Thermotolerant Cells Research Articles

Heat protection by deuterium oxide of heat sensitive and wild-type Chinese hamster ovary cells.

The ability of deuterium oxide (D2O) to protect a heat-sensitive and thermotolerance-impaired Chinese hamster ovary (CHO) mutant cell line, HS-36 (Harvey and Bedford 1988), from heat killing was examined and compared to the parent CHO 10B cell line (WT). Both non-thermotolerant (NT) and thermotolerant (TT) G1 populations were examined. D2O differentially protected the NT cell lines from heat killing, with thermal protection ratios (D0) of 2 x 5 and 4 x 3 for HS-36 and WT cells, respectively. D2O provided additional protection to TT cells, but now protected the TT HS-36 cells more than the TT WT cells when the thermal protection ratios of TT cells are compared with those of NT cells (1.15 versus 0.82). The differential protection from heat of the mutant and wild-type lines by D2O may be useful in studies of induced lesions in proteinaceous cellular systems (e.g. the nuclear matrix, cytoskeleton and plasma membrane) using these two paired cell lines.

Regulation of hsp70 induction in thermotolerant HeLa cells

Upon exposure to heat shock, non-thermotolerant (NT) HeLa cells transiently synthesize a large amount of 70-kDa heat-shock protein (hsp70), whereas thermotolerant (TT) cells synthesize a small amount of hsp70. When the hsp70 mRNA of HeLa cells was analyzed, it became apparent that hsp70 mRNA in TT cells did not increase following heat shock, whereas hsp70 mRNA in NT cells did increase dramatically. A further analysis of the activation of the heat-shock transcription factor (HSF) showed that significant activation of HSF was observed immediately after heat shock in both NT and TT cells. However, activated HSF was rapidly repressed in the TT cells, but not in the NT cells. Thus, the decreased induction of hsp70 synthesis observed in the TT HeLa cells may be due to the immediate repression of activated cellular HSF, which probably results in the reduced induction of hsp70 mRNA. The hsp70 content in the TT cells was usually higher than in the NT cells. However, after heat-shock treatment, the hsp70 content of the NT cells increased to nearly the level of the TT cells concomitant with the repression of hsp70 synthesis. The association of activated HSF with hsp70 was observed in both NT and TT cells, and the amount of HSF-hsp70 complex within the cell increased in proportion to the increase in hsp70 in the cells. These findings strongly suggest that the activity of HSF is negatively regulated by the intracellular content of hsp70 in these cells. Furthermore, in vitro experiments on the activation of HSF suggest that HSFs of NT and TT cells may have different properties, or an unknown factor may exist which regulates HSF activation in these cells.

Regulation of hsp70 synthesis induced by cupric sulfate and zinc sulfate in thermotolerant HeLa cells.

Upon exposure to the heavy metals copper and zinc, a large amount of 70,000-Da heat shock protein (hsp70) was synthesized in normal, non-thermotolerant (NT) HeLa cells, whereas only a little increase of hsp70 synthesis was observed in thermotolerant (TT) cells. To determine the inhibition mechanism of hsp70 induction in the TT cells, we first analyzed the hsp70 mRNA of these cells. Hsp70 mRNA in the NT cells increased immediately after exposure to these metals. In TT cells, however, the increase of hsp70 mRNA was delayed, even though it eventually increased to a similar or slightly lower level compared with NT cells. Further analysis of the activation of heat shock transcription factor (HSF) showed that a significant activation of HSF was observed immediately after exposure to these metals in the NT cells, whereas the activation of HSF was initially repressed in the TT cells. Thus, the decreased induction of hsp70 synthesis observed in the TT cells seemed to be due to the reduced translation of hsp70 mRNA and also in part to the reduced activation of HSF. Furthermore, by the gel mobility shift assay using anti-hsp70 antibody, the association of copper- and zinc-activated HSF with hsp70 was observed in both NT and TT cells. The amount of HSF-hsp70 complex was prominent in TT cells, in which the hsp70 content was 5 to 10 times higher than that in the NT cells. These findings strongly suggest that the activity of HSF is negatively regulated by hsp70 in the TT cells.

Reduced Adriamycin Concentration in Rat Mammary Tumors Treated Previously with Hyperthermia

The effect of prior hyperthermia on the concentrations of adriamycin in tumors was examined. Chemically induced (9,10-dimethyl-1,2-benzanthracene) rat mammary adenocarcinomas were not heated or were heated one or two times (43.5 degrees C, 1 h; 24 h apart when two times) using an Nd:YAG laser. Rats were administered adriamycin (5 mg/kg, iv) 3 h after hyperthermia treatment. The concentrations of adriamycin in plasma and tissue were determined using high-performance liquid chromatography. Although plasma pharmacokinetics was unchanged by prior local hyperthermia, the concentration of adriamycin was significantly lower in tumors that had been heated previously compared to that in unheated tumors. Tumor blood flow rate was determined after an identical heating protocol using a reference sample technique with 113Sn microspheres. Tumor blood flow rate increased slightly during hyperthermia and then rapidly returned to control levels after hyperthermia. Decreased concentrations of adriamycin in tumors may result from biochemical changes induced by hyperthermia in neoplastic cells and may explain the adriamycin resistance in thermotolerant cells reported previously.

Heat shock in human neutrophils: superoxide generation is inhibited by a mechanism distinct from heat-denaturation of NADPH oxidase and is protected by heat shock proteins in thermotolerant cells.

Independently of the stimulating agent used, generation of O2- by human neutrophils is transiently inhibited when the cells have been exposed to elevated temperatures. This phenomenon is concomitant with the synthesis of heat shock proteins (HSPs). We have investigated a possible relationship between HSPs and modulation of NADPH oxidase activity in human neutrophils exposed to heat. HSPs were not involved in the inhibition of O2- generation since 1) in enucleated cytoplasts, which are unable to synthesize proteins, the generation of O2- was inhibited after exposure to 43 degrees C, 2) using actinomycin D (Act D) in intact cells, it was possible selectively to inhibit the synthesis of HSPs without modifying the inhibition of NADPH oxidase activity that followed HS. Furthermore, the recovery of NADPH oxidase activity was not under the control of HSPs because the enzyme recovered as well in Act D-treated neutrophils. The NADPH oxidase activity was reconstituted in a cell-free assay by combining the cytosol with the plasma membrane-enriched fraction in the presence of arachidonic acid (AA) and NADPH. Subcellular fractions obtained from control or heated neutrophils exhibited similar oxidase activities suggesting that heat exposure did not induce denaturation of the oxidase components but rather altered the mechanisms of translocation and/or assembly of these components with the plasma membrane. This hypothesis was supported by the inhibition of the granule release in heated cells, a process which also requires translocation and association fusion with the plasma membrane. On the other hand, preexposure of neutrophils to HS prevented the inhibition of O2- generation during a second challenging HS. This acquired thermotolerance was abolished when the synthesis of HSPs was inhibited during the first HS with Act D, indicating a direct relationship between protection of O2- generation and synthesis of HSP. Here we demonstrate that synthesis of HSPs and inhibition or recovery of NADPH oxidase activity are concomitant but unrelated phenomena. In contrast, accumulation of HSPs in thermotolerant neutrophils appeared to play an important role in the prevention of NADPH oxidase inhibition. These results provide further insights into the behavior of human neutrophils and NADPH oxidase upon heat injury.

Poly(A) tail length of a heat shock protein RNA is increased by severe heat stress, but intron splicing is unaffected

The small heat shock proteins (sHSPs) are induced in all eukaryotes in response to high temperature stress, but are most abundant among members of the plant kingdom where they accumulate in multiple subcellular compartments. We have analyzed the expression of the chloroplast-localized sHSP from Arabidopsis thaliana, HSP21, and characterized the structure of the gene encoding this protein to facilitate future genetic studies on the function of HSP21 in the heat shock response. HSP21 is encoded in Arabidopsis by a single gene whose coding region is interrupted by a single intron. Previous studies have shown that intron processing is disrupted by severe, abrupt heat stress but is protected by pretreatments that induce thermotolerance. The processing of the HSP21 transcript was investigated in response to an abrupt heat stress regime and a gradual heat stress regime, the latter of which is known to confer thermotolerance in plants. Under abrupt stress conditions the HSP21 transcript is somewhat longer than under gradual heat stress conditions. However, the molecular basis for the size difference is not impaired intron splicing, but rather a difference in the length of the poly(A) tail depending on the heat stress regime. The results suggest that an increase in poly(A) tail length may be a generalized response to severe, abrupt heat stress and that poly(A) tail metabolism may be one of numerous cellular processes normally protected in thermotolerant cells from the otherwise damaging effects of high temperature stress.

Employment of a turbidimetric assay system to study the biochemical role of HSP70 in heat-induced protein aggregation

1. 1.|The biochemical role of the 70 kDa constitutive heat shock protein (cHSP70) in heat-induced protein aggregation was studied by a turbidimetric assay system. 2. 2.|When rhodanese (2.5 mg/ml) was diluted 100-fold into heated cytoplasmic proteins (500 μg/ml), turbidity caused by protein-rhodanese aggregation rapidly increased and reached maximum within 5 min. 3. 3.|The amount of protein aggregates was reduced 2-fold by adding cHSP70 (20 μg/ml), but not bovine serum albumin, during heating at 4.5°C. Similar results were observed when cHSP70 was added to cytoplasmic proteins from cells heated at 4.5°C for 10 min. 4. 4.|The reduction of protein aggregation by treatment with HSP70 was dependent on ATP content. The aggregation was also significantly reduced when cytoplasmic proteins from thermotolerant cells that contain high level of HSP70 were used. The reduction effects were markedly decreased by adding anti-SHp70 antibody (0.1 mg/ml) to cytoplasms before heating at 45.5°C for 20 min. 5. 5.|Thus, data from the turbidimetric assay system indicate that HSP70 plays an important role in reduction of protein aggregation, and perhaps dissociation of protein aggregates in the presence of ATP.

Similar Heat-Induced Cell Cycle Delays in the Clonogenic and Nonclonogenic Fractions of a Thermotolerant Population

We have tested the theory that a faster rate of recovery from thermal damage in thermotolerant cells relative to nontolerant cells is the critical factor which confers heat resistance. We measured the rate of recovery from the heat-induced cell cycle delay in Chinese hamster ovary cells, since this delay is shortened in thermotolerant cells (i.e., exhibits thermotolerance) and can be measured in individual cells. Individual thermotolerant cells were followed by discontinuous microscopic observation from shortly after heating, through the first mitosis after heating, until a colony formed or failed to form by 8 days. The heat-induced delay in the clonogenic and nonclonogenic fractions was the same. This shows that the rate of recovery from the cell cycle delay was not the determining factor as to whether or not a cell survived to form a colony. Either additional factors are involved or the rate of recovery from the cell cycle delay plays no role in cell survival. These data show the importance of determining whether a faster rate of repair of thermal damage is specific for the clonogenic fraction, since most if not all types of thermal damage are likely to be repaired more quickly when thermotolerant and nontolerant cells are compared at isodose.

Effect of amino acid analogs on the development of thermotolerance and on thermotolerant cells

Exposure of HA-1 Chinese hamster fibroblasts to amino acid analogs has been shown to have a heat-sensitizing effect as well as inducing the heat shock response (Li and Laszlo, 1985a). In this study, we have examined the effect of amino acid analogs on the development of thermotolerance after a brief heat shock or exposure to sodium arsenite and the effect of amino acid analogs on cells that are already thermotolerant. Exposure of HA-1 cells to amino acid analogs inhibited the development of thermotolerance following a mild heat shock or treatment with sodium arsenite. However, cells that were already thermotolerant were resistant to the sensitizing action of amino acid analogs. The refractoriness of thermotolerant cells to amino acid analog treatment developed in parallel with thermotolerance. The uptake of the arginine analog, canavanine, and its incorporation into proteins was not altered in the thermotolerant cells. Furthermore, another biological consequence of exposure to amino acid analogs, sensitization to ionizing radiation, also was not altered in the thermotolerant cells. The inhibition of the development of thermotolerance by amino acid analogs and the refractoriness of thermotolerant cells to the heat-sensitizing action of amino acid analogs lend further support the role of heat-shock proteins in the phenomenon of thermotolerance.

Changes in intracellular free calcium during hyperthermia: effects of local anesthetics and induction of thermotolerance.

We wished to determine if local anesthetics (LAs) induced changes in intracellular free calcium ([fCa2+i]) that could have an effect on cell killing by hyperthermia. Flow cytometry was used to measure [fCa2+i] of mouse NIH 3T3 cells during heating at 45.5 degrees C. In both non-tolerant and thermotolerant cells, heating caused a rapid increase (within 1 min) in [fCa2+i] of approximately 100 nM, which remained relatively constant during 25 min of continued heating; however, survival was higher in thermotolerant cells. Procaine, lidocaine, and tetracaine had no effect on survival or [fCa2+i] of cells kept at 37 degrees C up to 25 min. Cells heated with procaine and lidocaine showed no difference in [fCa2+i] compared to cells heated without LAs but were greatly sensitized to killing. Cells heated with tetracaine became permeable to trypan blue within 10-15 min of heating. We conclude that heat sensitization by LAs does not involve changes in [fCa2+i]. Furthermore, these studies reject the hypothesis that changes in [fCa2+i] are involved in heat-induced cell killing.

An immunoassay for heat shock protein 73/72: use of the assay to correlate HSP73/72 levels in mammalian cells with heat response.

An enzyme-linked immunosorbent assay (ELISA) for measurement of levels of heat shock proteins 73 and 72 (HSP73/72) in cultured cells and tissues is described. The assay involves detection of HSP73/72 in cell homogenates in 96-well plates using a specific monoclonal antibody. The assay has been used to explore the relationship between the amount of HSP73/72 in a cell and its response to heat shock, both before and after the development of thermotolerance. Six mammalian cell lines with differing responses to heat were characterized with respect to their response to heat treatments at 44 degrees C and concentrations of HSP73/72. Contrary to the widely expressed idea that the amount of HSP73/72 dictates the degree of heat resistance, no positive correlation between levels of HSP73/72 and heat resistance was found for the six lines tested here: if one particular line, a mutant selected for heat resistance, was excluded from the analysis, there was a negative correlation between HSP73/72 levels and heat resistance. A different result was, however, obtained when thermotolerant (transiently resistant) cells were compared to control cells. Here, we found a good correlation between the extent of thermotolerance and the amount of HSP73/72, suggesting that an increase in HSP73/72 level is important for the development of thermotolerance. The validity of the ELISA technique was checked using a second method for quantifying levels of HSP73/72. This involved uniform radiolabeling of cellular proteins, separation on two-dimensional gels and radioscanning to quantify radioactivity in each protein. The second technique is more powerful in that different isoforms of HSP73/72 can be distinguished, but it is more difficult to perform, is more labour intensive and requires an expensive device for gel scanning. The results using the second technique agreed well with those from the immunoassay and indicated that the level of the highly inducible HSP72 correlated best with the extent of thermotolerance.

Association of HSP72 with the nuclear (TX-100-insoluble) fraction upon heating tolerant and non-tolerant HeLa S3 cells.

HSP72 levels in the cellular and the nuclear (TX-insoluble) fraction before and after heating of heat- and sodium arsenite-induced thermotolerant and non-tolerant HeLa S3 cells have been investigated by 1D- and 2D-electrophoresis, followed by Western blotting and immunostaining, using a newly developed monoclonal antibody that specifically detects HSP72 (Heine et al. 1991). HSP72 was constitutively expressed in HeLa S3 cells and elevated upon heat or arsenite stress. Immediate association of HSP72 with the nuclear fraction was induced by heat but not arsenite. However, at the time of maximal thermotolerance, elevated levels of HSP72 were found associated with nuclei isolated from both heat- and arsenite-induced thermotolerant cells. After (test) heat treatments (0-60 min at 45 degrees C) translocation of HSP72 to the nuclear fraction in all cells was observed, albeit with different kinetics and to different plateau values. When tolerant and non-tolerant cells were allowed to recover from a heat stress (at 37 degrees C) before isolation of the nuclei, no dissociation of HSP72 from the nuclear fraction was observed within a 5 h time period. Our data indicate that association/dissociation of HSP72 with/from the nuclear fraction is not related to the recovery from heat-induced intranuclear protein aggregation (Kampinga et al. 1992), nor to the extent of thermotolerance in the human HeLa S3 cell line.

Heat induced protein denaturation in the particulate fraction of HeLa S3 cells: effect of thermotolerance.

In this study we investigated the effect of heat on the proteins of the particulate fraction (PF) of HeLa S3 cells using electron spin resonance (ESR) and thermal gel analysis (TGA). ESR detects overall conformational changes in proteins, while TGA detects denaturation (aggregation due to formation of disulfide bonds) in specific proteins. For ESR measurements the -SH groups of the proteins were labelled with a maleimido bound spin label (4-maleimido-tempo). The sample was heated inside the ESR spectrometer at a rate of 1 degree C/min. ESR spectra were made every 2-3 degrees C between 20 degrees C and 70 degrees C. In the PF of untreated cells conformational changes in proteins were observed in three temperature stretches: between 38 and 44 degrees C (transition A, TA); between 47 and 53 degrees C (transition B, TB); and above 58 degrees C (transition C, TC). With TGA, using the same heating rate, we identified three proteins (55, 70, and 90 kD) which denatured during TB. No protein denaturation was observed during TA, while during TC denaturation of all remaining proteins in the PF occurred. When the ESR and TGA measurements were done with the PF of (heat-induced) thermotolerant cells, TA was unchanged while TB and TC started at higher temperatures. The temperature shift for the onset of these transitions correlated with the degree of thermotolerance that was induced in the cells. These results suggest that protection against heat-induced denaturation of proteins in the PF is involved in heat induced thermotolerance.

The relationship between hsp 70 localization and heat resistance

Using indirect immunofluorescence we have investigated the kinetics of nuclear accumulation and removal of hsp 70 in HA-1 Chinese hamster fibroblasts exposed to elevated temperatures. The kinetics of accumulation of hsp 70 in the nuclei were found to be time/temperature dependent at all temperatures tested (42–45 °C). At a given temperature, the fraction of cells manifesting nuclear localization of hsp 70 increased with exposure time. For a given duration of heating, the fraction of cells manifesting nuclear localization of hsp 70 increased with the temperature. The kinetics of the nuclear accumulation of hsp 70 were similar for normal HA-1 cells, their heat-resistant variants, and transiently thermotolerant cells (triggered by prior exposure to a brief heat shock or to sodium arsenite). Upon return to 37 °C after heat shock, the kinetics of removal of the hsp 70 associated with the nucleus was dependent on the severity of the initial heat challenge. However, for a given heat dose, the decay of nuclear localization of hsp 70 was more rapid in thermotolerant and heatresistant cells than in their normal counterparts. These results suggest that the increased levels of hsp 70 associated with the transient or permanently heat-resistant state may play a direct role in restoring and/or repairing heat-induced nuclear and nucleolar alterations associated with heat-induced cell killing. Furthermore, they also suggest that the heat-resistant state may involve ameliorated repair of heat-induced cellular alterations.

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 °C) after abrief pretreatment at a milder temperature (35 °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 °C was sufficient to provide full splicing thermotolerance to a 30-min severe heat shock of 38 °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 °C incubation and is completely lost by 8 h after the pretreatment. The kinetics experiments of pre-mRNAs synthesized during the 38 °C treatment in splicing thermotolerant cells indicate that one or more splicing thermotolerance factors are synthesized during the 35 °C pretreatment which interact with pre-mRNA-eontaining 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 °C incubation are protected, whereas those synthesized late are not. In the absence of splicing thermotolerant factors, the pre-mRNA-eontaining complexes leave the normal splicing pathway and are allowed to exit to the Cytoplasm.

The thermoresistant state: Protection from initial damage or better repair?

The induction of and recovery from heat-induced perturbations in several cellular parameters were examined in normal, transiently thermotolerant, and permanently heat-resistant HA-1 Chinese hamster fibroblasts. The initial heat-induced perturbations in total cellular protein synthesis, RNA synthesis, vimentin-containing intermediate filaments, and nuclear protein mass were similar in the three different cell types which display various levels of thermal resistance as determined by clonogenic survival. The posthyperthermia recovery from the heat-induced perturbations in all of the cellular parameters was more rapid in both the permanently heat-resistant cells and in the transiently thermotolerant cells. This response was observed in cells in which transient thermotolerance was induced by either a mild heat shock or exposure to sodium arsenite. The development and decay of the capacity for more rapid recovery from the initial heat-induced perturbations in total cellular protein and RNA synthesis paralleled the development and decay of clonogenic thermotolerance. Overall, these results support the notion that more rapid recovery from similar levels of heat-induced perturbations in various cellular parameters are a salient feature of both the transiently and permanently heat-resistant state.

Induction of thermotolerance in T cells protects nuclear DNA topoisomerase I from heat stress

In this study, we have demonstrated that topoisomerase I DNA relaxing activity is protected against a severe heat shock in T cells made thermotolerant by a prior modest heat treatment. However, following a severe heat-shock challenge and incubation at 37 degrees C, topoisomerase activity in the control population eventually returned to levels similar to those detected in thermotolerant cells. This recovery of topoisomerase activity appears to result from the renaturation of heat-inactivated enzyme rather than from synthesis of new protein because the rate of recovery of catalytic activity was not inhibited by the presence of the protein synthesis inhibitor, cycloheximide.

Induced thermotolerance to apoptosis in a human T lymphocyte cell line.

A brief exposure to elevated temperatures elicits, in all organisms, a transient state of increased heat resistance known as thermotolerance. The mechanism for this thermotolerant state is unknown primarily because it is not clear how mild hyperthermia leads to cell death. The realization that cell death can occur through an active process of self destruction, known as apoptosis, led us to consider whether thermotolerance provides protection against this mode of cell death. Apoptosis is a common and essential form of cell death that occurs under both physiological and pathological conditions. This mode of cell death requires the active participation of the dying cell and in this way differs mechanistically from the alternative mode of cell death, necrosis. Here we show that mild hyperthermia induces apoptosis in a human leukemic T cell line. This is evidenced by chromatin condensation, nuclear fragmentation and the cleavage of DNA into oligonucleosome size units. DNA fragmentation is a biochemical hallmark of apoptosis and requires the activation of an endogenous endonuclease. The extent of DNA fragmentation was proportional to the severity of heat stress for cells heated at 43 degrees C from 30 to 90 minutes. A brief conditioning heat treatment induced a resistance to apoptosis. This was evident as a resistance to DNA fragmentation and a reduction in the number of apoptotic cells after a heat challenge. Resistance to DNA fragmentation developed during a recovery period at 37 degrees C and was correlated with enhanced heat shock protein (hsp) synthesis. This heat-induced resistance to apoptosis suggests that thermotolerant cells have gained the capacity to prevent the onset of this pathway of self-destruction. An examination of this process in heated cells should provide new insights into the molecular basis of cellular thermotolerance.

Inhibition of heat shock gene expression does not block the development of thermotolerance.

After cells have been exposed to a nonlethal heat shock, they develop an enhanced resistance to subsequent prolonged heat shock. This process, termed thermotolerance, correlates with the expression of a group of proteins called the heat shock proteins. When cells are exposed to heat, protein synthesis is rapidly turned off and takes 5-6 hr to recover. In thermotolerant cells, protein synthesis is not blocked by heat. The heat shock proteins are thought to be responsible for the development of thermotolerance and the protection of the protein synthesis machinery from heat inactivation. To test the hypothesis that the heat shock proteins are involved in the heat shock response, we used two inhibitors to block their transcription and expression during heating and then monitored the effect on the development of thermotolerance and on protein synthesis. Camptothecin inhibits DNA topoisomerase I and blocks transcription of all actively transcribed genes, whereas dichloro-D-ribofuranosylbenzimidazole (DRB) inhibits only those genes transcribed by RNA polymerase II. Both DRB and camptothecin blocked the heat-induced expression of the heat shock proteins, but the absence of these proteins did not block either the development of thermotolerance or the protection of protein synthesis after heating. The data indicate that thermotolerance can develop in the absence of new protein synthesis.

Acquisition of thermotolerance induced by heat and arsenite in HeLa S3 cells: multiple pathways to induce tolerance?

Recent data indicate that cells may acquire thermotolerance via more than one route. In this study, we observed differences in thermotolerance development in HeLa S3 cells induced by prior heating (15 minutes at 44 degrees C) or pretreatment with sodium-arsenite (1 hour at 37 degrees C, 100 microM). Inhibition of overall protein and heat shock protein (HSP) synthesis (greater than 95%) by cycloheximide (25 micrograms/ml) during tolerance development nearly completely abolished thermotolerance induced by arsenite, while significant levels of heat-induced thermotolerance were still apparent. The same dependence of protein synthesis was found for resistance against sodium-arsenite toxicity. Toxic heat, but not toxic arsenite treatments caused heat damage in the cell nucleus, measured as an increase in the protein mass of nuclei isolated from treated cells (intranuclear protein aggregation). Recovery from this intranuclear protein aggregation was observed during post-heat incubations of the cells at 37 degrees C. The rate of recovery was faster in heat-induced tolerant cells than in nontolerant cells. Arsenite-induced tolerant cells did not show an enhanced rate of recovery from the heat-induced intranuclear protein aggregation. In parallel, hyperthermic inhibition of RNA synthesis was the same in tolerant and nontolerant cells, whereas post-heat recovery was enhanced in heat-induced, but not arsenite-induced thermotolerant cells. The more rapid recovery from heat damage in the nucleus (protein aggregation and RNA synthesis) in cells made tolerant by a prior heat treatment seemed related to the ability of heat (but not arsenite) to induce HSP translocations to the nucleus.