How is the immune response affected by hyperthermia and heat shock proteins?

There is growing body of evidence linking the cellular response to heat stress with the response of the immune system to cancer. The anti-tumor immune response can be markedly enhanced by treatment with hyperthermia particularly in the fever range. In addition, the heat shock proteins (hsp) which are produced in abundant quantities in cells exposed to heat are potent immune modulators and can lead to stimulation of both the innate and adaptive immune responses to tumors. Immunostimulation by hyperthermia involves both direct effects of heat on the behavior of immune cells as well as indirect effects mediated through hsp release. In addition, the hsp can be deployed as components of antitumor vaccines in protocols that do not include hyperthermia. Understanding these process may permit the effective deployment of hyperthermia and hsp based vaccines in tumor treatment.     

Suppression of heat-induced hsp72 accumulation by cisplatin in human glioblastoma cells

The accumulation of the inducible hsp72 (72-kDa heat shock protein) after hyperthermia and/or cisplatin treatment in human glioblastoma cell line (A-172) was studied by Western blot analysis. The level of hsp72 increased to eight-fold 10 h after hyperthermia alone (44 °C for 20 min, D₅₀) and to three-fold 10 h after cisplatin treatment (5 μg/ml) at 37 °C for 15 min (D₅₀). In contrast, when the cells were simultaneously heated with cisplatin, the accumulation of hsp72 was suppressed. The level of hsp72 increased to about six-fold and two-fold 10 h after hyperthermia (44 °C, 15 min) in the presence of 1 and 10 μg/ ml (D₅₀ or D₁₀) of cisplatin, respectively. In addition, we found both the enhancement of thermosensitivity and the suppression of thermotolerance by the simultaneously combined treatment of hyperthermia and cisplatin. It has been reported that the enhancement of cisplatin cytotoxicity by hyperthermia is due to increase of both cisplatin uptake and DNA damage by hyperthermia. Our results suggest that the interactive cytotoxic enhancement by the combination of hyperthermia and cisplatin may be also due to the suppression of heat-induced hsp72 accumulation by cisplatin.     

Enhancement of heat-induced heat shock protein (hsp)72 accumulation by doxorubicin (Dox) in vitro

Previous studies have shown that cellular thermotolerance develops in response to exposure to doxorubicin (Dox) and treatment with hyperthermia. In the present study, we evaluated the induction of thermotolerance and the accumulation of heat shock protein (hsp)72 after treatment with Dox and/or hyperthermia at 44 degrees C in Chinese hamster V-79 cells. Thermotolerance developed after exposure to Dox at 37 degrees C for 2 h in a dose-dependent manner. Western blot analysis showed no accumulation of hsp72 after exposure to Dox (0.2 microg/ml) for 2 h (lethal dose (LD)(15)). Hsp72 accumulated 12 h after hyperthermia at 44 degrees C for 35 min (LD(15)). However, when the cells were exposed to Dox (0.1 microg/ml) followed by heating at 44 degrees C for 25 min (LD(15)), accumulation of hsp72 was observed after 6 h. These results suggest that enhancement of induction of hsp72 accumulation by Dox may be involved in the development of thermotolerance induced by sequential treatment with Dox and hyperthermia.     

Molekulare und immunologische Effekte der Hyperthermie auf Tumorprogression und Metastasierung

Hyperthermia treatment in the temperature range 39–43°C can influence the adaptive and natural immune system in addition to the direct toxic effect of temperatures above 41°C on cells. The type of cell destruction (necrosis or apoptosis) plays an important role in whether a pro-inflammatory or anti-inflammatory immune response is triggered by hyperthermia. In addition danger signals, such as high mobility group B1 protein, adenosine-5-triphosphate (ATP) and heat-shock protein, which are released from necrotic tumor cells, can stimulate cells of the natural immune system and therefore assist the anti-tumor response. In this review article the various molecular and immunological mechanisms which are regulated by hyperthermia treatment will be described in detail.     

Re-induction of hsp70 synthesis: an assay for thermotolerance

Of the many heat shock proteins (HSPs), hsp70 appears to correlate best with heat resistance, either permanent or transient. We have investigated various approaches to quantify the concentration of hsp 70, and examined the relationship between hsp70 and cells' thermal sensitivity during the development and decay of thermotolerance in model systems. Here, experiments were performed to determine the possibility of using the rate of synthesis of hsp70 after a second test heat shock to predict the kinetics of thermotolerance. Specifically, we studied the relationship between the retained thermotolerance in a murine tumor cell line SQ-1 and a human tumor cell line, HCT-8, after fractionated heat doses and the cells' ability to re-initiate synthesis of hsp70 in response to an additional test heat dose in vitro. Monolayers of cells were exposed to a first heat treatment (e.g., 41 degrees C, 4 h) and then incubated at 37 degrees C for 0-72 h. At various times after the first heat treatment, cells were either challenged with a 45 degrees C, 45 min heat shock to assess the residual thermotolerance by colony formation, or labelled with [35S]methionine before or after an additional test heat dose (e.g. 43.5 degrees C, 15 min). We found that the cells' ability to re-initiate hsp70 synthesis in response to the test heat shock inversely correlated with retained thermotolerance. Our data suggest the level of hsp70 in thermotolerant cells regulates the rate of synthesis of additional hsp70 in response to the subsequent heat challenge. Furthermore, the results showed that the rate of re-induction of hsp70 synthesis after a test shock can be used as a rapid measure of retained thermotolerance. This study suggests an approach for quantifying the level of retained thermotolerance during a course of fractionated hyperthermia.     

Hsp27 protects the cytoskeleton and nucleus from the effects of 42°C at pH 6.7 in CHO cells adapted to growth at pH 6.7

CHO cells are normally sensitized to hyperthermia by acidic pH. However, CHO cells adapted to growth in pH 6.7 medium become less sensitive to heat killing at the reduced pH. The adapted cells maintain their ability to develop thermotolerance at pH 6.7 and their steady state intracellular pH is elevated. Furthermore, the small molecular weight stress chaperone, hsp27, is elevated in unheated cells maintained at pH 6.7. This report documents that the cytoskeletal and nuclear components of the low pH adapted CHO cells are resistant to 42°C-induced collapse and protein accretion, respectively. Hyperthermia induced a perinuclear collapse of the microtubular cytoskeleton and an increase in the amount of insoluble protein associated with the nuclei and nuclear matrix fractions in the control cells heated at pH 7.3 or heated after acute acidification to pH 6.7. Protection from these effects was observed in the low pH adapted cells heated at pH 6.7. Hsp70 does not appear to play a dominant role in the response of the adapted cells to 42°C. The induction of hsp70 during heating is abrogated by pH 6.7 in cells cultured at either pH 7.3 or pH 6.7. The resistance of the microtubular cytoskeleton to perinuclear collapse and the absence of protein aggregation in the nucleus during 42°C may be due to the elevated levels of hsp27 both before heating and during the heat treatment. In summary, the phenotype of CHO cells adapted to growth at low pH includes resistance of the cytoskeleton to 42°C-induced perinuclear collapse and resistance to 42°C-induced aggregation of nuclear proteins, in addition to the reduction in heat cytotoxicity, upregulation of intracellular pH and upregulation of hsp27.     

Hyperthermia classic article commentary: ‘Re-induction of hsp70 synthesis: An assay for thermotolerance’ by Gloria C. Li and Johnson Y. Mak,International Journal of Hyperthermia1989;5:389-403

Of the many heat shock proteins (HSPs), hsp70 appears to correlate best with heat resistance, either permanent or transient. We have investigated various approaches to quantify the concentration of hsp70, and examined the relationship between hsp70 and cells’ thermal sensitivity during the development and decay of thermotolerance in model systems. Specifically, experiments were performed to determine the possibility of using the rate of synthesis of hsp70 after a second test heat shock to predict the kinetics of thermotolerance in tumor cells in vitro and in animal tumor models. We found that the cells’ ability to re-initiate hsp70 synthesis in response to the test heat shock inversely correlated with retained thermotolerance. These data suggest the level of hsp70 in thermotolerant cells regulates the rate of synthesis of additional hsp70 in response to the subsequent heat challenge. Furthermore, the results showed that the rate of re-induction of hsp70 synthesis after a test heat shock can be used as a rapid measure of retained thermotolerance. This study suggests an approach for quantifying the level of retained thermotolerance during fractionated hyperthermia.     

Hsp27 protects the cytoskeleton and nucleus from the effects of 42 degrees C at pH 6.7 in CHO cells adapted to growth at pH 6.7.

CHO cells are normally sensitized to hyperthermia by acidic pH. However, CHO cells adapted to growth in pH 6.7 medium become less sensitive to heat killing at the reduced pH. The adapted cells maintain their ability to develop thermotolerance at pH 6.7 and their steady state intracellular pH is elevated. Furthermore, the small molecular weight stress chaperone, hsp27, is elevated in unheated cells maintained at pH 6.7. This report documents that the cytoskeletal and nuclear components of the low pH adapted CHO cells are resistant to 42 degrees C-induced collapse and protein accretion, respectively. Hyperthermia induced a perinuclear collapse of the microtubular cytoskeleton and an increase in the amount of insoluble protein associated with the nuclei and nuclear matrix fractions in the control cells heated at pH 7.3 or heated after acute acidification to pH 6.7. Protection from these effects was observed in the low pH adapted cells heated at pH 6.7. Hsp70 does not appear to play a dominant role in the response of the adapted cells to 42 degrees C. The induction of hsp70 during heating is abrogated by pH 6.7 in cells cultured at either pH 7.3 or pH 6.7. The resistance of the microtubular cytoskeleton to perinuclear collapse and the absence of protein aggregation in the nucleus during 42 degrees C may be due to the elevated levels of hsp27 both before heating and during the heat treatment. In summary, the phenotype of CHO cells adapted to growth at low pH includes resistance of the cytoskeleton to 42 degrees C-induced perinuclear collapse and resistance to 42 degrees C-induced aggregation of nuclear proteins, in addition to the reduction in heat cytotoxicity, upregulation of intracellular pH and upregulation of hsp27.     

Re-induction of hsp70 synthesis: An assay for thermotolerance

Of the many heat shock proteins (HSPs), hsp70 appears to correlate best with heat resistance, either permanent or transient. We have investigated various approaches to quantify the concentration of hsp70, and examined the relationship between hsp70 and cells' thermal sensitivity during the development and decay of thermotolerance in model systems. Here, experiments were performed to determine the possibility of using the rate of synthesis of hsp70 after a second test heat shock to predict the kinetics of thermotolerance. Specifically, we studied the relationship between the retained thermotolerance in a murine tumor cell line SQ-1 and a human tumor cell line, HCT-8, after fractionated heat doses and the cells' ability to re-initiate synthesis of hsp70 in response to an additional test heat dose in vitro. Monolayers of cells were exposed to a first heat treatment (e.g., 41°C, 4 h) and then incubated at 37°C for 0–72 h. At various times after the first heat treatment, cells were either challenged with a 45°C, 45 min heat shock to assess the residual thermotolerance by colony formation, or labelled with [³⁵S]methionine before or after an additional test heat dose (e.g. 43.5°C, 15 min). We found that the cells' ability to re-initiate hsp70 synthesis in response to the test heat shock inversely correlated with retained thermotolerance. Our data suggest the level of hsp70 in thermotolerant cells regulates the rate of synthesis of additional hsp70 in response to the subsequent heat challenge. Furthermore, the results showed that the rate of re-induction of hsp70 synthesis after a test shock can be used as a rapid measure of retained thermotolerance. This study suggests an approach for quantifying the level of retained thermotolerance during a course of fractionated hyperthermia.     

Re-induction of hsp70 synthesis: An assay for thermotolerance

Of the many heat shock proteins (HSPs), hsp70 appears to correlate best with heat resistance, either permanent or transient. We have investigated various approaches to quantify the concentration of hsp70, and examined the relationship between hsp70 and cells' thermal sensitivity during the development and decay of thermotolerance in model systems. Here, experiments were performed to determine the possibility of using the rate of synthesis of hsp70 after a second test heat shock to predict the kinetics of thermotolerance. Specifically, we studied the relationship between the retained thermotolerance in a murine tumor cell line SQ-1 and a human tumor cell line, HCT-8, after fractionated heat doses and the cells' ability to re-initiate synthesis of hsp70 in response to an additional test heat dose in vitro. Monolayers of cells were exposed to a first heat treatment (e.g., 41 °C, 4 h) and then incubated at 37°C for 0–72 h. At various times after the first heat treatment, cells were either challenged with a 45 °C, 45 min heat shock to assess the residual thermotolerance by colony formation, or labelled with [³⁵S]methionine before or after an additional test heat dose (e.g. 43.5°C, 15 min). We found that the cells' ability to re-initiate hsp70 synthesis in response to the test heat shock inversely correlated with retained thermotolerance. Our data suggest the level of hsp70 in thermotolerant cells regulates the rate of synthesis of additional hsp70 in response to the subsequent heat challenge. Furthermore, the results showed that the rate of re-induction of hsp70 synthesis after a test shock can be used as a rapid measure of retained thermotolerance. This study suggests an approach for quantifying the level of retained thermotolerance during a course of fractionated hyperthermia.     

Cancer immunotherapy: stress proteins and hyperthermia

Heat shock proteins (hsps) can induce anti-cancer immune responses by targeting associated tumour antigens to the immune system. Hsps are not merely carriers of antigen but can also induce maturation of dendritic cells (DCs), resulting in a more efficient antigen presentation. However, improvement of hsp-based vaccines is still desirable if one is to realize their full therapeutic potential. Since the immune system consists of different elements functioning together in a highly integrated way, a combination therapy utilizing important immunomodulators together with hsp-based vaccination may improve therapeutic response. Hyperthermia has been shown to have important stimulatory effects on several cellular and organismal endpoints related to the immune system. This review highlights advantages and disadvantages of various ways of using stress proteins in cancer immunotherapy. It also overviews the interaction of hyperthermia with heat shock protein therapy and the related effects on the host's immune response.     

Enhanced efficacy of tumor cell vaccines transfected with secretable hsp70

Tumor immunotherapy has exploited the ability of heat shock proteins to chaperone precursors of antigenic peptides to antigen-presenting cells and to activate efficiently an immune response against tumor-associated antigens. The most common strategy is based on the purification of heat shock protein-peptide complexes from tumor cell lines or from tumor surgical samples for in vivo administration. In this article, we have modified the murine-inducible hsp70 into a secreted protein and engineered tumor cells to secrete constitutively their antigenic repertoire associated with the hsp70 protein. In vitro studies showed that the relocalization of hsp70 from the cytoplasm to the secretory pathway did not modify the ability of hsp70 to interact with peptides derived either from natural tumor-associated antigens or model antigens, and that antigen-presenting cells specifically took up the secreted hsp70 and presented the chaperoned epitopes to T cells. In vivo studies showed that tumors secreting hsp70 displayed increased immunogenicity, with induction of a strong and specific CTL response. Mice injected with hsp70-secreting tumors showed increased survival and impaired tumor take compared with mice bearing parental tumors. More than 70% of mice rejected tumor cells secreting hsp70 through mechanisms that involve T lymphocytes and natural killer cells, with the induction of a memory response in the case of T lymphocytes. Moreover, hsp70 secretion increased the immunogenic potential of tumor cell vaccines.     

Cancer immunotherapy: stress proteins and hyperthermia.

Heat shock proteins (hsps) can induce anti-cancer immune responses by targeting associated tumour antigens to the immune system. Hsps are not merely carriers of antigen but can also induce maturation of dendritic cells (DCs), resulting in a more efficient antigen presentation. However, improvement of hsp-based vaccines is still desirable if one is to realize their full therapeutic potential. Since the immune system consists of different elements functioning together in a highly integrated way, a combination therapy utilizing important immunomodulators together with hsp-based vaccination may improve therapeutic response. Hyperthermia has been shown to have important stimulatory effects on several cellular and organismal endpoints related to the immune system. This review highlights advantages and disadvantages of various ways of using stress proteins in cancer immunotherapy. It also overviews the interaction of hyperthermia with heat shock protein therapy and the related effects on the host's immune response.     

Effect of hyperthermia on selective expression of HL-60 heat shock proteins

Hyperthermia is used experimentally to treat human malignancy. The effect of heat delivery rate and thermotolerance on the expression of heat shock proteins (hsp) by the human HL-60 cell line before and after differentiation was studied. This leukemia cell synthesized multiple hsp in response to elevated temperatures. The most obvious and consistent proteins were within the highly conserved stress-inducible family of polypeptides hsp70 which resolved as a hsp69/72 doublet. Cells which were made thermotolerant by gradual heating selectively failed to express the hsp70 doublet even though other hsp were synthesized. Mature HL-60 cells induced to differentiate by incubation in retinoic acid expressed a full complement of hsp when exposed to immediate heat, but there was selective deletion of hsp70 with gradual hyperthermia. This model for selective induction of hsp confirms that synthesis of hsp and thermotolerance can be dissociated in the HL-60. It suggests that the hsp70 does not play an obligatory role in thermotolerance of this human leukemia cell.     

Response of human breast cancer cells to heat shock and chemotherapeutic drugs.

Previous studies have shown that certain chemotherapeutic drugs are less effective on tumor cells when cells have been previously exposed to hyperthermia. In the present study, we have evaluated whether specific modifications in heat shock protein (hsp) expression are associated with resistance to anticancer drugs. RNA levels for hsp90, hsp70, and hsp27 were studied by Northern and slot blots, while proteins were studied by two-dimensional gel electrophoresis, in MCF-7/BK and MDA-MB-231 breast cancer cells. The sensitivities of these cells to doxorubicin, colchicine, 5-fluorouracil, cisplatin, actinomycin D, and methotrexate were tested by clonogenic assays. These techniques were applied to both cell lines before (control) and after heat shock. The study revealed that elevated hsp70 and hsp27 levels were associated with doxorubicin resistance. In addition, the presence of phosphorylated hsp27 isoforms was also associated with doxorubicin resistance. The study showed that elevated hsps were not associated with multidrug resistance. Heat shock did not induce P170 glycoprotein mRNA overexpression or resistance to the other drugs tested. We also found that the level of doxorubicin protection conferred by the overexpression of hsp was lower than that obtained in cells expressing a multidrug resistance phenotype (MDA-A1R cells). In these cells, heat shock did not confer additional doxorubicin resistance and hsp27 phosphorylation was deficient. Our studies suggest that specific hsps are associated with doxorubicin resistance in certain human breast cancer cells and that this mechanism seems to be independent of the multidrug resistance system.     

Screening of stress enhancer based on analysis of gene expression profiles: enhancement of hyperthermia-induced tumor necrosis by an MMP-3 inhibitor

To improve the therapeutic benefit of hyperthermia, we examined changes of global gene expression after heat shock using DNA microarrays consisting of 12 814 clones. HeLa cells were treated for 1 h at 44°C and RNA was extracted from the cells 0, 3, 6, and 12 h after heat shock. The 664 genes that were up or down-regulated after heat shock were classified into 7 clusters using fuzzy adaptive resonance theory (fuzzy ART). There were 41 genes in two clusters that were induced in the early phase after heat shock. In addition to shock response genes, such as hsp70 and hsp40 , the stress response genes c-jun, c-fos and egr-1 were expressed in the early phase after heat shock. We also found that expression of matrix metalloproteinase 3 (MMP-3) was enhanced during the early response. We therefore investigated the role of MMP-3 in the heat shock response by examining HeLa cell survival after heat treatment in the presence and absence of an MMP-3 inhibitor, N-isobutyl-N-(4-methoxyphenyl-sulfonyl)glycylhydroxamic acid (NNGH) or N-hydroxy-2(R)-[[4-methoxysulfonyl] (3-picolyl)amino]-3-methylbutaneamide hydro-chloride (MMI270). The number of surviving cells 3 days after heat treatment significantly decreased, reaching 3.5% for NNGH and 0.2% for MMI270. These results indicate that the MMP-3 inhibitors enhanced heat shock-induced cell death and behaved as stress enhancers in cancer cells. This valuable conclusion was reached as a direct result of the gene expression profiling that was performed in these studies.     

Heating the patient: a promising approach?

There is a clear rationale for using hyperthermia in cancer treatment. Treatment at temperatures between 40 and 44 degrees C is cytotoxic for cells in an environment with a low pO(2) and low pH, conditions that are found specifically within tumour tissue, due to insufficient blood perfusion. Under such conditions radiotherapy is less effective, and systemically applied cytotoxic agents will reach such areas in lower concentrations than in well perfused areas. Therefore, the addition of hyperthermia to radiotherapy or chemotherapy will result in at least an additive effect. Furthermore, the effects of both radiotherapy and many drugs are enhanced at an increased temperature. Hyperthermia can be applied by several methods: local hyperthermia by external or internal energy sources, regional hyperthermia by perfusion of organs or limbs, or by irrigation of body cavities, and whole body hyperthermia. The use of hyperthermia alone has resulted in complete overall response rates of 13%. The clinical value of hyperthermia in addition to other treatment modalities has been shown in randomised trials. Significant improvement in clinical outcome has been demonstrated for tumours of the head and neck, breast, brain, bladder, cervix, rectum, lung, oesophagus, vulva and vagina, and also for melanoma. Additional hyperthermia resulted in remarkably higher (complete) response rates, accompanied by improved local tumour control rates, better palliative effects and/or better overall survival rates. Generally, when combined with radiotherapy, no increase in radiation toxicity could be demonstrated. Whether toxicity from chemotherapy is enhanced depends on sequence of the two modalities, and on which tissues are heated. Toxicity from hyperthermia cannot always be avoided, but is usually of limited clinical relevance. Recent developments include improvements in heating techniques and thermometry, development of hyperthermia treatment planning models, studies on heat shock proteins and an effect on anti-cancer immune responses, drug targeting to tumours, bone marrow purging, combination with drugs targeting tumour vasculature, and the role of hyperthermia in gene therapy. The clinical results achieved to date have confirmed the expectations raised by results from experimental studies. These findings justify using hyperthermia as part of standard treatment in tumour sites for which its efficacy has been proven and, furthermore, to initiate new studies with other tumours. Hyperthermia is certainly a promising approach and deserves more attention than it has received until now.     

Antitumor Immunity Induction by Intracellular Hyperthermia Using Magnetite Cationic Liposomes

Induction of antitumor immunity to T-9 rat glioma by intracellular hyperthermia using functional magnetic particles was investigated. Magnetite cationic liposomes (MCLs), which have a positive surface charge, were used as heating mediators for intracellular hyperthermia. Solid T-9 glioma tissues were formed subcutaneously on both femurs of female F344 rats, and MCLs were injected via a needle only into the left solid tumors (treatment side). The rats were then divided into two groups, which received no irradiation, or irradiation for 30 min given three times at 24-h intervals with an alternating magnetic field (118 kHz, 384 Oe). On the treatment side, the tumor tissue disappeared completely in many rats exposed to the magnetic field. The tumor tissue on the opposite side also disappeared completely, even though MCLs were not injected into the right solid tumors. To examine whether a long-lasting and tumor-specific immunity could be generated, the rats that had been cured by the hyperthermia treatment were rechallenged with T-9 cells 3 months later. After a period of transient growth, all tumors disappeared. Furthermore, immuno-cytochemical assay revealed that the immune response induced by the hyperthermia treatment was mediated by both CD8⁺ and CD4⁺ T cells and accompanied by a marked augmentation of tumor-selective cytotoxic T lymphocyte activity. These results suggest that our magnetic particles are potentially effective tools for hyperthermic treatment of solid tumors, because in addition to killing of the tumor cells by heat, a host immune response is induced.     

Heat shock response by cells treated with azetidine-2-carboxylic acid

The purpose of this study is to reinvestigate the heat shock response in cells treated with the antimetabolite azetidine-2-carboxylic acid (azetidine), an analogue of proline. Previous studies could not clearly discriminate between the progressive thermosensitization caused by amino acid analogues and a parallel induction of thermotolerance by heat shock. Incubation of H35 cells with 2.5 mm azetidine causes an increasing thermosensitization which achieves a maximum after approximately 18-22 h. At this point, azetidine does not prevent the development of acute thermotolerance following a heat shock at 42.5 C, or the simultaneous induction of chronic thermotolerance during mild hyperthermia at 38-41 C. However, for both the acute and chronic heating conditions thermotolerance levels are reduced in proportion with azetidine-thermosensitization. Incorporation of azetidine causes an apparent downward temperature shift of approximately 1 C relative to the time-temperature relationships for normal, or following heat shocks, for thermotolerant cells. After 18h of incubation with azetidine, protein synthesis is reduced by a factor of 4 and cells show a preferential synthesis of heat shock proteins (hsp). A heat shock then, although inducing thermotolerance, is not followed by any noticeable effect on the synthesis of hsps. It is shown that the combination of prolonged azetidine treatment and heat shock causes a persistent inhibition of protein synthesis. This is hypothesized to result in the development of hsp synthesis independent thermotolerance. Additional treatment following heat shock in azetidine-treated cells with the protein synthesis inhibitor cycloheximide does not affect the induction of thermotolerance. In contrast to the heat shock response, no thermotolerance induction is observed in azetidine-treated cells after an exposure to sodium arsenite.