Development Of Thermotolerance Research Articles

A new model of thermal inactivation and its application to clonogenic survival data for WiDr human colonic adenocarcinoma cells.

Based on the analysis of clonogenic survival data for human colonic adenocarcinoma cells (WiDr) after a single heating, a new model is proposed to describe cell survival after hyperthermia quantitatively. The effects of heat are explained as heat-induced cell damage assuming a first-order (single-hit) and a second-order (cumulative damage) process. Thus cell survival at a specified temperature can be described by the linear-quadratic (LQ) model. The proposed model is based on an alternative definition of the (single) thermal dose, given as the (normalized) product of heating time and a specified nonlinear function of the increase in temperature (relative to a threshold temperature) to be interpreted as the thermal dose rate. In further analogy to the modeling of the effects of low-dose-rate radiation, an inherent capacity of the cells to repair sublethal damage is assumed, and these effects are quantified by the usual g factor measuring incomplete repair effects. The model defines thermal dose-response and isoeffect dose relationships, enabling a direct (i. e. single-step) analysis of the available thermal response data. Additionally, the analysis of our data based on heating times in the range from 0 to 360 min and temperatures from 41 to 46 degrees C and covering a broad spectrum of different densities of cells seeded for colony formation did not yield any evidence of the existence of a breaking point usually derived from Arrhenius plots based on the single-hit, multitarget model and the Arrhenius equation. The model includes no specific assumptions describing the development of thermotolerance, which can be assumed to be negligible under our experimental conditions. The proposed thermal dose-response model correlates satisfactorily with the in vitro survival data for WiDr adenocarcinoma cells.

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°C in Chinese hamster V-79 cells. Thermotolerance developed after exposure to Dox at 37°C for 2 h in a dose-dependent manner. Western blot analysis showed no accumulation of hsp72 after exposure to Dox (0.2 μg/ml) for 2 h (lethal dose (LD) 15). Hsp72 accumulated 12 h after hyperthermia at 44°C for 35 min (LD 15). However, when the cells were exposed to Dox (0.1 μg/ml) followed by heating at 44°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.

Hsp72-mediated suppression of c-Jun N-terminal kinase is implicated in development of tolerance to caspase-independent cell death.

Pretreatment with mild heat shock is known to protect cells from severe stress (acquired thermotolerance). Here we addressed the mechanism of this phenomenon by using primary human fibroblasts. Severe heat shock (45 degrees C, 75 min) of the fibroblasts caused cell death displaying morphological characteristics of apoptosis; however, it was caspase independent. This cell death process was accompanied by strong activation of Akt, extracellular signal-regulated kinase 1 (ERK1) and ERK2, p38, and c-Jun N-terminal (JNK) kinases. Suppression of Akt or ERK1 and -2 kinases increased cell thermosensitivity. In contrast, suppression of stress kinase JNK rendered cells thermoresistant. Development of thermotolerance was not associated with Akt or ERK1 and -2 regulation, and inhibition of these kinases did not reduce acquired thermotolerance. On the other hand, acquired tolerance to severe heat shock was associated with downregulation of JNK. Using an antisense-RNA approach, we found that accumulation of the heat shock protein Hsp72 is necessary for JNK downregulation and is critical for thermotolerance. The capability of naive cells to withstand moderate heat treatment also appears to be dependent on the accumulation of Hsp72 induced by this stress. Indeed, exposure to 45 degrees C for 45 min caused only transient JNK activation and was nonlethal, while prevention of Hsp72 accumulation prolonged JNK activation and led to massive cell death. We also found that JNK activation by UV irradiation, interleukin-1, or tumor necrosis factor was suppressed in thermotolerant cells and that Hsp72 accumulation was responsible for this effect. Hsp72-mediated suppression of JNK is therefore critical for acquired thermotolerance and may play a role in tolerance to other stresses.

Heat shock-induced arrests in different cell cycle phases of rat C6-glioma cells are attenuated in heat shock-primed thermotolerant cells.

The response kinetics of rat C6 glioma cells to heat shock was investigated by means of flow cytometric DNA measurements and western blot analysis of HSP levels. The results showed that the effects on cell cycle progression are dependent on the cell cycle phase at which heat shock is applied, leading to either G1 or G2/M arrest in randomly proliferating cells. When synchronous cultures were stressed during G0 they were arrested with G1 DNA content and showed prolongation of S and G2 phases after release from the block. In proliferating cells, HSC70 and HSP68 were induced during the recovery and reached maximum levels just before cells were released from the cell cycle blocks. Hyperthermic pretreatment induced thermotolerance both in asynchronous and synchronous cultures as evidenced by the reduced arrest of cell cycle progression after the second heat shock. Thermotolerance development was independent of the cell cycle phase. Pre-treated cells already had high HSP levels and did not further increase the amount of HSP after the second treatment. However, as in unprimed cells, HSP reduction coincided with the release from the cell cycle blocks. These results imply that the cell cycle machinery can be rendered thermotolerant by heat shock pretreatment and supports the assumption that HSP70 family members might be involved in thermotolerance development.

HSP16.6 is involved in the development of thermotolerance and thylakoid stability in the unicellular cyanobacterium, Synechocystis sp. PCC 6803.

The low molecular weight (LMW) heat shock protein (HSP), HSP16.6, in the unicellular cyanobacterium, Synechocystis sp. PCC 6803, protects cells from elevated temperatures. A 95% reduction in the survival of mutant cells with an inactivated hsp16.6 was observed after exposure for 1 h at 47 degrees C. Wild-type cell survival was reduced to only 41%. HSP16.6 is also involved in the development of thermotolerance. After a sublethal heat shock at 43 degrees C for 1 h and subsequent challenge exposure at 49 degrees C for 40 min, mutant cells did not survive, while 64% of wild-type cells survived. Ultrastructural changes in the integrity of thylakoid membranes of heat-shocked mutant cells also are discussed. These results demonstrate an important protective role for HSP16.6 in the protection of cells and, in particular, thylakoid membrane against thermal stress.

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.

Inhibition of thermotolerance observed on tsAF8 cells

The characteristic development of thermotolerance was examined in tsAF8 cells, which were temperature sensitive cell line derived from BHK21. tsAF8 cells lose RNA polymerase II activity and arrest in the G1 phase of the cell cycle at the nonpermissive temperature of 39.5-40.6°C. tsAF8 cells were pre-heated at 45°C for 20 min, incubated at 34 or 39.7°C for different durations, and then heated again at 45°C for 30 min. The cells were trypsinized immediately after the second heating to evaluate cell survival. As a result, thermotolerance was developed rapidly in the cells incubated at 34°C. However, the survival rate of the cells incubated at 39.7°C was almost constant. Thermotolerance of BHK21 cells, which were wild-type cells of tsAF8 cells, developed at 39.7°C. These results suggest that thermotolerance may be inhibited in tsAF8 cells incubated at 39.7°C, and that some metabolism which is inhibited in tsAF8 cells at 39.7°C may be related to thermotolerance development.

The Escherichia coli heat shock protein ClpB restores acquired thermotolerance to a cyanobacterial clpB deletion mutant

In both prokaryotes and eukaryotes, the heat shock protein ClpB functions as a molecular chaperone and plays a key role in resisting high temperature stress. ClpB is important for the development of thermotolerance in yeast and cyanobacteria but apparently not in Escherichia coli. We undertook a complementation study to investigate whether the ClpB protein from E coli (EcClpB) differs functionally from its cyanobacterial counterpart in the unicellular cyanobacterium Synechococcus sp. PCC 7942. The EcClpB protein is 56% identical to its ClpB1 homologue in Synechococcus. A plasmid construct was prepared containing the entire E coli clpB gene under the control of the Synechococcus clpB1 promoter. This construct was transformed into a Synechococcus clpB1 deletion strain (deltaclpB1) and integrated into a phenotypically neutral site of the chromosome. The full-length EcClpB protein (EcClpB-93) was induced in the transformed Synechococcus strain during heat shock as well as the smaller protein (EcClpB-79) that arises from a second translational start inside the single clpB message. Using cell survival measurements we show that the EcClpB protein can complement the Synechococcus deltaclpB1 mutant and restore its ability to develop thermotolerance. We also demonstrate that both EcClpB-93 and -79 appear to contribute to the degree of acquired thermotolerance restored to the Synechococcus complementation strains.

Increased thermosensitivity associated with topoisomerase I deletion and promoter mutations in Escherichia coli.

An Escherichia coli mutant with three of the promoters for the topoisomerase I gene (topA) deleted, such that only the sigma 32-dependent promoter (P1) remained, had a decreased level of topoisomerase I at 30 degrees C and showed increased thermosensitivity at 52 degrees C. However, it could still develop thermotolerance and had a wild-type level of resistance to 52 degrees C treatment if exposed first to 42 degrees C. This indicated that newly synthesized topoisomerase I from transcription initiated at P1 was important for development of thermotolerance. Two other E. coli mutants lacking topA were > 100 times more sensitive to high temperature than their wild-type isogenic strains.

Increased thermosensitivity associated with topoisomerase I deletion and promoter mutations in Escherichia coli

An Escherichia coli mutant with three of the promoters for the topoisomerase I gene ( topA) deleted, such that only the σ 32-dependent promoter (P1) remained, had a decreased level of topoisomerase I at 30°C and showed increased thermosensitivity at 52°C. However, it could still develop thermotolerance and had a wild-type level of resistance to 52°C treatment if exposed first to 42°C. This indicated that newly synthesized topoisomerase I from transcription initiated at P1 was important for development of thermotolerance. Two other E. coli mutants lacking topA were >100 times more sensitive to high temperature than their wild-type isogenic strains.

Is radiosensitive cell line cross-sensitive to heat?: Effect of heat on two rat yolk sac tumor cell lines with different radiosensitivity

The differences between two rat yolk sac tumor cell lines, which are of the same origin but differ in their response to irradiation, in thermal sensitivity and development of thermotolerance were investigated. A radiosensitive cell line NMT-1 is consistently less heat sensitive than the radioresistant cell line NMT-1R. The thermotolerances in NMT-1 and in NMT-1R preheated at 43°C for 30 min, respectively, reached peaks at 6 h and at 12 h, but did not completely disappear at 48 h. Thermotolerance with a good correlation between both cell lines with HSP70 accumulation was observed in a different time course after a primary heating.

Stimulation of survival capacity in heat shocked cells by subsequent exposure to minute amounts of chemical stressors; role of similarity in hsp-inducing effects.

A brief and moderate heat shock to Reuber H35 hepatoma cells causes a rapid increase in the synthesis of heat shock proteins (hsp) and initiates the development of thermotolerance, which results in an increased ability to survive exposure to otherwise lethal temperatures. We now demonstrate that low doses of various chemical stressors (arsenite, cadmium, mercury, lead, copper, menadione and diethyldithiocarbamate (ddtc)), at concentrations that do not exert any effect in control cultures, are able to enhance the synthesis of hsps and to stimulate the development of thermotolerance when applied to cultures which were pretreated with a mild heat shock. The degree of stimulation appears to be stressor-specific, which is not only observed in the ensuing development of thermotolerance but also in the enhancement of the heat shock-induced synthesis of stress proteins. The different hsps that show an enhanced induction when heat shocked cultures are exposed to the various secondary applied low doses of chemical stressors, were found to resemble the hsp pattern that is characteristic for the secondary stressor and not for the initial heat shock. In other words, the nature of the post-treatment determines the observed pattern of enhanced synthesis of hsps. In order to analyze the origin of the stimulation of survival capacity by low doses of the mentioned stressors, we studied whether the degree of stimulation is determined by the degree of similarity between the overall stress response to heat shock and to the second stress condition when applied singly. The degree in which low doses of chemical stressors stimulate tolerance development and enhance the synthesis of hsps in cells that were previously heat shocked, appears to be related to the degree of similarity in the hsp pattern induced by both stressors. Our results support the notion that low doses of toxic compounds may, under certain conditions, have beneficial effects related to a stimulation of endogenous cytoprotective mechanisms.

Cepharanthin enhances thermosensitivity without a resultant reduction in the thermotolerance of a murine mammary carcinoma.

Cepharanthin (Ce) is a biscoclaurine alkaloid extracted from Stephania cepharantha Hayata. The results of our previous in vitro study indicated that Ce reduces thermotolerance by enhancing thermosensitivity. In the present study, we investigated the in vitro and in vivo effects of Ce on thermosensitivity and thermotolerance using a murine mammary carcinoma, MCa, and C3H/HeN mice. Ce enhanced the thermosensitivity of MCa cells for heating at 44 degrees C not only in vitro but also in vivo. The in vivo enhancement ratio +/- SD of Ce at 100 mg/kg for heating at 44 degrees C was 1.3+/-0.3. The fractionated heat treatments at 44 degrees C for 30 and 60 min with an interval time of 0-6 days resulted in the development of remarkable thermotolerance and the expression of heat shock protein 70 in MCa tumors after the first heating. Ce at 100 mg/kg given immediately after the first heating increased the expression of heat shock protein 70 in MCa tumors, and did not reduce the development of thermotolerance. Ce given immediately before the first or second heating also did not inhibit the thermotolerance. The results of this study suggest that Ce enhances the thermosensitivity of MCa tumors as a thermosensitizer, but that this mild thermosensitizing property of Ce might be insufficient to conquer the remarkable thermotolerance in MCa tumors that develops after the first heating.

332 Effects of Heat Stress on Spinach Seed Germination

Seed germination of spinach (Spinacia oleracea L.) is partially inhibited by a high germination temperature (35 °C). Tolerance of high germination temperatures varies widely depending on the variety used. We ascertained that seed germination of these spinach varieties was thermoinhibited at 35 °C and secondary dormancy was not induced as seeds germinated when transferred to optimum germination conditions (20 °C). Treatment with 99% oxygen and 10 ppm kinetin significantly increased germination of thermoinhibited varieties at 35 °C. During heat stress, all organisms produce heat shock proteins (HSPs), which may function as molecular chaperons, are possibly required for the development of thermotolerance, and may be crucial for cell survival during heat stress. Western blotting of SDS-PAGE gels using antibodies to various heat shock proteins indicated that spinach varieties with the highest degree of thermotolerance have higher levels of HSP expression than varieties with the lowest degree of thermotolerance during germination. These results suggest that thermotolerance could be further improved, either through a breeding program or possibly by genetic engineering.

The Bacillus subtilis htpG gene is not involved in thermal stress management.

To study the influence of the htpG gene on thermal stress management in Bacillus subtilis, two different kinds of htpG mutation were constructed. In one case, the gene was inactivated by insertion of a cat cassette in to the coding region; htpG was thus found to be non-essential. In the second case, the htpG gene was fused to a xylose-dependent promoter, allowing expression of the gene to be controlled. In the absence of HtpG protein, recovery of cells from a heat shock at 53 degrees C was retarded, and this delay could be eliminated by overproduction of HtpG. While htpG is not involved in the development of induced thermotolerance, DnaK and GroE proteins are absolutely required. Overproduction of class I heat-shock proteins prior to shifting cells to a lethal temperature is important but not sufficient for the development of intrinsic thermotolerance. It could be shown that the HtpG protein does not act as a cellular thermometer in B. subtilis.

Calcium is involved in regulation of the synthesis of HSPs in suspension‐cultured sugar beet cells under hyperthermia

Exposure of plants to elevated temperatures induces a complex set of changes that enable plants to adapt following heat stress. In order to test the effect of Ca2+ on heat shock‐induced changes in cell protein synthesis the incorporation of [ S]methionine into protein was studied in cultured sugar beet (Beta vulgaris L.) cells incubated in media containing different calcium concentrations. Heat shock inhibited the synthesis of non‐heat shock proteins (non‐HSPs) and promoted the synthesis of a set of HSPs, typical of plants. The synthesis of non‐HSPs was greatly inhibited by external Ca2+ removal by treatment of the cells with ethylene glycol‐bis(β‐aminoethylether)‐N,N,N′,N′‐tetraacetic acid. In contrast, extracellular Ca2+ appeared not to be strictly required for the de novo production of HSPs, but this cation exerted different effects on the synthesis of individual HSPs. Cell injury increased if the cells were exposed simultaneously to high temperature and Ca2+‐deficient medium. Recovery of HSP synthesis and reduced cell injury were observed after addition of exogenous calcium to Ca2+‐depleted cells. These findings are consistent with a Ca2+ requirement for the survival of the cells under heat shock, and likely for the development of cell thermotolerance.

Impaired induction of heat shock protein implicated in decreased thermotolerance in a temperature-sensitive multinucleated cell line.

Cells of a temperature-sensitive mutant line (tsFT101) derived from a mouse mammary carcinoma cell line (FM3A) become multinucleated at a non-permissive temperature of 39 degrees C because of disturbed cytokinesis. To explore how this relates to thermotolerance, we examined the proliferative activity of, and heat shock protein (HSP) expression in, FM3A and tsFT101 cells cultured at 37 degrees C and 39 degrees C after heat shock pretreatment (15 min exposure at 45 degrees C). FM3A cells developed thermotolerance when cultured at both 37 degrees C and 39 degrees C, but whereas tsFT101 cells developed thermotolerance at 37 degrees C, this was markedly reduced at 39 degrees C. Western blot analysis showed similar degrees of expression of constitutive HSP70 (HSP73) in FM3A and tsFT101 cells after heat shock pretreatment at both 37 degrees C and 39 degrees C. However, expression of inducible HSP70 (HSP72) was reduced in tsFT101 cells at 39 degrees C compared to 37 degrees C and to FM3A cells at both 37 degrees C and 39 degrees C. Heat shock pretreatment activated DNA binding of heat shock transcription factor (HSF) in FM3A cells at 37 degrees C and 39 degrees C, but only at 37 degrees C in tsFT101 cells. These results indicate that (1) multinucleation caused by disturbed cytokinesis increases temperature sensitivity, (2) HSP70 is critical for the development of thermotolerance in both FM3A and tsFT101 cells, and (3) decreased expression of inducible HSP70 parallels deficient development of thermotolerance in tsFT101 cells cultured at a non-permissive temperature.

Postharvest heat treatments

Abstract Postharvest heat treatments are being used for disinfestation and disinfection of an increasing variety of crops, including flowers, fruits and vegetables. This review focuses on the effects of heat on the commodity and its postharvest quality. The effects of a postharvest heat treatment on aspects of ripening and development of thermotolerance is discussed. Ethylene production, respiration, softening, color change and taste components such as soluble solids, acidity and volatiles are dealt with. Heat treatment induction of thermotolerance, both to high and low temperatures, so as to prevent heat injury and chilling injury is discussed, as well as the possible mechanisms of action of this response. Heat damage manifestations from unsuccessful treatments are described.

Suppression of heat-induced HSF activation by CDDP in human glioblastoma cells

Purpose: The kinetics of the accumulation of inducible 72-kD heat shock protein (hsp72) and the activation of heat shock transcriptional factor (HSF) after hyperthermia and/or CDDP treatment in two human glioblastoma cell lines, A-172 having the wild-type p53 gene and T98G having the mutated p53 gene were evaluated. Methods and Materials: Western blot analysis of hsp72, gel-mobility shift assay of HSF, cell survival, and development of thermotolerance were examined. Results: The prominent suppression of heat-induced hsp72 accumulation by CDDP was seen in A-172 cells, but not in T98G cells. This was due to the p53-dependent inhibition of heat-induced HSF activation by CDDP. The interactive hyperthermic enhancement of CDDP cytotoxicity was observed in A-172 cells, but not in T98G cells. In addition, the heat-induced thermotolerance was suppressed by the presence of CDDP in the pretreatment. Conclusion: Suppression of heat-induced hsp72 accumulation by CDDP contributes to an interactive hyperthermic enhancement of CDDP cytotoxicity in the cells bearing the wild-type p53 gene.

Heat Shock Protein 72 Modulates Pathways of Stress-induced Apoptosis

The resistance to stress-induced apoptosis conferred by the thermotolerant state or by exogenous expression of HSP72 was measured in mouse embryo fibroblasts. The induction of thermotolerance protects cells from heat, tumor necrosis factor alpha (TNFalpha), and ceramide-induced apoptosis but not from ionizing radiation. Because the development of thermotolerance is associated with increased levels of heat shock proteins, we determined whether constitutive expression of one of the major inducible heat shock proteins, HSP72, could also protect cells from stress-induced apoptosis. Cells expressing constitutive HSP72 were shown to have significantly reduced levels of apoptosis after heat, TNFalpha, and ceramide but not after ionizing radiation. Activation of stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) was found to be strongly inhibited in thermotolerant cells after heat shock but not after other stresses. Cells that constitutively express HSP72 did not demonstrate decreased SAPK/JNK activation after any of these stresses. Thus, factors other than HSP72 that are induced in the thermotolerant state are able to reduce activation of SAPK/JNK after heat stress. Notably, the level of activation of SAPK/JNK did not correlate with the amount of apoptosis detected after different stresses. Constitutive HSP72 expression inhibited poly(ADP-ribose) polymerase cleavage in cells after heat shock and TNFalpha but not after ceramide or ionizing radiation. The results suggest either that SAPK/JNK activation is not required for apoptosis in mouse embryo fibroblasts or that HSP72 acts downstream of SAPK/JNK. Furthermore, the data support the concept that caspase activity, which can be down-regulated by HSP72, is a crucial step in stress-induced apoptosis. Based on data presented here and elsewhere, we propose that the heat shock protein family can be classified as a class of anti-apoptotic genes, in addition to the Bcl-2 and inhibitor of apoptosis protein families of genes.