effect of ethanol was an effective temperature shift of circa 6.5 degrees C, i.e., the cell survival curve at 37 degrees C in 1 M ethanol was equivalent to that at 43.6 degrees C in medium without ethanol. In addition to the effective temperature shift, ethanol also resulted in sensitization to "heat" with a temperature dependence that was similar to the stepdown heating effect. When thermotolerance was induced with acute ethanol exposures (25 min, 37 degrees C or 60 min, 35.5 degrees C), the kinetics and the magnitude of tolerance were similar to those after isotoxic conditioning treatments with heat alone (10 min, 45 degrees C). In contrast, equimolar ethanol at 22 degrees C did not induce thermotolerance. These data provide a rationale for conflicting results in the literature regarding thermotolerance induction by ethanol. Both heat sensitization and the induction of thermotolerance are interpreted as the effect of ethanol on the solution properties of intracellular water. These solvent alterations reduce the temperature necessary to elicit cytotoxicity and the development of thermotolerance.     

Induction of heat shock proteins in Chinese hamster ovary cells and development of thermotolerance by intermediate concentrations of puromycin

During 4 hr after puromycin (PUR: 20 micrograms/ml) treatment, the synthesis of three major heat shock protein families (HSPs: Mr = 110,000, 87,000, and 70,000) was enhanced 1.5-fold relative to that of untreated cells, as studied by one-dimensional gel electrophoresis. The increase of unique HSPs, if studied with two-dimensional gels, would probably be much greater. In parallel, thermotolerance was observed at 10(-3) isosurvival as a thermotolerance ratio (TTR) of either 2 or greater than 5 after heating at either 45.5 degrees C or 43 degrees C, respectively. However, thermotolerance was induced by only intermediate concentrations (3-30 micrograms/ml) of puromycin that inhibited protein synthesis by 15-80%; a high concentration of PUR (100 micrograms/ml) that inhibited protein synthesis by 95% did not induce either HSPs or thermotolerance. Also, thermotolerance was never induced by any concentration (0.01-10 micrograms/ml) of cycloheximide that inhibited protein synthesis by 5-94%. Furthermore, after PUR (20 micrograms/ml) treatment, the addition of cycloheximide (CHM: 10 micrograms/ml), at a concentration that reduces protein synthesis by 94%, inhibited both thermotolerance and synthesis of HSP families. Thus, thermotolerance induced by intermediate concentrations of PUR correlated with an increase in newly synthesized HSP families. This thermotolerance phenomenon was compared with another phenomenon termed heat resistance and observed when cells were heated at 43 degrees C in the presence of CHM or PUR immediately after a 2-hr pretreatment with CHM or PUR. Heat protection increased with inhibition of synthesis of both total protein and HSP families. Moreover, this heat protection decayed rapidly as the interval between pretreatment and heating increased to 1-2 hr, and did not have any obvious relationship to the synthesis of HSP families. Therefore, there are two distinctly different pathways for developing thermal resistance. The first is thermotolerance after intermediate concentrations of PUR treatment, and it requires incubation after treatment and apparently the synthesis of HSP families. The second is resistance to heat after CHM or PUR treatment immediately before and during heating at 43 degrees C, and it apparently does not require synthesis of HSP families. This second pathway not requiring the synthesis of HSP families also was observed by the increase in thermotolerance at 45.5 degrees C caused by heating at 43 degrees C after cells were incubated for 2-4 hr following pretreatment with an intermediate concentration of PUR.     

Differences in thermosensitization among cloned cell lines isolated from a single human melanoma xenograft

Differences in thermosensitization (effect of step-down heating) among one uncloned and five cloned cell lines isolated from a single tumor of a human melanoma xenograft were studied. Cells from passages 7-12 in vitro were exposed to graded heat treatments at 41.5 degrees C immediately, 1 h, and 2 h after a conditioning treatment of 43.5 degrees C (90 min). The thermosensitization was largest immediately after the conditioning treatment and then decayed exponentially. The differences among the cell lines were reflected in the maximum magnitude as well as in the rate of decay of the thermosensitization. The maximum thermosensitization ratios (TSR), calculated as the ratio of the D0 values at 41.5 degrees C for single-heated and preheated cells, ranged from 5.3 +/- 1.5 to 14.9 +/- 5.2 and were not correlated to the surviving fractions after the conditioning treatment. The half-times for the decay of the thermosensitization ranged from 1.5 +/- 0.3 h to 3.1 +/- 0.5 h and were not correlated to the maximum TSR. Moreover, there was no correlation between the magnitude of the maximum thermosensitization at 41.5 degrees C and the magnitude of the maximum thermotolerance at 43.5 degrees C, as induced by the same treatment (43.5 degrees C for 90 min).     

Rapidly reversible enzyme inhibition in a temperature-sensitive mammalian cell mutant lacks thermotolerance

The temperature-sensitive (ts) Chinese hamster ovary (CHO) cell mutant tsH1 contains a thermolabile leucyl-tRNA synthetase. Upon incubation at the nonpermissive temperature of 39.5 degrees C, the enzyme became reversibly inhibited over a period of minutes, and the cells lost viability over a period of many hours. However, killing of tsH1 by acute heating at 45 degrees C was identical to that of wild-type (SC) cells. In addition, the heat-induced inhibition of protein synthesis was similar for both cell types, as measured after acute heating at 45 degrees C. Furthermore, both killing and inhibition of protein synthesis showed thermotolerance in both cell types. In contrast to the effects at 45 degrees C, at 39.5 degrees C, neither the inhibition of leucyl-tRNA synthetase activity nor the killing of tsH1 expressed thermotolerance. Also, treatment of tsH1 at 39.5 degrees C did not induce thermotolerance to killing at 45 degrees C. The inhibition of leucyl-tRNA synthetase activity in tsH1 at 39.5 degrees C was further distinguished from the 45 degrees C-induced inhibition of protein synthesis in SC cells by a much more rapid reversal of the inhibition of leucyl-tRNA synthetase activity. Also, the rate of reversal of the inhibition of protein synthesis by 45 degrees C in SC cells was decreased by increased heat dose. Such was not true for the 39.5 degrees C inhibition of leucyl-tRNA synthetase activity in tsH1. The data indicate that there exist two distinct types of thermal inhibition--one slowly reversible type which was observed during and after heating at 45 degrees C and both induced and expressed thermotolerance, and a second, rapidly reversible type, which was evident only during heating of tsH1 at 39.5 degrees C and neither induced nor expressed thermotolerance.     

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.     

Relationship between thermal tolerance and protein degradation in temperature-sensitive mouse cells.

The induction of thermotolerance was studied in a temperature sensitive mouse cell line, ts85, and results were compared with those for the wild-type FM3A cells. At the nonpermissive temperature of 39 degrees C, ts85 cells are defective in the degradation of short-lived abnormal proteins, apparently because of loss of activity of a ubiquitin-activating enzyme. The failure of the ts85 cells to develop thermotolerance to 41-43 degrees C after incubation at the nonpermissive temperature of 39 degrees C correlated with the failure of the cells to degrade short-lived abnormal proteins at 39 degrees C. However, the failure of the ts85 cells to develop thermotolerance to 43 degrees C during incubation at 33 degrees C after either arsenite treatment or heating at 45.5 degrees C for 6 or 10 min did not correlate with protein degradation rates. Although the rate of degrading abnormal protein was reduced after heating at 45.5 degrees C for 10 min, the rates were normal after arsenite treatment or heating at 45.5 degrees C for 6 min. In addition, when protein synthesis was inhibited with cycloheximide both during incubation at 33 degrees C or 39 degrees C and during heating at 41-43 degrees C, resistance to heating was observed, but protein degradation rates at 39 degrees C or 43 degrees C were not altered by the cycloheximide treatment. Therefore, there is apparently no consistent relationship between rates of degrading abnormal proteins and the ability of cells to develop thermotolerance and resistance to heating in the presence of cycloheximide.     

Splicing thermotolerance maintains pre-mRNA transcripts in the splicing pathway during severe heat shock

Thermotolerance, the ability of cells and organisms to withstand severe elevated temperatures after brief exposure to mild elevated temperatures, has been studied in numerous laboratories. Survival thermotolerance is defined as the increase in cell or organism survival at severe elevated temperatures after a pretreatment at mild elevated temperatures. This study examines splicing thermotolerance in Drosophila melanogaster, the ability to splice pre-mRNAs made at the severe temperature (38 degrees C) after a brief pretreatment at a milder temperature (35 degrees C). It is probably one of a number of mechanisms by which cells adapt to heat shock. These experiments demonstrate that pre-mRNAs synthesized at the severe temperatures in splicing thermotolerant cells, although protected in splicing-competent complexes, are not actually processed to mature mRNAs until the cells are returned to their normal temperature. We have also studied the kinetics of acquisition and loss of splicing thermotolerance. As little as 10 min of pretreatment at 35 degrees C was sufficient to provide full splicing thermotolerance to a 30-min severe heat shock of 38 degrees C. Pretreatments of less than 10 min provide partial splicing thermotolerance for a 30-min severe heat shock. Full splicing thermotolerance activity begins to decay about 4 h after the cessation of the 35 degrees C incubation and is completely lost by 8 h after the pretreatment. The kinetics experiments of pre-mRNAs synthesized during the 38 degrees C treatment in splicing thermotolerant cells indicate that one or more splicing thermotolerance factors are synthesized during the 35 degrees C pretreatment which interact with pre-mRNA-containing complexes to keep them in a splicing-competent state. These kinetic experiments also indicate that in cells which are partially splicing thermotolerant, the pre-mRNAs synthesized early during the 38 degrees C incubation are protected, whereas those synthesized late are not. In the absence of splicing thermotolerant factors, the pre-mRNA-containing complexes leave the normal splicing pathway and are allowed to exit to the cytoplasm.     

Influence of temperature on the development and decay of thermotolerance and heat shock proteins

The development of thermotolerance and its decay in plateau-phase Chinese hamster cells are shown to be temperature-dependent phenomena. Development of tolerance, after an initial dose of 10 min at 45 degrees C, is appreciably slower between 20 and 28 degrees C than it is at 37 degrees C. Decay of tolerance is also slower in that temperature range; at 4-23 degrees C, it does not decay at all during the 96-h interval of the experiment. At 41 degrees C, thermotolerance decay, "step-down" cell killing, and thermotolerance induction apparently all occur and affect cell survival. The decay of HSP 70 mirrors that of thermotolerance, except at 41 degrees C. At that temperature very likely de novo synthesis of that protein becomes important in determining protein concentration. Our data show that care must be taken when extrapolating from kinetic data obtained with surface tissues in vivo to those in depth. The former are usually at a temperature between 25 and 32 degrees C; the latter are at 37 degrees C.     

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

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

Relationship between cell survival and heat-stress protein synthesis in a Drosophila cell line

Heat-stress protein (hsp) kinetics and clonogenic survival were studied at 33, 37 and 42 degrees C in a continuous Drosophila cell line, WR69-DM-1. Induction and repression of hsp were temperature-dependent and independently modulated. The subsequent cell-survival curves were complex; however, survival generally decreased in a time- and temperature-dependent manner during continuous heating at 33, 37 or 42 degrees C. Constant 33 degrees C heating induced five hsp at 90, 72, 70, 24 and 19 kilodaltons (kDa). A 30 min 33 degrees C heat dose led to thermotolerance after 1, 3 or 6 h incubations at 28 degrees C. The hsp synthesized after this dose were quickly repressed, suggesting the cells were able to respond to this stress. Increasing the challenge temperature to 37 degrees C induced three additional hsp at 34, 22 and 14 kDa, but hsp synthesis did not lead to thermotolerance over the 6 h interval. The number and intensity of hsp synthesized was higher and repression was much slower than at 33 degrees C. Heating at 42 degrees C inhibited all protein synthesis, and thermotolerance was not observed. Direct survival data are critical to understanding the role and function of hsp in Drosophila thermotolerance since the relevance of information on number and kinetics of hsp synthesis and their subsequent localization is dubious without it.     

Thermotolerance in preirradiated intestine and its influence on time-temperature relationships

The crypt compartment of mouse jejunum showed a transient increase in thermal susceptibility approximately 10 days after moderate X-ray doses to the abdomen (9-10 Gy). The increase in response was manifest as an increase in slope of the crypt dose-response curve but was limited to temperatures below 43 degrees C. As a result, the 43 degrees C inflexion in the Arrhenius plot (the relationship between treatment time and temperature) for thermal sensitivity of crypts was eliminated in preirradiated tissue, and the curve became monophasic over the range 42.0-44.5 degrees C. At temperatures below 42 degrees C, the curve again deviated. At supranormal temperatures of 42 degrees C and below, the durations of hyperthermia needed for measurable effect were sufficient to allow thermotolerance to be expressed within the heating period. Neither the threshold heating times nor this thermotolerance were affected by prior irradiation. In the temperature range 42-43 degrees C, an earlier development of thermotolerance could be demonstrated in control tissue by challenging with an acute high-temperature heat treatment. This thermotolerance was eliminated in preirradiated tissue, resulting in the apparent increase in sensitivity. The findings support the view that the complex nature of the time-temperature relationship seen in normal tissue in vivo is a manifestation of the ability of the tissue to progressively acquire a thermotolerant state during treatment at temperatures below approximately 43 degrees C, so that the "intrinsic" sensitivity is modulated while being assessed.     

A generalized concept for cell killing by heat

Based on the analysis of many survival curves obtained after hyperthermic treatments of CHO cells at various temperatures, or after consecutive exposure to two different temperatures, a generalized concept has been developed for the action of heat on cell survival. The basic idea of this concept is that cellular inactivation by heat is a two step process. In the first step, heating produces nonlethal lesions. In the second step, the nonlethal lesions are converted into lethal events upon further heating. The conversion of one of the nonlethal lesions in a cell leads to cell death. Based on the assumption that both production and conversion of nonlethal lesions occur at random and depend only on temperature, a mathematical model has been worked out that quantitatively describes cell killing by single heating as well as by step-down or step-up heating. After the cells are heated at a certain temperature for a time t, the surviving fraction is given by the equation S(t) = exp [(p/c) X [1 - c X t - exp(-c X t)]) where p is the rate constant for the production of nonlethal lesions per cell and per unit of time, and c is the rate constant for the conversion of one nonlethal lesion into a lethal event per unit of time. When heating is performed consecutively at two different temperatures; i.e., when a pretreatment at the temperature T1 for the time t1 is followed by a graded exposure to the temperature T for the time t, the surviving fraction is given by the equation S(t1,t) = exp [(p1/c1) X exp(-c X t) X [1 - c1 X t1 X exp (c X t) - exp(-c1 X t1) + (p/c) X [1 - c X t - exp(-c X t)]) where p1 and c1 are the production rate and the conversion rate at the temperature T1 of the pretreatment, and p and c are the corresponding values at the temperature of the second treatment. By fitting the equations given above to the experimental data of many heat survival curves, the values of p and c were determined for the temperature range 39 to 45 degrees C. In this range, the conversion rate c increases exponentially with temperature; the slope corresponds to an activation energy of Ea = 86 +/- 6 kcal/mol. The Arrhenius plot of the production rate p shows an inflection point at 42.5 degrees C. Above that temperature, the activation energy is 185 +/- 14 kcal/mol; below, Ea = 370 +/- 30 kcal/mol was obtained.(ABSTRACT TRUNCATED AT 400 WORDS)     

Differences in thermotolerance induced by heat or sodium arsenite: cell killing and inhibition of protein synthesis

Chinese hamster ovary (CHO) cells became thermotolerant after treatment with either heat for 10 min at 45.5 degrees C or incubation in 100 microM sodium arsenite for 1 h at 37 degrees C. Thermotolerance was tested using heat treatment at 45 degrees C or 43 degrees C administered 6-12 h after the inducing agent. At 45 degrees C thermotolerance ratios at 10(-2) isosurvival levels were 4.2 and 3.8 for heat and sodium arsenite, respectively. Recovery from heat damage as measured by resumption of protein synthesis was more rapid in heat-induced thermotolerant cells than in either sodium arsenite-induced thermotolerant cells or nonthermotolerant cells. Differences in inhibition of protein synthesis between heat-induced thermotolerant cells and sodium arsenite-induced thermotolerant cells were also evident after test heating at 43 degrees C for 5 h. At this temperature heat-induced thermotolerant cells were protected immediately from inhibition of protein synthesis, whereas sodium arsenite-induced thermotolerant cells, while initially suppressed, gradually recovered within 24 h. Furthermore, adding cycloheximide during the thermotolerance development period greatly inhibited sodium arsenite-induced thermotolerance (SF less than 10(-6] but not heat-induced thermotolerance (SF = 1.7 X 10(-1] when tested with 43 degrees C for 5 h. Our results suggest that both the development of thermotolerance and the thermotolerant state for the two agents, while similar in terms of survival, differed significantly for several parameters associated with protein synthesis.     

Effect of cycloheximide or puromycin on induction of thermotolerance by sodium arsenite in Chinese hamster ovary cells: involvement of heat shock proteins

After sodium arsenite (100 microM) treatment, the synthesis of three major heat shock protein families (HSPs; Mr = 110,000, 87,000, and 70,000), as studied with one-dimensional gels, was enhanced twofold relative to that of unheated cells. The increase of unique HSPs, if studied with two-dimensional gels, would probably be much greater. In parallel, thermotolerance was observed as a 100,000-fold increase in survival from 10(-6) to 10(-1) after 4 hr at 43 degrees C, and as a thermotolerance ratio (TTR) of 2-3 at 10(-3) isosurvival for heating at 45.5 degrees C. Cycloheximide (CHM: 10 micrograms/ml) or puromycin (PUR: 100 micrograms/ml), which inhibited total protein synthesis and HSP synthesis by 95%, completely suppressed the development of thermotolerance when either drug was added after sodium arsenite treatment and removed prior to the subsequent heat treatment. Therefore, thermotolerance induced by arsenite treatment correlated with an increase in newly synthesized HSPs. However, with or without arsenite treatment, CHM or PUR added 2-6 hr before heating and left on during heating caused a 10,000-100,000-fold enhancement of survival when cells were heated at 43 degrees C for 4 hr, even though very little synthesis of heat shock proteins occurred. Moreover, these cells manifesting resistance to heating at 43 degrees C after CHM treatment were much different than those manifesting resistance to 43 degrees C after arsenite treatment. Arsenite-treated cells showed a great deal of thermotolerance (TTR of about 10) when they were heated at 45 degrees C after 5 hr of heating at 43 degrees C, compared with less thermotolerance (TTR of about 2) for the CHM-treated cells heated at 45 degrees C after 5 hr of heating at 43 degrees C. Therefore, there are two different phenomena. The first is thermotolerance after arsenite treatment (observed at 43 degrees C or 45.5 degrees C) that apparently requires synthesis of HSPs. The second is resistance to heat after CHM or PUR treatment before and during heating (observed at 43 degrees C with little resistance at 45.5 degrees C) that apparently does not require synthesis of HSPs. This phenomenon not requiring the synthesis of HSPs also was observed by the large increase in thermotolerance to 45 degrees C caused by heating at 43 degrees C, with or without CHM, after cells were incubated for 6 hr following arsenite pretreatment. For both phenomena, a model based on synthesis and redistribution of HSPs is presented.