Inhibition of heat shock protein synthesis and thermotolerance by cycloheximide

Chinese hamster ovary (CHO) cells were exposed to a 43 degrees C, 15-min heat shock to study the relationship between protein synthesis and the development of thermotolerance. The 43 degrees C heat shock triggered the synthesis of three protein families having molecular weights of 110,000, 90,000, and 65,000 (HSP). These proteins were synthesized at 37 and 46 degrees C. This heat shock also induced the development of thermotolerance, which was measured by incubating the cells at 46 degrees C 4 h after the 43 degrees C heat treatment. CHO cells were also exposed to 20 micrograms/ml of cycloheximide for 30 min at 37 degrees C, 15 min at 43 degrees C, and 4 h at 37 degrees C. This treatment inhibited the enhanced synthesis of the Mr 110,000, 90,000, and 65,000 proteins. The cycloheximide was then washed out and the cells were incubated at 46 degrees C. HSP synthesis did not recover during the 46 degrees C incubation. This cycloheximide treatment also partially inhibited the development of thermotolerance. These results suggest that for CHO cells to express thermotolerance when exposed to the supralethal temperature of 46 degrees C protein synthesis is necessary.     

Hyperthermia-induced heat shock response and thermotolerance in postimplantation rat embryos

Postimplantation stage rat embryos (6-10 somites) undergo abnormal development after exposure to a temperature of 43 degrees C for 30 min. A heat shock of 43 degrees C for 30 min also induces the synthesis of a set of eight heat shock proteins (hsps) with molecular masses ranging from 28,000 to 82,000 Da. The synthesis of these hsps is rapidly induced after the heat shock is applied and rapidly decays after embryos are returned to 37 degrees C. A heat shock of 42 degrees C for 30 min has no effect on rat embryo growth and development, but does induce the synthesis of three hsps. The most prominent of these three is believed to be the typical mammalian 70 kDa hsp. Furthermore, a 42 degrees C, 30-min heat shock followed by a 43 degrees C 30-min heat shock leads to partial protection from the embryotoxic effects of a single exposure at 43 degrees C, i.e., thermotolerance.     

Characterization of the thermotolerant cell. I. Effects on protein synthesis activity and the regulation of heat-shock protein 70 expression

Exposure of mammalian cells to a nonlethal heat-shock treatment, followed by a recovery period at 37 degrees C, results in increased cell survival after a subsequent and otherwise lethal heat-shock treatment. Here we characterize this phenomenon, termed acquired thermotolerance, at the level of translation. In a number of different mammalian cell lines given a severe 45 degrees C/30-min shock and then returned to 37 degrees C, protein synthesis was completely inhibited for as long as 5 h. Upon resumption of translational activity, there was a marked induction of heat-shock (or stress) protein synthesis, which continued for several hours. In contrast, cells first made thermotolerant (by a pretreatment consisting of a 43 degrees C/1.5-h shock and further recovery at 37 degrees C) and then presented with the 45 degrees C/30-min shock exhibited considerably less translational inhibition and an overall reduction in the amount of subsequent stress protein synthesis. The acquisition and duration of such "translational tolerance" was correlated with the expression, accumulation, and relative half-lives of the major stress proteins of 72 and 73 kD. Other agents that induce the synthesis of the stress proteins, such as sodium arsenite, similarly resulted in the acquisition of translational tolerance. The probable role of the stress proteins in the acquisition of translational tolerance was further indicated by the inability of the amino acid analogue, L-azetidine 2-carboxylic acid, an inducer of nonfunctional stress proteins, to render cells translationally tolerant. If, however, analogue-treated cells were allowed to recover in normal medium, and hence produce functional stress proteins, full translational tolerance was observed. Finally, we present data indicating that the 72- and 73-kD stress proteins, in contrast to the other major stress proteins (of 110, 90, and 28 kD), are subject to strict regulation in the stressed cell. Quantitation of 72- and 73-kD synthesis after heat-shock treatment under a number of conditions revealed that "titration" of 72/73-kD synthesis in response to stress may represent a mechanism by which the cell monitors its local growth environment.     

Induction of heat shock protein and thermal sensitivity of mammalian cells maintained at low culture temperature.

Effects of low culture temperature on the induction of heat shock proteins in FM3A cells by a heat shock and on the thermal sensitivity of the cells were examined. FM3A cells maintained at 33 degrees C could not induce hsp70 during continuous heating or after a short heat shock at either 39, 42, or 45 degrees C, although FM3A cells maintained at a normal culture temperature of 37 degrees C can induce the synthesis of hsp70. Furthermore, the cells maintained at 33 degrees C were more sensitive to the subsequent heat shock than the cells maintained at 37 degrees C. Thus, the culture temperature of the mammalian cells may be an important factor for the induction of hsp70, and hsp70 may play an important role to protect or repair the thermal damage of cells.     

Thermoinduced radioresistance of Escherichia coli cells and heat shock proteins

Radioresistance of E. coli cells is slightly increased (dose modification factor (DMF) = 1.2) with temperature elevated from 4 degrees to 43 degrees C at the time of gamma-irradiation. However, an appreciable effect of the thermoinduced radioresistance (DMF = 1.7) was observed when the wild-type cells were exposed to gamma-radiation at 15-43 degrees C (but not at 4 degrees C) after 30-min preincubation at 43 degrees C. This effect was absent in htpR mutants, defective in induction of heat shock proteins, and coupled with the decreased post-irradiation DNA degradation in gamma-irradiated htpR+ cells. It is suggested that heat shock proteins are involved in the thermoinduced radioresistance.     

Effect of heat shock on the intracellular distribution of proteins

Exposure of cells to heat induces thermotolerance, a transient resistance to subsequent heat challenges. It has been shown that thermotolerance is correlated in time with the enhanced synthesis of heat shock proteins. In this study, the association of induced heat shock proteins with various cellular fractions was investigated and the heat-induced changes in skeletal protein composition in thermotolerant and control cells was compared. All three major heat shock proteins induced in Chinese hamster fibroblasts after a 46 degrees C, 4-min heat treatment (70, 87, and 110 kDa) were purified with the cytoplasmic fraction, whereas only the 70-kDa protein was also found in other cell fractions, including that containing the cellular skeleton. Immediately after a second heat treatment at 45 degrees C for 45 min, the 110-kDa protein from thermotolerant cells also purified extensively with the cellular skeletal fraction. In this regard, the 110-kDa protein behaved similarly to many other cellular proteins, since we observed an overall temperature-dependent increase in the total labeled protein content of the high-salt-resistant cellular skeletal fraction after heat shock. Pulse-chase studies demonstrated that this increased protein content gradually returned to normal levels after a 3-hr incubation at 37 degrees C. The alteration or recovery kinetics of the total labeled protein content of the cellular skeletal fraction after heat shock did not correlate with the dramatic increase in survival observed in thermotolerant cells. The relationship between heat shock proteins and thermotolerance, therefore, does not correlate directly with changes in the heat-induced cellular alterations leading to differences in protein fractionation.     

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.     

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.     

Heat-shock proteins in membrane vesicles of Bacillus subtilis

Fractionation of B. subtilis cells after heat shock, from 37 degrees C to 54 degrees C, shows an increase in synthesis of proteins localized in cell membranes and a decrease in synthesis of proteins localized in cytosol. There is no such effect of heat shock at temperature of 45 degrees C. Autoradiograms of electrophoretically separated proteins, labelled during heat shock at 54 degrees C, reveal 26 heat-shock proteins (hsps) in membrane vesicles and 11 hsps in cytosol, five of which are common to both fractions. Heat shock at 45 degrees C induces 18 hsps localized in membrane vesicles and 13 hsps localized in cytosol, six of which are common to both fractions. Results are interpreted as showing a relevant role of membrane proteins in cell response to shock at high temperature, pointing to two steps of defense against heat stress.     

Heat-induced modulation of lamin B content in two different cell lines

Previous work in our laboratory indicates that the nuclear matrix protein lamin B is a "prompt" heat shock protein, which increases significantly when human U-1 melanoma and HeLa cells are exposed to 45.5 degrees C for 5-40 min. Using Western blotting, we found that the lamin B content in U-1 and HeLa cells increased to a greater extent during post-heat incubation at 37 degrees C than during the heat dose itself. When HeLa cells were heated at 45.5 degrees C for 30 min, and then incubated at 37 degrees C for up to 7 h, lamin B content was increased significantly (1.69-fold maximum increase at 3 h) compared to unincubated heated cells. Also, thermotolerant HeLa cells showed a greater increase (up to 1.72-fold) in lamin B content during subsequent heating compared to nontolerant cells. The increase in lamin B content in thermotolerant cells, or when heated cells were incubated at 37 degrees C, was also observed in U-1 cells. HeLa cells heated in the presence of glycerol (a heat protector) showed a 1.21-1.72-fold increase in lamin B content compared to cells heated for 10-30 min without glycerol. In contrast, lamin B content decreased 1.23-1.85-fold when cells were heated for 10-30 min in the presence of procaine (a heat sensitizer) compared to cells heated without procaine. These data suggest that lamin B may play an important role in the heat shock response, and that modulation of lamin B content by heat sensitizers or protectors may play a role in regulation of heat sensitivity.     

Dephosphorylation of S6 and expression of the heat shock response in Drosophila melanogaster.

A basic ribosomal phosphoprotein of 30,000 molecular weight was rapidly dephosphorylated in cultured Drosophila melanogaster cells heat shocked at 37 degrees C. The protein was associated with the 40S ribosomal subunit and had an electrophoretic mobility similar to that of purified rat liver protein S6 on basic two-dimensional polyacrylamide gels as well as a similar partial proteolysis peptide map. In logarithmically growing cultures, this D. melanogaster S6 protein appeared to have a single phosphorylated species consisting of 30 to 40% of the total cellular S6. Thus, the nearly complete dephosphorylation of this protein observed in heat shock involves a large fraction of the cellular S6. The significance of this dephosphorylation in the expression of the heat shock response was investigated by examining the phosphorylation status of S6 in recovery from heat shock and in response to chemical inducers of the heat shock response. During recovery from a 30-min heat shock, the recovery of normal protein synthesis was almost complete in 2 to 4 hr, whereas there was no significant rephosphorylation of S6 for 8 h. Two chemical inducers of the heat shock response, canavanine and sodium arsenite, induced the synthesis of heat shock proteins in D. melanogaster cells. Sodium arsenite also caused an inhibition of normal protein synthesis similar to that observed in heat shock. Neither agent, however, caused significant dephosphorylation of S6. These results suggest that the dephosphorylation of S6, although invariably observed in heat-shocked cells, may in some cases be dissociated from both the induction of heat shock protein synthesis and the turnoff of normal protein synthesis which occur in a heat shock response.     

Thermal tolerance and heat shock protein synthesis during development in the anuran Lepidobatrachus laevis

Development of the Paraguayan anuran Lepidobatrachus laevis is unusual in that the larvae are obligate carnivores, facultative cannibals and apparently exist at high environmental temperatures in their natural habitat. In the present study, the effect of environmental temperature on the rate of anuran development was investigated. The larvae have a thermotolerance range of 18°C for normal development between 19 and 37°C. The effect of temperature on the rate of development was dramatic; larvae that were incubated at 36.8°C develop to stage 24 (Gosner) in approximately 9 h compared with 24 h for larvae incubated at 19°C. The ability of larvae to survive heat shock was also examined; larvae did not survive a shock of 45°C for 15 min when it was administered at stages 3, 5, 9, 10 or 20. However, using the same heat shock conditions, 50% survival was observed when larvae were shocked at stage 16. To study protein synthesis during heat shock, larvae were pulsed with [³⁵S]-methionine during heat shock and labeled proteins were analyzed by electrophoresis under reducing and denaturing conditions. Larvae synthesized two sets of heat-shock proteins at doublet molecular weights of 83/78 and 62/59 kDa. These proteins were synthesized independently of the stage of development at which the shock was administered or the magnitude of the heat shock.     

Heat-shocked A20 lymphoma cells fail to induce degranulation of cytotoxic T lymphocytes: possible mechanism of resistance

A20 lymphoma cells were subjected to heat shock for 2 h at 42 and 43 +/- 0.1 degrees C and then evaluated at 37 degrees C for sensitivity to lysis by intact allo-specific cytotoxic T lymphocytes (CTLs), perforin-containing granules isolated from CTLs, and Fas-mediated apoptosis. Heat shock at 42 degrees C caused little change in sensitivity of the lymphoma cell line to lysis by intact CTLs or their isolated cytotoxic granules, but caused increased sensitivity to Fas-mediated apoptosis. However, A20 cells shocked at 43 degrees C declined significantly in sensitivity to lysis by intact CTLs, while remaining very sensitive to perforin granules and to Fas-mediated apoptosis. Expression of the inducible heat shock protein was observed in A20 cells incubated at 43 degrees C, but not in those incubated at 42 degrees C, suggesting a role for heat shock proteins. Furthermore, A20 cells shocked at 43 degrees C did not provoke degranulation and secretion of granzymes by antigen-specific CTLs, although formation of CTL-target conjugates and levels of MHC class I molecules remained unchanged. These observations demonstrate that hyperthermia or febrile conditions may reduce susceptibility of target cells to CTL attack due to failure of antigen presentation and the inability of CTLs to recognize heat stressed targets, thus enabling targets to escape CTL attack.     

Stress tolerance in a yeast lipid mutant: membrane lipids influence tolerance to heat and ethanol independently of heat shock proteins and trehalose.

The response of a yeast unsaturated fatty acid auxotroph, defective in delta 9-desaturase activity, to heat and ethanol stresses was examined. The most heat- and ethanol-tolerant cells had membranes enriched with oleic acid (C18:1), followed in order by cells enriched with linoleic (C18:2) and linolenic (C18:3) acids. Cells subjected to a heat shock (25-37 degrees C for 30 min) accumulated trehalose and synthesized typical heat shock proteins. Although there were no obvious differences in protein profiles attributable to lipid supplementation of the mutant, relative protein synthesis as determined by densitometric analysis of autoradiograms suggested that hsp expression was different. However, there was no consistent relationship between the synthesis of heat shock proteins and the acquisition of thermotolerance in the lipid supplemented auxotroph or related wild type. Furthermore, trehalose accumulation was also not closely related to stress tolerance. On the other hand, the data presented indicated a more consistent role for membrane lipid composition in stress tolerance than trehalose, heat shock proteins, or ergosterol. We suggest that the sensitivity of C18:3-enriched cells to heat and ethanol may be attributable to membrane damage associated with increases in membrane fluidity and oxygen-derived free radical attack of membrane lipids.