Heat shock gene expression in Xenopus laevis A6 cells in response to heat shock and sodium arsenite treatments

Continuous exposure of a Xenopus laevis kidney epithelial cell line, A6, to either heat shock (33 degrees C) or sodium arsenite (50 microM) resulted in transient but markedly different temporal patterns of heat-shock protein (HSP) synthesis and HSP 70 and 30 mRNA accumulation. Heat-shock-induced synthesis of HSPs was detectable within 1 h and reached maximum levels by 2-3 h. While sodium arsenite induced the synthesis of some HSPs within 1 h, maximal HSP synthesis did not occur until 12 h. The pattern of HSP 70 and 30 mRNA accumulation was similar to the response observed at the protein level. During recovery from heat shock, a coordinate decline in HSPs and HSP 70 and 30 mRNA was observed. During recovery from sodium arsenite, a similar phenomenon occurred during the initial stages. However, after 6 h of recovery, HSP 70 mRNA levels persisted in contrast to the declining HSP 30 mRNA levels. Two-dimensional polyacrylamide gel electrophoresis revealed the presence of 5 HSPs in the HSP 70 family, of which two were constitutive, and 16 different stress-inducible proteins in the HSP 30 family. In conclusion, heat shock and sodium arsenite induce a similar set of HSPs but maximum synthesis of the HSP is temporally separated by 12-24 h.     

Changes in intracellular levels of Ap3A and Ap4A in cysts and larvae of Artemia do not correlate with changes in protein synthesis after heat-shock.

Artemia larvae respond to a brief heat-shock between 28 degrees and 40 degrees C with an increase in the synthesis of two groups of proteins of Mr 68,000 and 89,000. At 40 degrees C synthesis of all other proteins is strongly repressed. Cysts, which are naturally thermotolerant, synthesise both heat-shock proteins at temperatures up to 47 degrees C but maintain normal protein synthesis. During pre-emergence development, Ap3A is present in cysts at a concentration twice that of Ap4A. The maximum level of 7.6 pmol/10(6) cells is reached shortly before hatching of the larvae. After hatching, the levels of both nucleotides decline. A 40 degrees C heat-shock produces a 1.8-fold increase in both nucleotides within 20 min in cysts and larvae. A 2.8-fold increase results from a 47 degrees C heat-shock to cysts. The rates of increase parallel but do not precede the increases in the heat-shock proteins. Since non-heat-shocked cysts possess higher levels of Ap3A and Ap4A than do heat-shocked larvae, the observed heat-induced changes in gene expression cannot be explained simply in terms of the intracellular concentrations of these nucleotides.     

Induction of acquired thermotolerance in Tetrahymena thermophila: effects of protein synthesis inhibitors.

When Tetrahymena thermophila cells growing at 30 degrees C are shifted to either 40 or 43 degrees C, the kinetics and extent of induction of heat shock mRNAs in both cases are virtually indistinguishable. However, the cells shifted to 40 degrees C show a typical induction of heat shock protein (HSP) synthesis and survive indefinitely (100% after 24 h), whereas those at 43 degrees C show an abortive synthesis of HSPs and die (less than 0.01% survivors) within 1 h. Cells treated at 30 degrees C with the drugs cycloheximide or emetine, at concentrations which are initially inhibitory to protein synthesis and cell growth but from which cells can eventually recover and resume growth, are after this recovery able to survive a direct shift from 30 to 43 degrees C (ca. 70% survival after 1 h). This induction of thermotolerance by these drugs is as efficient in providing thermoprotection to cells as is a prior sublethal heat treatment which elicits the synthesis of HSPs. However, during the period when drug-treated cells recover their protein synthesis ability and simultaneously acquire the ability to subsequently survive a shift to 43 degrees C, none of the major HSPs are synthesized. The ability to survive a 1-h, 43 degrees C heat treatment, therefore, does not absolutely require the prior synthesis of HSPs. But, as extended survival at 43 degrees Celsius depends absolutely on the ability of cells to continually synthesize HSPs, it appears that a prior heat shock as well as the recovery from protein synthesis inhibition elicits a change in the protein synthetic machinery which allows the translation of HSP mRNAs at what would otherwise be a nonpermissive temperature for protein synthesis.     

Thermotolerance and the heat-shock response in Candida albicans

At elevated temperatures, yeast cells of Candida albicans synthesized nine heat-shock proteins (HSPs) with apparent molecular masses of 98, 85, 81, 76, 72, 54, 34, 26 and 18 kDa. The optimum temperature for the heat-shock response was 45 degrees C although HSPs were detected throughout the range 41-46 degrees C. Protein synthesis was not observed in cells kept at 48 degrees C. Yeast cells survived exposure to an otherwise lethal temperature of 55 degrees C when they had previously been exposed to 45 degrees C. The thermotolerance induced during incubation at 45 degrees C required protein synthesis, since protection was markedly reduced by trichodermin. Mercury ions induced a set of three stress proteins, one of which corresponded in size to an HSP, and cadmium ions evoked one stress protein seemingly unrelated to the HSPs observed after temperature shift.     

Heat and sodium arsenite act synergistically on the induction of heat shock gene expression in Xenopus laevis A6 cells

Heat shock protein (HSP) synthesis was studied in the Xenopus epithelial cell line A6 in response to heat and sodium arsenite, either singly or together. Temperatures of 33-35 degrees C consistently brought about the synthesis of HSPs at 87, 73, 70, 54, 31, and 30 kilodaltons (kDa), whereas sodium arsenite at 25-100 microM induced the synthesis of HSPs at 73 and 70 kDa. In cultures exposed to 10 microM sodium arsenite at 30 degrees C, HSP synthesis in the 68- to 73-kDa and 29- to 31-kDa regions was much greater than the HSP synthesis in response to each treatment individually. RNA dot blot analysis using homologous genomic subclones revealed that heat shock induced the accumulation of HSP 70 and 30 mRNAs. The sizes of the HSP 70 and 30 mRNAs determined by Northern hybridization were 2.7 and 1.5 kilobases, respectively. Sodium arsenite (10-100 microM) also induced the accumulation of both HSP 70 and 30 mRNAs. Finally, a mild heat shock (30 degrees C) plus a low concentration of sodium arsenite (10 microM) acted synergistically on HSP 70 and 30 mRNA accumulation in A6 cells. Thus sodium arsenite and heat act synergistically at the level of both HSP synthesis and HSP mRNA accumulation.     

Differences in preferential synthesis and redistribution of HSP70 and HSP28 families by heat or sodium arsenite in Chinese hamster ovary cells

Since both heat and sodium arsenite induce thermotolerance, we investigated the differences in synthesis and redistribution of stress proteins induced by these agents in Chinese hamster ovary cells. Five major heat shock proteins (HSPs; Mr 110, 87, 70, 28, and 8.5 kDa) were preferentially synthesized after heat for 10 min at 45.5 degrees C, whereas four major HSPs (Mr 110, 87, 70, and 28 kDa) and one stress protein (33.3 kDa) were preferentially synthesized after treatment with 100 microM sodium arsenite (ARS) for 1 hr. Two HSP families (HSP70a,b,c, and HSP28a,b,c) preferentially relocalized in the nucleus after heat shock. In contrast, only HSP70b redistributed into the nucleus after ARS treatment. Furthermore, the kinetics of synthesis of each member of HSP70 and HSP28 families and their redistribution were different after these treatments. The maximum rates of synthesis of HSP70 and HSP28 families, except HSP28c, were 6-9 hr after heat shock, whereas those of HSP70b and HSP28b,c were 0-2 hr after ARS treatment. In addition, the maximum rates of redistribution of HSP70 and HSP28 families occurred 3-6 hr after heat shock, whereas that of HSP70b occurred immediately after ARS treatment. The degree of redistribution of HSP70b after ARS treatment was significantly less than that after heat treatment. These results suggest that heat treatment but not sodium arsenite treatment stimulates the entry of HSP70 and HSP28 families into the nucleus.     

Evidence for a role of heat-shock proteins in proliferation after heat treatment of synchronized mouse neuroblastoma cells

A role for heat-shock proteins (HSPs) in proliferation after heat treatment was considered in synchronized mouse neuroblastoma cells. For this purpose enhancement of HSP synthesis after heat treatment was inhibited by actinomycin D and the effect of this on cell cycle progression into mitosis and on cell survival was studied both in thermoresistant G1- and in thermosensitive late S/G2-phase cells. In G1-phase cells expression of basal and heat-induced HSP synthesis was the same as that in late S/G2-phase cells, which suggests that regulation of thermoresistance throughout the cell cycle is not directly linked with HSP synthesis. The synthesis of HSP36, HSP68, and HSP70 was enhanced after a 30-min treatment at 41-43 degrees C. Increase of HSP synthesis after heat shock was partly suppressed by the presence of 0.1 microgram/ml actinomycin D during heat treatment, while 0.2 micrograms/ml prevented enhancement of HSP synthesis completely. Suppression of heat-induced HSP synthesis by actinomycin D had the same concentration dependency in G1- and late S/G2-phase cells. Actinomycin D potentiated induction of mitotic delay by heat treatment (30 min, 42.5 degrees C) but only under conditions where it actually inhibited heat-induced enhancement of HSP synthesis. Heat-induced cell killing was also potentiated by actinomycin D. The potentiating effect of actinomycin D on heat-induced mitotic delay and on heat-induced cell killing was more pronounced in G1-phase cells than in late S/G2-phase cells. These results give evidence for a role of HSPs in the resumption of proliferation after heat treatment and suggest that heated G1-phase cells are more dependent on HSP synthesis for recovery of proliferation after heat treatment than heated late S/G2-phase cells.     

Protection of Chinese hamster ovary cells from heat killing by treatment with cycloheximide or puromycin: involvement of HSPs?

Cycloheximide (CHM) or puromycin (PUR) added for 2 h before heating at 43 degrees C followed by either PUR or CHM during heat greatly protected cells from heat killing. This protection increased with inhibition of protein synthesis. Since treatment with a drug both before and during heating was required for heat protection, and since one drug could be exchanged for the other after the 2-h pretreatment without affecting the heat protection, a common mode of action involving inhibition of protein synthesis is suggested for the two drugs. Drug treatment reduced the synthesis of heat-shock proteins (HSPs) as studied by one-dimensional gel electrophoresis by 80-98% relative to 37 degrees C untreated controls. Synthesis of large molecules (greater than 30 kDa) was preferentially inhibited by PUR but not by CHM. Also for CHM, but not for PUR treatment, a 42 kDa band appeared along with a great reduction in the 43 kDa actin band during CHM treatment at both 37 and 43 degrees C. Furthermore, during CHM or PUR treatment, incorporation of [35S]methionine into HSP families 70, 87, or 110 was not increased relative to incorporation into total protein. However, synthesis of the 70 kDa HSP family was selectively suppressed when cells were incubated at 37 degrees C after CHM treatment, but when cells were incubated at 37 degrees C after treatment at 43 degrees C with CHM, synthesis of the 70 kDa HSP family resumed. When cells were labeled for 3 days, there was no preferential accumulation or turnover of HSP families during heating with or without CHM. Therefore, heat protection caused by treatment with CHM or PUR apparently involves a common mode of action not associated with changes in either total levels or synthesis of HSP families during drug treatment before and during heating. The significance of the changes observed in the synthesis of the HSP 70 family after heat is unknown. As thermotolerance developed during 5 h at 42 degrees C without drugs, synthesis of HSP families 70, 87, and 110, as studied with one-dimensional gels, increased 1.4-fold relative to synthesis of total protein, but compared to HSP families in cells labeled for 5 h at 37 degrees C incorporation was reduced by 40%. The increase of unique HSPs, if studied with two-dimensional gels, would probably be much greater.(ABSTRACT TRUNCATED AT 400 WORDS)     

Influences of temperature, oxidative stress, and phosphorylation on binding of heat shock proteins in skeletal muscle fibers

Heat shock proteins (HSPs) help maintain cellular function in stressful situations, but the processes controlling their interactions with target proteins are not well defined. This study examined the binding of HSP72, HSP25, and αB-crystallin in skeletal muscle fibers following various stresses. Rat soleus (SOL) and extensor digitorum longus (EDL) muscles were subjected in vitro to heat stress or strongly fatiguing stimulation. Superficial fibers were "skinned" by microdissection and HSP diffusibility assessed from the extent of washout following 10- to 30 min exposure to a physiological intracellular solution. In fibers from nonstressed (control) SOL muscle, >80% of each HSP is readily diffusible. However, after heating a muscle to 40°C for 30 min ～95% of HSP25 and αB-crystallin becomes tightly bound at nonmembranous myofibrillar sites, whereas HSP72 bound at membranous sites only after heat treatment to ≥44°C. The ratio of reduced to oxidized cytoplasmic glutathione (GSH:GSSG) decreased approximately two- and fourfold after heating muscles to 40° and 45°C, respectively. The reducing agent dithiothreitol reversed HSP72 binding in heated muscles but had no effect on the other HSPs. Intense in vitro stimulation of SOL muscles, sufficient to elicit substantial oxidation-related loss of maximum force and approximately fourfold decrease in the GSH:GSSG ratio, had no effect on diffusibility of any of the HSPs. When skinned fibers from heat-treated muscles were bathed with additional exogenous HSP72, total binding increased approximately two- and 10-fold, respectively, in SOL and EDL fibers, possibly reflective of the relative sarco(endo)plasmic reticulum Ca(2+)-ATPase pump densities in the two fiber types. Phosphorylation at Ser59 on αB-crystallin and Ser85 on HSP25 increased with heat treatment but did not appear to determine HSP binding. The findings highlight major differences in the processes controlling binding of HSP72 and the two small HSPs. Binding was not directly related to cytoplasmic oxidative status, but oxidation of cysteine residues influenced HSP72 binding.     

Inhibition of heat shock protein synthesis and protein glycosylation by stepdown heating

Mammalian cells exhibit increased sensitivity to hyperthermic temperatures of 38-43 degrees C after an acute high-temperature heat shock; this phenomenon is known as the stepdown heating (SDH) effect. We characterized the SDH effect on (1) the synthesis of major heat shock proteins, HSP110, 90, 72/70, 60 (35S-amino acids label), (2) on heat-induced protein glycosylation (3H-D-mannose label), and (3) on thermotolerance expression, using cell survival as an endpoint. Partitioning of label between soluble and insoluble cell fractions was separately examined. Synthesis of high molecular weight HSPs (HSP110, 90, and 72/70) was increased both by acute (10 min, 45 degrees C) and chronic (1-6 h, 41.5 degrees C) hyperthermia, primarily in the soluble cytosol fraction. SDH (10 min, 45 degrees C + 1 to 6 h, 41.5 degrees C) completely inhibited labeling of HSP110, partially inhibited HSP90 labeling, and had virtually no effect on HSP72/70 synthesis, when compared with chronic hyperthermia alone. At the cell survival level, SDH increased sevenfold the rate of cell killing at 41.5 degrees C, but reduced the expression of thermotolerance by only a factor of two. This suggests that SDH sensitization did not result from changes in HSP72/70 synthesis, nor solely from inhibition of thermotolerance. 35S-labeled HSP60 and HSP50 were found primarily in the cellular pellet fraction after both acute and chronic hyperthermia. SDH completely inhibited 35S-labeling of both HSP60 and HSP50. Labeling of GP50 with 3H-D-mannose was also completely inhibited by SDH. Moreover, SDH progressively reduced N-acetylgalactosaminyl-transferase activity. The data demonstrate that heat sensitization by SDH is accompanied by complex and selectively inhibitory patterns of HSP synthesis and protein glycosylation. Profound inhibition of HSP110, HSP60, and HSP50/GP50 labeling suggests that these may be associated with mechanisms of SDH sensitization.     

Proteomic analysis of altered protein expression in skeletal muscle of rats in a hypermetabolic state induced by burn sepsis

mRNA profiling has been extensively used to study muscle wasting. mRNA level changes may not reflect that of proteins, especially in catabolic muscle where there is decreased synthesis and increased degradation. As sepsis is often associated with burn injury, and burn superimposed by sepsis has been shown to result in significant loss of lean tissues, we characterized changes in the skeletal-muscle proteome of rats subjected to a cutaneous burn covering 20% of the total body surface area, followed 2 days later by sepsis induced by CLP (caecal ligation and puncture). EDL (extensor digitorum longus) muscles were dissected from Burn-CLP animals (n=4) and controls (sham-burned and sham-CLP-treated, n=4). Burn-CLP injury resulted in a rapid loss of EDL weight, increased ubiquitin-conjugated proteins and increased protein carbonyl groups. EDL protein profiles were obtained by two-dimensional gel electrophoresis using two immobilized pH gradient strips with overlapping pH range covering a pH 3-8 range. Seventeen spots were significantly altered in the Burn-CLP compared with the control group, representing 15 different proteins identified by peptide mass fingerprinting. The identities of three proteins including transferrin were further confirmed by liquid chromatography-tandem MS. The significant changes in transferrin and HSP27 (heat-shock protein 27) were verified by Western-blot analysis. HSP60, HSP27 and HSPbeta6 were down-regulated, along with HSP70, as detected by Western blotting. Six metabolic enzymes related to energy production were also down-regulated. A simultaneous decrease in chaperone proteins and metabolic enzymes could decrease protein synthesis. Furthermore, decreased HSPs could increase oxidative damage, thus accelerating protein degradation. Using cultured C2C12 myotubes, we showed that H2O2-induced protein degradation in vitro could be partially attenuated by prior heat-shock treatment, consistent with a protective role of HSP70 and/or other HSPs against proteolysis.     

Developmental regulation of heat shock protein synthesis and HSP 70 RNA accumulation during postimplantation rat embryogenesis

Exposure of postimplantation rat embryos on days 9, 10, 11, and 12 of gestation to an in vitro heat shock of 43 degrees C for 30 min results in the induction of heat shock proteins (HSPs) in day 9 and 10 embryos, a severely attenuated response in day 11 embryos, and no detectable response in day 12 embryos. The heat shock response in day 9 embryos (presomite stage) is characterized by the synthesis of HSPs with molecular weights of 28-78 kDa. In heat shocked day 10 embryos, two additional HSPs are induced (34 and 82 kDa). In addition, two HSPs present on day 9 are absent on day 10. In day 11 heat shocked embryos, only three HSPs (31, 39, and 69 kDa) are induced, while in day 12 embryos no detectable HSPs are induced. Northern blot analysis of HSP 70 RNA levels indicates that the accumulation of this RNA, but not actin RNA, varies depending on developmental stage at the time of exposure to heat as well as the duration of the heat shock. Day 9 embryos exhibit the most pronounced accumulation of HSP 70 RNA while embryos on days 10-12 exhibit an increasingly attenuated accumulation of HSP 70 RNA, particularly after the more acute exposures (43 degrees C for 30 or 60 min). Thus, the ability to synthesize HSP 70 and to accumulate HSP 70 RNA changes dramatically as rat embryos develop from day 9 to day 12 (presomite to 31-35 somite stages).     

The synergistic effect of hyperthermia and ethanol on changing gene expression of mouse lymphocytes

Cultured mouse lymphocytes respond to a brief incubation at an elevated temperature (41-43 degrees C) with the new and (or) enhanced synthesis of a select group of polypeptides (known as heat-shock proteins, HSPs) having relative molecular masses of 110, 100, 90, 70, and 65 kilodaltons (kDa). Expression of these HSPs is dependent on new RNA synthesis. Because the synthesis of any particular HSP is dependent on the temperature and the length of time cells remain at a particular elevated temperature, synthesis of each HSP is not necessarily coordinated with the synthesis of the other HSPs. Cultured mouse lymphocytes treated with arsenite or ethanol exhibit new and (or) enhanced synthesis of HSPs with molecular masses of 110, 90, 70, and 65 kDa but do not exhibit enhanced synthesis of the 100-kDa HSP. Short-term concurrent exposure of mouse lymphocytes to an elevated temperature and a level of ethanol, which individually do not induce detectable HSP synthesis, results in the pronounced synthesis of HSPs similar to those seen following exposure to higher levels of either stress applied separately. Thus, in this study we demonstrate that hyperthermia and ethanol stress can act synergistically to affect a dramatic change in the gene expression of mouse lymphocytes.     

Inhibition of the ubiquitin-proteasome pathway induces differential heat-shock protein response in cardiomyocytes and renders early cardiac protection

The effects of proteasome inhibition (PI) on heat-shock protein (HSP) expression in cardiomyocytes were investigated. Neonatal rat cardiomyocytes were incubated with MG132 (0.1-10 microM) for 1 h. Induction of various HSPs was determined by real-time PCR and Western blotting. PI induced a 2- to 3-fold increase in HSP27, HSP60, and HSP90, and a 18-fold increase in HSP70 mRNA expression, whereas HSP40 levels were unaffected. Western blotting revealed increased protein expression for HSP70 after PI. Similar results were obtained with MG262. HSP induction correlated with enhanced survival of neonatal cardiomyocytes after sublethal heat stress in XTT testing. In papillary muscles, pretreatment with MG132 (10 microM, 90 min) was associated with enhanced recovery of the contractile parameters after a 40-min hypoxia. In these proof-of-principle experiments, we show that PI induces differential heat-shock response in cardiomyocytes, accompanied by enhanced cell survival and functional recovery after various forms of stress.     

Direct evidence for the intracellular localization of Hsp104 in Saccharomyces cerevisiae by immunoelectron microscopy

To reveal the intracellular localization of Hsp104 in the yeast Saccharomyces cerevisiae before and after heat-shock, we performed immunoelectron microscopy after immunogold labeling with anti-Hsp104 antibody. At normal temperature (25 degrees C), a small amount of Hsp104 was located in the cytoplasm and nucleus. On exposure to mild heat-shock at 40 degrees C, protein aggregates appeared in the cytoplasm and nucleus, and Hsp104 increased around the aggregates with increasing time of the mild heat-shock treatment. Moreover, at lethal heat-shock temperature (51 degrees C) for 20 min after mild heat treatment at 40 degrees C, the intracellular localization of Hsp104 and intracellular structures were similar to those of the mild heat-shocked cells. However, in the lethally heat-shocked cells, certain intracellular structures were destroyed, and Hsp104 was not expressed. In the hsp104 null mutant strain Deltahsp104 which was treated at 40 degrees C, Hsp104 was not localized around the aggregates. Additionally, in the Deltahsp104 strain, even mild heat-shocked cells at 37 degrees C or 40 degrees C, showed destruction of intracellular structure compared to the wild-type strain. Our data suggest the following: (1) Hsp104 is associated closely with protein aggregates during heat-shock treatment, (2) Hsp104 is important for maintenance of the intracellular structure under lethal heat-shock conditions, (3) acquisition of thermotolerance depends on the amount of Hsp104 produced during mild heat-shock treatment.     

Characterization of a cDNA clone, encoding a 70 kDa heat shock protein from the dermatophyte pathogen Trichophyton rubrum

Trichophyton rubrum is an anthropophilic fungus causing up to 90% of chronic cases of dermatophytosis. To characterize T. rubrum proteins at the molecular level, we established a cDNA library of this pathogen. Here we describe a recombinant cDNA clone identical to eukaryotic 70kDa heat-shock proteins (HSPs). Western blot analysis using an anti HSP70 monoclonal antibody detected a recombinant fusion protein in Escherichia coli transformed with the expression vector containing the cloned cDNA insert. Southern blot analysis of T. rubrum genomic DNA detected no other members of the HSP70 gene family. Further analysis revealed the presence of two introns within the ORF of the HSP70 gene. In Northern blot analysis, the cDNA clone was hybridized to a RNA species of about 3.5kb which was constitutively expressed by cells cultured at 27 degrees C and was strongly up-regulated after culture at 37 degrees C. In summary, we have cloned the first member of the HSP family of dermatophytes and characterized it as a member of the Dnak subfamily of 70kDa HSPs.     

Tissue-Type-Specific Heat-Shock Response and Immunolocalization of Class I Low-Molecular-Weight Heat-Shock Proteins in Soybean

A monospecific polyclonal antibody was used to study the tissue-type specificity and intracellular localization of class I low-molecular-weight (LMW) heat-shock proteins (HSPs) in soybean (Glycine max) under different heat-shock regimes. In etiolated soybean seedlings, the root meristematic regions contained the highest levels of LMW HSP. No tissue-type-specific expression of class I LMW HSP was detected using the tissue-printing method. In immunolocalization studies of seedlings treated with HS (40[deg]C for 2 h) the class I LMW HSPs were found in the aggregated granular structures, which were distributed randomly in the cytoplasm and in the nucleus. When the heat shock was released, the granular structures disappeared and the class I LMW HSPs became distributed homogeneously in the cytoplasm. When the seedlings were then given a more severe heat shock following the initial 40[deg]C -> 28[deg]C treatment, a large proportion of the class I LMW HSPs that originally localized in the cytoplasm were translocated into the nucleus and nucleolus. Class I LMW HSPs may assist in the resolubilization of proteins denatured or aggregated by heat and may also participate in the restoration of organellar function after heat shock.     

Effect of cycloheximide or puromycin on induction of thermotolerance by sodium arsenite in Chinese hamster ovary cells: involvement of heat shock proteins

After sodium arsenite (100 microM) treatment, the synthesis of three major heat shock protein families (HSPs; Mr = 110,000, 87,000, and 70,000), as studied with one-dimensional gels, was enhanced twofold relative to that of unheated cells. The increase of unique HSPs, if studied with two-dimensional gels, would probably be much greater. In parallel, thermotolerance was observed as a 100,000-fold increase in survival from 10(-6) to 10(-1) after 4 hr at 43 degrees C, and as a thermotolerance ratio (TTR) of 2-3 at 10(-3) isosurvival for heating at 45.5 degrees C. Cycloheximide (CHM: 10 micrograms/ml) or puromycin (PUR: 100 micrograms/ml), which inhibited total protein synthesis and HSP synthesis by 95%, completely suppressed the development of thermotolerance when either drug was added after sodium arsenite treatment and removed prior to the subsequent heat treatment. Therefore, thermotolerance induced by arsenite treatment correlated with an increase in newly synthesized HSPs. However, with or without arsenite treatment, CHM or PUR added 2-6 hr before heating and left on during heating caused a 10,000-100,000-fold enhancement of survival when cells were heated at 43 degrees C for 4 hr, even though very little synthesis of heat shock proteins occurred. Moreover, these cells manifesting resistance to heating at 43 degrees C after CHM treatment were much different than those manifesting resistance to 43 degrees C after arsenite treatment. Arsenite-treated cells showed a great deal of thermotolerance (TTR of about 10) when they were heated at 45 degrees C after 5 hr of heating at 43 degrees C, compared with less thermotolerance (TTR of about 2) for the CHM-treated cells heated at 45 degrees C after 5 hr of heating at 43 degrees C. Therefore, there are two different phenomena. The first is thermotolerance after arsenite treatment (observed at 43 degrees C or 45.5 degrees C) that apparently requires synthesis of HSPs. The second is resistance to heat after CHM or PUR treatment before and during heating (observed at 43 degrees C with little resistance at 45.5 degrees C) that apparently does not require synthesis of HSPs. This phenomenon not requiring the synthesis of HSPs also was observed by the large increase in thermotolerance to 45 degrees C caused by heating at 43 degrees C, with or without CHM, after cells were incubated for 6 hr following arsenite pretreatment. For both phenomena, a model based on synthesis and redistribution of HSPs is presented.     

Altered synthesis of the 26-kDa heat stress protein family and thermotolerance in cell lines with elevated levels of calcium-binding proteins

Using a bovine papilloma virus-based vector, mouse mammary adenocarcinoma cells have been transformed to express elevated amounts of functional calmodulin (CaM) (Rasmussen and Means, 1987) and another Ca2(+)-binding protein, parvalbumin (PV) (Rasmussen and Means, 1989) that is not normally synthesized in these cells. Parental cells (C127) and cells transformed by the vector alone (BPV-1), the vector containing a CaM gene (CM-1), or the vector containing parvalbumin (PV-1) were used to study the effect of increased synthesis of Ca2(+)-binding proteins on heat-stress protein (HSP) synthesis and cell survival following heating at 43 degrees C. The induction, stability, and repression of the synthesis of most HSPs after 43 degrees C heating was not significantly affected by increased amounts of Ca2(+)-binding proteins, but the rate of synthesis of all three isoforms of the 26-kDa HSP (HSP26) was greatly reduced. C127 cells, which have about one half as much CaM as do BPV-1 cells, synthesized the most HSP26. CM-1 cells, which have more than fourfold higher levels of CaM than do BPV-1 cells, had a rate of synthesis of HSP26 approaching that of unheated cells. BPV-1 cells, with a two-fold increase in CaM, were intermediate in HSP26 synthesis. This effect on HSP26 synthesis may be largely related to the Ca2(+)-binding capacity of CaM rather than to a specific CaM-regulated function, since PV-1 cells also showed reduced rates of HSP26 synthesis. Survival experiments showed that reduced HSP26 synthesis in cells with increased amounts of Ca2(+)-binding proteins did not significantly alter intrinsic resistance to continuous 43 degrees C heating. Thermotolerance was not reduced and appeared to develop more rapidly in CM-1 and PV-1 cells. These results suggest that (1) the signal for HSP26 synthesis can be largely abrogated by elevated Ca2+ binding protein levels, and (2) if these HSPs are involved in thermotolerance development, that function may be associated with intracellular Ca2+ homeostasis.