Effects of low culture temperature on the induction of hsp70 mRNA and the accumulation of hsp70 and hsp105 in mouse FM3A cells.

We have shown that heat shock does not induce the synthesis of hsp70 in FM3A cells maintained at a low culture temperature of 33 degrees C although it does so in cells maintained at 37 degrees C [T. Hatayama et al. (1991) Biochem. Int. 24, 467-474]. In this paper, we show that FM3A cells maintained at 37 degrees C produced hsp70 mRNA during continuous heating at 42 degrees C or during postincubation at either 37 or 33 degrees C after being heated at 45 degrees C for 15 min, whereas cells maintained at 33 degrees C did not produce hsp70 mRNA during continuous heating at 37, 39, 42, or 45 degrees C, or during postincubation after being heated at any temperature. Thus the lack of hsp70 synthesis in cells maintained at 33 degrees C seemed to be due to the absence of hsp70 mRNA induction. Also, hsp70 was accumulated in cells maintained at 37 degrees C during continuous heating at 42 degrees C and during postincubation at 37 degrees C after heat shock at 45 degrees C, but not during postincubation at 33 degrees C. The cellular level of the constitutive hsp73 as well as the mRNA level were both similar in cells maintained at 33 and 37 degrees C. On the other hand, the cellular level of the constitutive hsp105 in cells maintained at 33 degrees C was only half of that in cells maintained at 37 degrees C. These hsp105 levels increased significantly in both types of cells after continuous heating at 39 degrees C. These findings indicate that the culture temperature affects not only the induction of hsp70 mRNA but also the accumulation of hsp70 and hsp105 in the cells.     

Analysis of the expression of the two major proteins of the 70 kilodalton mammalian heat shock family

This study compares the expression after heat shock of the two major variants of the mammalian 70 kilodalton heat shock family in three separate systems. The ability of wild type and temperature sensitive mutant (ts85) FM3A cells to elicit a heat shock response following a 45 degrees C, 12 min exposure was examined. The ts85 cells were found to be both significantly more thermosensitive than parent FM3A cells and to induce a 66kDa heat shock protein (hsp66) not visibly synthesized in the parent line by this exposure. However, a constitutive (synthesized at 37 degrees C) 68kDa heat shock protein (hsp68) is comparably induced in both cell lines after heat. A relationship between the severity of the heat exposure as seen by the cell and hsp66 expression is suggested and tested in Chinese hamster ovary cells. In CHO cells a brief 45 degrees C heat shock induces the constitutive hsp68 (but not hsp66), while longer and more severe exposures are required for the expression of hsp66. The induction of these two proteins is also examined in situ in mouse skeletal muscle. In this case both hsp66 and hsp68 are induced following comparatively mild heat treatments, and the 'threshold' for hsp66 induction observed in cultured cells either does not occur or is greatly reduced. However, once again, hsp68 is naturally synthesized at 37 degrees C while hsp66 appears to be de novo synthesized after heat shock.     

Dissociation of 68,000 Mr heat shock protein synthesis from thermotolerance expression in rat fibroblasts

Rat embryonic fibroblasts growing exponentially at either 35, 37, or 39 degrees C were exposed to 42 degrees C for times up to 6 hr. Cell survival was unaffected by this heat shock in cultures growing at 39 degrees C but survival was decreased in a temperature dependent manner in cells growing at 37 or 35 degrees C. Exposure to 42 degrees C of cells previously adapted to 35 or 37 degrees C resulted in the induction of heat shock proteins (hsps) with apparent molecular weights of 68,000 (hsp 68), 70,000 (hsp 70), and 89,000 (hsp 89); cells previously adapted to 39 degrees C expressed all hsps except hsp 68. Inasmuch as the synthesis of certain hsps may function to protect cells from thermal damage, these data indicate that hsp 68 may not be required for this adaptation-related thermotolerant survival response. Hsp 68 may only be expressed in cells destined to die.     

Examination of heat shock protein mRNA accumulation in early Xenopus laevis embryos

Elevation of the incubation temperature of Xenopus laevis neurulae from 22 to 33-35 degrees C induced the accumulation of heat shock protein (hsp) 70 mRNA (2.7 kilobases (kb)) and a putative hsp 87 mRNA (3.2 kb). While constitutive levels of both hsp mRNAs were detectable in unfertilized eggs and cleavage-stage embryos, heat-induced accumulation was not observed until after the mid-blastula stage. Exposure of Xenopus laevis embryos to other stressors, such as sodium arsenite or ethanol, also induced a developmental stage-dependent accumulation of hsp 70 mRNA. To characterize the effect of temperature on hsp 70 mRNA induction, neurulae were exposed to a range of temperatures (27-37 degrees C) for 1 h. Heat-induced hsp 70 mRNA accumulation was first detectable at 27 degrees C, with relatively greater levels at 30-35 degrees C and lower levels at 37 degrees C. A more complex effect of temperature on hsp 70 mRNA accumulation was observed in a series of time course experiments. While continuous exposure of neurulae to heat shock (27-35 degrees C) induced a transient accumulation of hsp 70 mRNA, the temporal pattern of hsp 70 mRNA accumulation was temperature dependent. Exposure of embryos to 33-35 degrees C induced maximum relative levels of hsp 70 mRNA within 1-1.5 h, while at 30 and 27 degrees C peak hsp 70 mRNA accumulation occurred at 3 and 12 h, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

T lymphocyte stress response. II. Protection of translation and DNA replication against some forms of stress by prior hyperthermic stress

We have compared the effects of a mild heat shock and febrile temperatures on heat-shock protein (hsp) synthesis and development of stress tolerance in T lymphocytes. Our previous studies demonstrated that febrile temperatures (less than or equal to 41 degrees C) induced the synthesis of hsp110, hsp90, and the constitutive or cognate form of hsp70 (hscp70; a weak induction of the strongly stress-induced hsp70 was also observed. In the studies reported herein, we demonstrate that a mild heat shock (42.5 degrees C) reverses this ratio; that is, hsp70 and not hscp70 is the predominate member of this family synthesized at this temperature. Modest heat shock also enhanced the synthesis of hsp110 and hsp90. In order to assess the relationship between hsp synthesis and the acquisition of thermotolerance, purified T cells were first incubated at 42.5 degrees C (induction temperature) and then subsequently subjected to a severe heat-shock challenge (45 degrees C, 30 min). T cells first incubated at a mild heat-shock temperature were capable of total protein synthesis at a more rapid rate following a severe heat shock than control cells (induction temperature 37 degrees C). This phenomenon, which has been previously termed translational tolerance, did not develop in cells incubated at the febrile temperature (induction temperature 41 degrees C). Protection of translation also extended to immunologically relevant proteins such as interleukin-2 and the interleukin-2 receptor. Because clonal expansion is a critical event during an immune response, the effects of hyperthermic stress on DNA replication (mitogen-induced T cell proliferation) was also evaluated in thermotolerant T cells. DNA synthesis in control cells (induction temperature 37 degrees C) was severely inhibited following heat-shock challenge at 44 degrees C or 45 degrees C; in contrast, T cells preincubated at 42.5 degrees C rapidly recovered their DNA synthetic capacity. T cells preincubated at a febrile temperature were moderately protected against hyperthermic stress. The acquisition of thermotolerance was also associated with enhanced resistance to chemical (ethanol)-induced stress but not to heavy metal toxicity (cadmium) or dexamethasone-induced immunosuppression. These studies suggest that prior hsp synthesis may protect immune function against some forms of stress (e.g., febrile episode) but would be ineffective against others such as elevated glucocorticoid levels which normally occur during an immune response.     

Differential temperature dependency of chemical stressors in HSF1-mediated stress response in mammalian cells

Expression of stress proteins is generally induced by a variety of stressors. To gain a better understanding of the sensing and induction mechanisms of stress responses, we studied the effects of culture temperature on responses to various stressors, since the induction of hsp70 in mammalian cells by heat shock is somehow modulated by culture temperature. Hsp70 was not induced by treatment with sodium arsenite, azetidine-2-carboxylic acid, or zinc sulfate at the level of heat shock factor (HSF) 1 activation in cells incubated at low temperature, although these treatments induced hsp70 in cells incubated at 37 degrees C. The repression of sodium arsenite or zinc sulfate-induced HSF1 activation by low temperature was not simply due to the inhibition of protein synthesis. On the other hand, heat shock and iodoacetamide induced HSF 1 activation in cells incubated at either temperature. Thus, there seem to be two kinds of stressors that induce HSF1 activation independently of or dependent on culture temperature. Furthermore, the reduction of glutathione level seemed to be essential for HSF1 activation by chemical stressors.     

Anti-proliferative effects of heating on the human prostatic carcinoma cells in culture

Prostatic cancers are well-known to be sensitive to heat stress. However, the mechanism by which the cancer cells are killed by high temperature remains poorly understood. The present study was undertaken to determine the anti-proliferative effects of heat stress on the prostatic cancer cells in culture. Heat shock at 43 degrees C inhibited the cell growth of three different prostatic cell lines. Flow cytometrical analysis using BrdU and PI showed a decrease in the proportion of cells in an S phase, accompanied by cell accumulation in G1 and G2, in both JCA-1 and PC-3 but not in LNcap. Both JCA-1 and PC-3 presented a strong expression of hsp70 at 37 degrees C. The heat shock caused apparent enhancement of the expression of hsp70 through the cell cycle. A treatment at 43 degrees C for 8 hours resulted in not only an apparent increment of positive hsp70 cells, but cells with subdiploid DNA content in LNcap. Flow cytometrical analysis by FITC-labeled Annexin V showed increment of apoptotic cells at 43 degrees C for 8 hours in LNcap cells. The results suggest that apoptosis is an important pathway of heat-induced killing of these cells. In conclusion, the cell growth of prostatic cancers may be affected by the temperature through relationship of the cell cycle and hsp70.     

The 70-kilodalton heat-shock proteins of the SSA subfamily negatively modulate heat-shock-induced accumulation of trehalose and promote recovery from heat stress in the yeast, Saccharomyces cerevisiae.

In the yeast, Saccharomyces cerevisiae, the disaccharide trehalose is a stress-related metabolite that accumulates upon exposure of cells to heat shock or a variety of non-heat inducers of the stress response. Here, we describe the influence of mutations in individual heat-shock-protein genes on trehalose metabolism. A strain mutated in three proteins of the SSA subfamily of 70-kDa heat-shock proteins (hsp70) overproduced trehalose during heat shock at 37 degrees C or 40 degrees C and showed abnormally slow degradation of trehalose upon temperature decrease from 40 degrees C to 27 degrees C. The mutant cells were unimpaired in the induction of thermotolerance; however, the decay of thermotolerance during recovery at 27 degrees C was abnormally slow. Since both a high content of trehalose and induced thermotolerance are associated with the heat-stressed state of cells, the abnormally slow decline of trehalose levels and thermotolerance in the mutant cells indicated a defect in recovery from the heat-stressed state. A similar albeit minor defect, as judged from measurements of trehalose degradation during recovery, was detected in a delta hsp104 mutant, but not in a strain deleted in the polyubiquitin gene, UB14. In all our experiments, trehalose levels were closely correlated with thermotolerance, suggesting a thermoprotective function of trehalose. In contrast, heat-shock proteins, in particular hsp70, appear to be involved in recovery from the heat-stressed state rather than in the acquisition of thermotolerance. Cells partially depleted of hsp70 displayed an abnormally low activity of neutral trehalase when shifted to 27 degrees C after heat shock at 40 degrees C. Trehalase activity is known to be under positive control by cAMP-dependent protein kinases, suggesting that hsp70 directly or indirectly stimulate these protein-kinase activities. Alternatively, hsp70 may physically interact with neutral trehalase, thereby protecting the enzyme from thermal denaturation.     

Heat shock-induced acquisition of thermotolerance at the levels of cell survival and translation in Xenopus A6 kidney epithelial cells.

In this study we have investigated the acquisition of thermotolerance in a Xenopus laevis kidney A6 epithelial cell line at both the level of cell survival and translation. In cell survival studies, A6 cells were incubated at temperatures ranging from 22 to 35 degrees degrees C for 2 h followed by a thermal challenge at 39 degrees degrees C for 2 h and a recovery period at 22 degrees C for 24 h. Optimal acquisition of thermotolerance occurred at 33 degrees degrees C. For example, exposure of A6 cells to 39 degrees degrees C for 2 h resulted in only 3.4% survival of the cells whereas prior exposure to 33 degrees C for 2 h enhanced the survival rate to 69%. This state of thermotolerance in A6 cells was detectable after 1 h at 33 degrees C and was maintained even after 18 h of incubation. Cycloheximide inhibited the acquisition of thermotolerance at 33 degrees C suggesting the requirement for ongoing protein synthesis. The optimal temperature for the acquisition of translational thermotolerance also occurred at 33 degrees C. Treatment of A6 cells at 39 degrees C for 2 h resulted in an inhibition of labeled amino acid incorporation into protein which recovered to approximately 14% of control after 19 h at 22 degrees C whereas cells treated at 33 degrees C for 2 h prior to the thermal challenge recovered to 58% of control levels. These translationally thermotolerant cells displayed relatively high levels of the heat shock proteins hsp30, hsp70, and hsp90 compared to pretreatment at 22, 28, 30, or 35 degrees C. These studies demonstrate that Xenopus A6 cells can acquire a state of thermotolerance and that it is correlated with the synthesis of heat shock proteins.     

Inhibition of glucose-regulated and heat shock protein induction by low temperature

The present study evaluating induction of the major stress proteins in the subphysiological temperature range (25-33 degrees C) shows that none of the agents used could effectively induce the heat shock proteins (hsp) or the glucose related protein grp95 at low temperature. However, grp82 was still induced by some amino acid analogs and by glucose deprivation while certain oxygen-regulated proteins were still induced by hypoxia at 25 degrees C. Analogs were incorporated and protein turnover was increased at low temperature even though most stress proteins were not induced. Synthesis of hsps, but not that of grps, was induced if cultures containing analog-substituted proteins were shifted to 37 degrees C. Temperature dependence of hsp induction by arsenite showed a sharp threshold between 30 degrees C and 33 degrees C. Low temperature inhibition of induction points to the existence of a temperature-dependent mechanism operating within the normal physiological temperature range and may be a useful parameter in evaluating proposed mechanisms of stress protein regulation.     

Effect of continuous heat stress on cell growth and protein synthesis in Aedes albopictus

Aedes albopictus (clone C6/36) cells, which normally grow at 28 degrees C, were maintained at a supraoptimal temperature of 37 degrees C. The effect of continuous heat stress (37 degrees C) on cell growth was analyzed as were the modifications occurring with protein synthesis during short- and long-term heat stress. We observed that cells in lag or exponential growth phase, present inhibition of cell growth, and cells in the lag phase showed more sensitivity to death than cells growing exponentially. During the first hour of exposing the cells to 37 degrees C, they synthesized two heat shock proteins (hsps) of 82 kd and 70 kd, respectively, concomitant with inhibition of normally produced proteins at control temperature (28 degrees C). However, for incubations longer than 2 hr at 37 degrees C, a shift to the normal pattern of protein synthesis occurred. During these transitions, two other hsps of 76 kd and 90 kd were synthesized. Pulse chase experiments showed that the 70-kd hsp is stable at least for 18 hr, when the cells are returned to 28 degrees C. However, if cells were incubated at 37 degrees C, the 70-kd hsp is stable for at least 48 hr. The 70-kd hsp was localized in the cytoplasmic and in the nuclear compartment. Our results indicate a possible role of hsp 70-kd protein in the regulation of cell proliferation.     

Loss of heat-shock acquisition of thermotolerance in yeast is not correlated with loss of heat-shock proteins

Yeast cells when subjected to a primary heat shock, defined as a temperature shift from 23 to 37 degrees C for 30 min, acquired tolerance to heat stress (52 degrees C/5 min). Primary heat shocked cells incubated at 23 degrees C for up to 3 h, progressively lost thermotolerance but retained high levels of the major heat-shock proteins as observed on polyacrylamide gels. On the other hand, a temperature shift back up to 37 degrees C for 30 min fully restored thermotolerance. The major high-molecular-mass heat-shock proteins (hsp) identified were of approximate molecular mass 100 kDa (hsp 100), 80 kDa (hsp 80) and 70 kDa (hsp 70). The results indicate that loss of heat-shock acquisition of thermotolerance is not correlated with loss of heat-shock proteins.     

Common antigenicity of mouse 42 degrees C-specific heat-shock protein with mouse HSP 105

Mammalian cells incubated at 42 degrees C synthesize a specific heat-shock protein at 42 degrees C (42 degrees C-hsp) that is not induced by heat-shock at 45 degrees C or by other stresses that induce major heat shock proteins (Hatayama et al. (1986) Biochem. Biophys. Res. Commun. 137, 957-963). Antibody raised against a heat-shock protein with molecular weight of 105,000 (hsp 105) purified from mouse FM 3A cells cross-reacted to the 42 degrees C-hsp of the same cells. The antibody reacted only weakly to hsp 105 and 42 degrees C-hsp of human HeLa cells. These results suggested that hsp 105 and 42 degrees C-hsp have the same antigenic determinant, and that 42 degrees C-hsp may have a structure similar to that of hsp 105.     

Acquired thermotolerance following heat shock protein synthesis prevents impairment of mitochondrial ATPase activity at elevated temperatures in Saccharomyces cerevisiae

The complex molecular response of cells to sudden temperature changes is a well-characterized phenomenon. Although it is clear that the induction of heat shock proteins provides protection from heat in all of the organisms so far tested, very little is known about the role that this set of proteins plays in cellular homeostasis. Recently, putative roles for hsp60 and hsp70-like proteins have been proposed in Saccharomyces cerevisiae. hsp70-like proteins have been shown to be necessary for translocation of precursor polypeptides into mitochondria and endoplasmic reticulum, while hsp60 is required for the assembly of precursor polypeptides into oligomeric complexes following incorporation into the mitochondrial matrix. In this paper, we report that a brief temperature shock (44 degrees C) impairs coupling of oxidative phosphorylation in S. cerevisiae as measured indirectly by the Cl-CCP/oligomycin assay. Furthermore, at high temperature oligomycin stimulates rather than inhibits oxygen uptake under nonthermotolerant conditions. Pretreatment of cells for a short period of time at 37 degrees C, prior to exposure to higher temperatures rescues the capacity to maintain coupling between oxidative phosphorylation and electron transport. Inhibition of cytoplasmic RNA or protein synthesis during heat shock prevents the protection of this mitochondrial activity. We propose that one of the roles of the induction of heat shock proteins (or related activities) is to protect mitochondrial ATPase activity under conditions of further increase in temperature.     

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 degrees 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 degrees 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 heat-resistant 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.