Dodecafluoropentane emulsion delays and reduces MRI markers of infarction in a rat stroke model: a preliminary report.

BACKGROUND: Dodecafluoropentane emulsion (DDFPe), an oxygen transport agent, has been shown to reduce infarct volume in animal models of acute ischemic stroke (AIS). Our study assesses the effect of DDFPe on MRI markers of infarct evolution in the early hours after vascular occlusion in a rat AIS model. We hypothesized that DDFPe will delay the development of MRI markers of AIS and/or reduce the extent of infarction. METHODS: Permanent, unilateral surgical occlusion of the middle cerebral and common carotid arteries was performed in control (n=4) and treatment (n = 10) rats. The treatment group received 1 IV dose of 2% w/v DDFPe at 0.6 mL/kg at 1 hour post-occlusion versus none. Diffusion-weighted (DWI) and inversion recovery (IR) MRI sequences were obtained over the 4 hours following occlusion. Infarct extent was quantified by number of abnormal MRI slices per sequence for each group and time point. Student's T-test was applied. RESULTS: DDFPe-treated rats demonstrated reduced infarct extent versus controls over combined time points on IR at 5.43 ± 0.40 (mean ± standard error) abnormal slices vs. 7.38 ± 0.58 (P = 0.01) and on DWI at 5.21 ± 0.54 vs. 9.00 ± 0.95 (P < 0.01). Development of abnormal MRI signal was delayed in the treatment group. CONCLUSIONS: DDFPe delays and reduces MRI markers of AIS in the early hours following vascular occlusion in a rat stroke model. Further investigation of DDFPe as a neuroprotectant is warranted.

Progress in dodecafluoropentane emulsion as a neuroprotective agent in a rabbit stroke model.

Dodecafluoropentane emulsion (DDFPe) in 250 nm nanodroplets seems to swell modestly to accept and carry large amounts of oxygen in the body at >29 °C. Small particle size allows oxygen delivery even into hypoxic tissue unreachable by erythrocytes. Using permanent cerebral embolic occlusion in rabbits, we assessed DDFPe dose response as a neuroprotectant at 7 and 24 h post-embolization without lysis of arterial obstructions and investigated blood pharmacokinetics. New Zealand White rabbits (N = 56) received cerebral angiography and embolic spheres (diameter = 700-900 µm) occluded middle and/or anterior cerebral arteries. Intravenous DDFPe dosing (2 % w/v emulsion) began at 60 min and repeated every 90 min until sacrifice at 7 or 24 h post-embolization. Seven-hour groups: (1) control (embolized without treatment, N = 6), and DDFPe treatment: (2) 0.1 ml/kg (N = 7), (3) 0.3 ml/kg (N = 9), (4) 0.6 ml/kg (N = 8). Twenty-four-hour groups: (5) control (N = 16), and DDFPe treatment: (6) 0.1 ml/kg (N = 10). Infarcts as percent of total brain volume were determined using vital stains on brain sections. Other alert normal rabbits (N = 8) received IV doses followed by rapid arterial blood sampling and GC-MS analysis. Percent infarct volume means significantly decreased for all DDFPe-treated groups compared with controls, p = <0.004 to <0.03. Blood DDFP (gas) half-life was 1.45 ± 0.17 min with R = 0.958. Mean blood clearance was 78.5 ± 24.9 ml/min/kg (mean ± SE). Intravenous DDFPe decreases ischemic stroke infarct volumes. Blood half-life values are very short. The much longer therapeutic effect, >90 min, suggests multiple compartments. Lowest effective dose and maximum effective therapy duration are not yet defined. Rapid development is warranted.

In vivo resolution of oligomers with fluorescence photobleaching recovery histograms.

Simple independent enzyme-catalyzed reactions distributed homogeneously throughout an aqueous environment cannot adequately explain the regulation of metabolic and other cellular processes in vivo. Such an unstructured system results in unacceptably slow substrate turnover rates and consumes inordinate amounts of cellular energy. Current approaches to resolving compartmentalization in living cells requires the partitioning of the molecular species in question such that its localization can be resolved with fluorescence microscopy. Standard imaging approaches will not resolve localization of protein activity for proteins that are ubiquitously distributed, but whose function requires a change in state of the protein. The small heat shock protein sHSP27 exists as both dimers and large multimers and is distributed homogeneously throughout the cytoplasm. A fusion of the green fluorescent protein variant S65T and sHSP27 is used to assess the ability of diffusion rate histograms to resolve compartmentalization of the 2 dominant oligomeric species of sHSP27. Diffusion rates were measured by multiphoton fluorescence photobleaching recovery. Under physiologic conditions, diffusion rate histograms resolved at least 2 diffusive transport rates within a living cell potentially corresponding to the large and small oligomers of sHSP27. Given that oligomerization is often a means of regulation, compartmentalization of different oligomer species could provide a means for efficient regulation and localization of sHsp27 activity.

Delaying S-phase progression rescues cells from heat-induced S-phase hypertoxicity.

The mechanism by which a cell protects itself from the lethal effects of heat shock and other stress-inducing agents is the subject of much research. We have investigated the relationship between heat-induced damage to DNA replication machinery and the lethal effects of heat shock, in S-phase cells, which are more sensitive to heat shock than either G1 or G2. We found that maintaining cells in aphidicolin, which prevents the passage of cells through S-phase, can rescue S-phase HeLa cells from the lethal effects of heat shock. When S-phase, HeLa cells were held for 5-6 h in 3 microM aphidicolin the measured clonogenic survival was similar to that for exponentially growing cells. It is known, that heat shock induces denaturation or unfolding of proteins, rendering them less soluble and more likely to co-isolate with the nuclear matrix. Here, we show that enhanced binding of proteins involved in DNA replication (PCNA, RPA, and cyclin A), with the nuclear matrix, correlates with lethality of S-phase cells following heat shock under four different experimental conditions. Specifically, the amounts of RPA, PCNA, and cyclin A associated with the nuclear matrix when cells resumed progression through S-phase correlated with cell killing. Heat-induced enhanced binding of nuclear proteins involved with other aspects of DNA metabolism, (Mrell, PDI), do not show this correlation. These results support the hypothesis that heat-induced changes in the binding of proteins associated with DNA replication factories are the potentially lethal lesions, which become fixed to lethal lesions by S-phase progression but are repairable if S-phase progression is delayed.

Heat-activated transgene expression from adenovirus vectors infected into human prostate cancer cells.

Replication-deficient adenovirus expression vectors were used to introduce a recombinant DNA construct containing enhanced green fluorescent protein (EGFP) under control of a truncated, human heat shock promoter into human prostate cancer cells growing either exponentially or in plateau phase. This was done to measure controlled, heat shock-induced EGFP expression under conditions relevant to treating human cancers with heat-activated gene therapy. Both the temporal duration and magnitude of EGFP expression increased proportionately with stronger heat shocks (time at temperature) up to maximum values that were induced by 4 h at 41.0 degrees C or 2 h at 42.0 degrees C. Longer heat shocks at either temperature yielded no additional EGFP expression and ultimately reduced it. Maximal EGFP expression was induced in exponential cultures by heat shocks delivered 12-24 h after virus infection. Induction at progressively later postinfection times induced increasingly lower, peak EGFP expression. Maximal EGFP expression could not be induced until 48 h after infection of plateau phase cultures but could still be induced 180 h after virus infection. However, peak EGFP levels in plateau cultures were approximately 25-50% of those observed in identically induced exponential cultures. Ostensibly, the differences in expression from the heat shock promoter observed in exponential and plateau cultures were attributable to cell division diluting the vector within exponential cultures and the lower metabolic activity in serum-starved plateau cultures. For all experimental conditions, EGFP expression induced from the heat shock promoter was comparable with or higher than that from the constitutively active cytomegalovirus promoter over any 24-h period. The experimental results demonstrated that EGFP expression from the heat shock promoter was controllable in both exponential and plateau phase cultures and support the plausibility of using controlled heat shock activation of this promoter as a means of regulating both the spatial and temporal expression of therapeutic DNA constructs within human tissues. The ability to localize and regulate expression from the heat shock promoter may prove particularly advantageous for many cancer applications, especially if the therapeutic products are highly toxic, e.g., proteotoxins or cytokines. However, the results of this study suggest that differential growth conditions within tumors could markedly affect the expression of recombinant DNA under control of both inducible and constitutive promoters. Consequently, inducing schemes may need to be spatially adjusted to obtain the desired therapeutic results in all tumor domains using heat-activated gene therapy.

Inhibition of the 26S proteasome induces expression of GLCLC, the catalytic subunit for gamma-glutamylcysteine synthetase.

The majority of short- and long-lived cellular proteins are degraded by the activities of the 26S proteasome, a large multi-catalytic protease. Its unique function places it as a central regulatory activity for many important physiological processes. Lactacystin is a very specific 26S proteasome inhibitor and represents an excellent tool for demonstrating that a pathway exhibits proteasome-dependent biochemical regulation. Exposure of HepG2 cells to lactacystin resulted in robust elevation of GLCLC mRNA levels, followed by an increase in GSH concentrations. GLCLC is the gene that encodes the catalytic subunit for gamma-glutamylcysteine synthetase, the rate-limiting enzyme for the synthesis of glutathione (GSH). Inhibition of non-proteasome, protease activities did not induce GLCLC. Gel mobility shift assays and expression of CAT activity from heterologous reporter vectors identified Nrf2 mediation of the GLCLC antioxidant response element, ARE4, as the mechanism by which lactacystin induced GLCLC. These studies have identified 26S proteasome activity as a central regulatory pathway for glutathione synthesis.

On the path to the heat shock response: destabilization and formation of partially folded protein intermediates, a consequence of protein thiol modification.

This review discusses the initial events that occur during oxidative stress that induce the synthesis of heat shock proteins. The focus is on non-native oxidation or modification of protein thiols and the destablization that can result. Proteins that contain non-native modified thiols can become destablized such that they unfold into molten globule-like intermediates at or below 37 degrees C, relieving Hsf-1 negative regulation, and inducing Hsp transcription.

Diamide-induced cytotoxicity and thermotolerance in CHO cells.

Treatment with the sulfhydryl oxidant diamide denatures and aggregates cellular proteins, which prior studies have implicated as an oxidative damage that activates the heat shock transcription factor and induces thermotolerance. This study was initiated to further characterize cellular response to diamide-denatured proteins, including their involvement in diamide cytotoxicity. Cytotoxic diamide exposures at 37.0 degrees C denatured and aggregated cellular proteins in a manner that was proportional to cell killing, but this correlation was different than that established for heated cells. Diamide exposures at 24.0 degrees C were orders of magnitude less cytotoxic, with little additional killing occurring after diamide was removed and cells were returned to 37.0 degrees C. Thus, protein denaturation that occurred at 37.0 degrees C, after proteins were chemically destabilized by diamide at 24.0 degrees C [Freeman et al., J. Cell. Physiol., 164:356-366 (1995); Senisterra et al., Biochemistry 36: 11002-11011 (1997)], had little effect on cell killing. Thermotolerance protected cells against diamide cytotoxicity but did not reduce the amount of denatured and aggregated protein observed immediately following diamide exposure. However, denatured/aggregated proteins in thermotolerant cells were disaggregated within 17 h following diamide exposure, while no disaggregation was observed in nontolerant cells. This more rapid disaggregation of proteins may be one mechanism by which thermotolerance protects cells against diamide toxicity, as it has been postulated to do against heat killing. As with heat shock, nontoxic diamide exposures induced maximal tolerance against heat killing; however, there was no detectable, increased synthesis of heat shock proteins. Thus, diamide treatment proved to be a reproducible procedure for inducing a phase of thermotolerance that does not require new heat shock protein (HSP) synthesis, without having to use transcription or translation inhibitors to suppress HSP gene expression. These results complement those from studies with other stresses to establish the importance of protein denaturation/aggregation as a cytotoxic consequence of stress and a trigger for thermotolerance induction. The data also illustrate that differences in how proteins are denatured and aggregated can affect their cytotoxicity and the manner in which thermotolerance is expressed.

Proteins containing non-native disulfide bonds generated by oxidative stress can act as signals for the induction of the heat shock response.

While oxidative stress can induce a heat shock response, the primary signals that initiate activation have not been identified. To identify such signals, HepG2 and V 79 cells were exposed to menadione, a compound that redox-cycles to generate superoxide. The oxidative stress generated by menadione resulted in oxidation of protein thiols in a dose-dependent manner. This was followed by protein destabilization and denaturation, as determined by differential scanning calorimetry of whole cells. To directly evaluate the effect of non-native disulfides on protein conformation, Ca2(+)-ATPase, isolated from rabbit sarcoplasmic reticulum, was chemically modified to contain non-native intermolecular or glutathione (GHS)-mixed disulfides. Differential scanning calorimetry profiles and 1-anilinonaphthalene-8-sulfonic acid fluorescence indicated that formation of non-native disulfides produced protein destabilization, denaturation, and exposure of hydrophobic domains. Cellular proteins shown to contain oxidized thiols formed detergent-insoluble aggregates. Cells treated with menadione exhibited activation of HSF-1, accumulated Hsp 70 mRNA, and increased synthesis of Hsp 70. This work demonstrates that formation of physiologically relevant, non-native intermolecular and GSH-mixed disulfides causes proteins to destabilize, unfold such that hydrophobic domains are exposed, and initiate a signal for induction of the heat shock response.

Thermotolerance expression in mitotic CHO cells without increased translation of heat shock proteins.

The objective of this study was to unequivocally demonstrate thermotolerance expression in mammalian cells in the absence of stress-induced synthesis of heat shock proteins (HSPs). Mitotic cells were selected as an experimental system since their genome was in the form of condensed chromosomes and ostensibly incapable of being transcribed; thus, obviating stress-induced HSP gene expression. Asynchronous Chinese hamster ovary (CHO) cells were treated with 0.2 microgram/ml nocodazole to accumulate cells in mitosis for harvest by mitotic shakeoff. Cells were maintained in mitosis with nocodazole during thermotolerance induction, thermotolerance development, and all challenge hyperthermia exposures. Although the heat shock transcription factor was activated by the thermotolerance inducing heat shock, as indicated by gel mobility shift assay, no increase in steady-state HSP mRNA levels was detected, as expected. Preferential synthesis of HSPs from extant mRNA was not detected during thermotolerance development and cellular levels of the 27 kDa, 70 kDa, and 90 kDa heat shock proteins remained constant, as determined by Western Blot analyses. The magnitude and induction threshold of expressed thermotolerance was not diminished when cells were incubated with 10.0 micrograms/ml cycloheximide during thermotolerance development confirming that new protein synthesis was not requisite. Parallel experiments were performed using nonmitotic cells in which protein synthesis was inhibited during thermotolerance development with 10.0 micrograms/ml cycloheximide. As with mitotic cells, high levels of thermotolerance were attained without detectable increases in the cellular content of the 27 kDa, 70 kDa, and 90 kDa heat shock proteins. The results of this study demonstrated that high levels of thermotolerance could be expressed in mitotic cells without stress-induced, preferential synthesis of HSPs, and support the contention that a substantial fraction of thermotolerance expressed in nonmitotic cells also occurs independently of induced HSP synthesis.

Excess protein in nuclei isolated from heat-shocked cells results from a reduced extractability of nuclear proteins.

An excellent correlation has been established between the quantity of protein associated with nuclei isolated from heat-shocked cells and the level of hyperthermic cell killing. However, controversy remains about whether increases in nuclear-associated protein result from a heat-induced migration of cytoplasmic proteins into the nucleus or because hyperthermia reduces the solubility of nuclear proteins in the detergent buffers commonly used to isolate nuclei. To address this controversy, the nuclear protein content was measured in whole and detergent-extracted cells before and following hyperthermia. It was found that hyperthermia caused no significant change in the nuclear protein content of whole, unextracted cells, and when fluorescently labeled proteins were microinjected into the cytoplasm no gross change in the selective permeability of the nuclear membrane to soluble proteins was observed during or following hyperthermia. Measurements in extracted cells showed that the detergent buffers removed protein from both the nucleus and cytoplasm of control, nonheated cells and that hyperthermia reduced the extractability of both nuclear and cytoplasmic proteins. The amount of protein found in nuclei isolated from heated cells approached that observed in nuclei within nonheated whole cells as the hyperthermic exposure was increased. Thus, the dose-dependent, two- to threefold increase in the protein content of nuclei isolated from heated cells represents a heat-induced reduction in the extractability of proteins normally present within cell nuclei and does not result from a mass migration of cytoplasmic proteins into the nucleus, although some specific proteins (e.g., the 70 KDa heat shock protein) do migrate to the nucleus following heat shock. Differential scanning calorimetry (DSC) measurements of whole cells, isolated nuclei, cytoplasts, and karyoplasts supported these conclusions and suggested that most of the detergent-insoluble proteins remaining in the nuclei and cytoplasm of heated cells are in their native state. Thus, a relatively small amount of denatured protein may be sufficient to initiate and sustain insoluble protein aggregates comprised of mostly native proteins. Analyses of the DSC data also implied that the previously identified critical target proteins, predicted to have a Tm of 46.0 degrees C, are present in both the nucleus and cytoplasm.

Characterization of a signal generated by oxidation of protein thiols that activates the heat shock transcription factor.

The diazenecarbonyl derivative, diamide, was used to produce nonnative protein disulfides in Chinese hamster ovary cells in order to characterize the events that occur during thiol oxidation-induced denaturation that trigger induction of Hsp 70. We limit the term protein denaturation to a process involving a conformational rearrangement by which the ordered native structure of a protein changes to a more disordered structure. Protein thiol oxidation resulted in immediate destabilization of proteins, as assessed by differential scanning calorimetry (DSC). The DSC profile indicated both a decrease in the onset temperature for detection of denaturation and destabilization of a class of proteins with an average transition temperature (Tm) of 60 degrees C. Concomitant with destabilization was an increase in proteins associated with isolated nuclei. Thiol oxidation also induced heat shock transcription factor (HSF) binding activity, however, this was nearly undetectable immediately following diamide treatment: maximum activation occurred 3 hr following exposure. In contrast, heat shock denatured thermolabile proteins which exhibited a Tm of < or = 48 degrees C. Heat shock also resulted in a rapid increase in proteins associated with isolated nuclei and produced immediate and maximum activation of HSF binding. The accumulation of Hsp and Hsc 70 mRNA following thiol oxidation reflected the delay in HSF binding. Acquisition of HSF binding activity occurred immediately if diamide-treated cells were subsequently exposed to a heat shock, indicating that HSF was not inactivated by the diamide treatment. Ostensibly, the cellular system for detecting denatured/abnormal proteins failed to immediately recognize the signal generated by thiol oxidation. These results suggest that at least two processes are involved in the induction of Hsp 70 by nonnative disulfide bond formation: destabilization of protein structure resulting in denaturation and recognition of denatured protein.

Protocol for freezing thermotolerant cells and maintaining thermotolerance following thawing.

Two independent laboratories have demonstrated that suspension-grown, Chinese hamster ovary (CHO) cells can be made thermotolerant, frozen and subsequently thawed such that they still express thermotolerance. Thermotolerance was determined as the ability to protect cells against hyperthermic cell killing (colony formation assay) and the ability to reduce protein aggregation within the nuclei of heated cells. Cells were frozen either following development of full or partial thermotolerance. In the former case frozen cells maintained thermotolerance upon thawing and in the latter case cells subsequently developed full thermotolerance following thawing and incubation at 37.0 degrees C. After thawing, frozen cells displayed a temporal course of thermotolerance development and decay that was similar to that for never-frozen cells. Success was obtained using either asynchronous or synchronous cell populations, and the heat sensitivity of the cells was not altered by the freezing procedure. The experimental results demonstrate the plausibility of utilizing a frozen stock of thermotolerant cells to make thermotolerance experiments more convenient.

Effect of 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) on HSP70 and HSP28 gene expression and thermotolerance development in human colon carcinoma cells.

The effect of 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), a potent protein kinase C (PKC) inhibitor, on the development of thermotolerance and expression of heat shock genes (HSP70 and HSP28) was investigated in human colon carcinoma HT-29 cells. After acute heating at 45 degrees for 15 min, cells became resistant to a challenge heat shock. The development of thermotolerance was suppressed by adding H-7 after heat shock. Northern blots show that the levels of HSP70 and HSP28 mRNA increased rapidly and reached maximal values within 6 hr. H-7 suppressed the accumulation of HSP70 and HSP28 mRNA as well as their protein synthesis, and the level of suppression was concentration dependent. However, little effect was observed if the drug was added 1 hr before and during heat shock. These results suggest that PKC is involved in the regulation of heat shock gene expression after acute heat shock.

Induction of tolerance to hypothermia and hyperthermia by a common mechanism in mammalian cells.

Pretreatment by hypothermic (25 degrees C) cycling (PHC) of attached exponential-phase V79 Chinese hamster cells by Method 4 (24 hr at 25 degrees C + 1.5 hr at 37 degrees C + 24 hr at 25 degrees C + trypsin + 3 hr at 37 degrees C) or by Method 3 (48 hr at 25 degrees C + trypsin + 3 hr at 37 degrees C) make mammalian V79 cells significantly more resistant to 43 degrees C hyperthermia. There is no significant difference in the 43 degrees C curves whether Method 3 or 4 is used for pre-exposure. If pre-exposure at 15 or 10 degrees C, the resistance to hyperthermia is significantly reduced. PHC by Method 4 significantly increases survival of cells exposed to 5 degrees C and, to a lesser extent, to 10 degrees C. The increase in hyper- and hypothermic survival after PHC cannot be accounted for by changes in cell cycle distribution. Heat-shock protein synthesis is not induced by PHC; hence, protection does not result from newly synthesized proteins. When cells are made tolerant to hyperthermia by a pretreatment in 2% DMSO for 24 hr at 37 degrees C (Method 8), the cells are not more resistant to subsequent exposures to hypothermia, either at 5 or 10 degrees C. The results imply that there may be two mechanisms of inducing resistance to hyperthermia, only one of which also confers resistance to hypothermia.

Effect of thermotolerance on heat-induced excess nuclear-associated proteins.

Earlier studies reported that thermotolerance had two effects on the heat-induced increase in nuclear-associated proteins (NAPs); reduction in NAP levels immediately following hyperthermia and facilitation of NAP recovery to control levels. It has also been demonstrated that there are two phases of thermotolerance; one that requires newly synthesized proteins (protein synthesis dependent thermotolerance; PSDT), and another that does not (protein synthesis independent thermotolerance; PSIT). This study was designed to determine if these two phases of thermotolerance affected NAP binding in a similar or different manner. The results demonstrated that protein synthesis during thermotolerance development was not required to reduce NAP levels measured immediately following hyperthermia, but was required to facilitate NAP recovery to control levels following hyperthermia. Reducing NAP levels was the predominant mechanism by which thermotolerance protected cells from this lesion at 43.0 degrees C while facilitated NAP recovery predominated in protecting against exposure to 45.5 degrees C. The facilitated recovery of NAPs required only proteins synthesized following thermotolerance induction and prior to the second heat challenge. Proteins synthesized following the second heat challenge were not requisite. Finally, the processes that facilitate NAP recovery were inhibited at 3 degrees C, suggesting that they are enzymatically mediated.

Cycloheximide protection against actinomycin D cytotoxicity.

Pretreatment plus concomitant treatment with 10 micrograms/ml cycloheximide protected Chinese hamster ovary cells and Swiss 3T3 cells against the cytotoxicity of actinomycin D. The cycloheximide treatment reduced the intracellular concentration of actinomycin D by reducing the level of actinomycin D bound to the acid precipitable fraction of the cell. Levels of unbound actinomycin D were unaffected by cycloheximide, indicating that the plasma membrane permeability to AD was not reduced. Actinomycin D inhibited total transcription but did not reduce cytoplasmic levels of rRNA nor of most tested mRNA; however, cytoplasmic levels of c-myc mRNA were reduced below detectability. Cycloheximide treatment further inhibited total transcription and had no effect on cytoplasmic levels of rRNA nor of most tested mRNA. Cytoplasmic levels of c-myc were elevated by cycloheximide and remained so even in the presence of actinomycin D. These data suggested that a reduction in cytoplasmic levels of short lived, essential mRNA, such as c-myc mRNA, was one lethal lesion of actinomycin D. Furthermore, cycloheximide's protection may result, in part, from its ability to stabilize and/or elevate cytoplasmic levels of these mRNA, thus counteracting their depletion by actinomycin D. Protection may also result from the cycloheximide-induced reduction of actinomycin D bound to the acid precipitable fraction of the cells.

Absence of HSP28 synthesis and phosphorylation during the development of chronic thermotolerance in murine L929 cells.

We investigated the correlation between chronic thermotolerance development and phosphorylation, synthesis, or expression of the HSP28 family in murine L929 cells. Chronic thermotolerance developed during heating at 41.5 degrees C as indicated by a biphasic survival curve. However, heat-induced phosphorylation of HSP28 was not detected. Furthermore, we failed to detect HSP28 synthesis during chronic heating by using two-dimensional polyacrylamide gel electrophoresis. The lack of HSP28 synthesis was also confirmed in acute thermotolerance. Similar results were observed in NIH 3T3 cells. Although Southern blots clearly demonstrated the presence of the HSP28 gene in genomic DNA, Northern blots failed to demonstrate its expression. Unlike HSP28, the expression of constitutive and inducible HSP70 genes, along with the synthesis of their proteins, were stimulated during chronic heating at 41.5 degrees C in L929 cells. These results suggest that HSP28 synthesis and its phosphorylation are not required to develop both chronic and acute thermotolerance in L929 cells.

Reduction of levels of nuclear-associated protein in heated cells by cycloheximide, D2O, and thermotolerance.

Hyperthermia increases levels of nuclear-associated proteins in a manner that correlates with cell killing. If the increase in nuclear-associated proteins represents a lethal lesion then treatments that protect against killing by heat should reduce and/or facilitate the recovery of levels of the proteins in heated cells. This hypothesis was tested using three heat protection treatments: cycloheximide, D2O, and thermotolerance. All three treatments reduced levels of the proteins measured immediately following hyperthermia at 43.0 or 45.5 degrees C, with the greatest reduction occurring at 43.0 degrees C. In addition to reducing the proteins, thermotolerance facilitated the recovery of the proteins to control levels following hyperthermia. Thus thermotolerance may protect cells by both reducing the initial heat damage and facilitating recovery from that damage. Cycloheximide and D2O did not facilitate recovery of nuclear-associated proteins, suggesting that their protection against cytotoxicity related to the proteins resulted solely from their reduction of increases in levels of the proteins. All three treatments have been shown to stabilize cellular proteins against thermal denaturation. The results of this study suggest that the increase in nuclear-associated proteins may result from thermally denatured proteins adhering to the nucleus and that it is the ability of cycloheximide, D2O, and thermotolerance to thermostabilize proteins that reduces the increase in levels of the proteins within heated cells.

Development of acute thermotolerance in L929 cells: lack of HSP28 synthesis and phosphorylation.

We investigated the correlation between the development of acute thermotolerance and the phosphorylation, synthesis, and expression of the HSP28 family in murine L929 cells. Following heating at 43 degrees C for 30 min, thermotolerance developed rapidly in exponential-phase cells and reached its maximum 4-9 h after heat shock. Maximal thermal resistance was maintained for 24 h and then gradually decayed. However, heat-induced phosphorylation of HSP28 was not detected. Furthermore, HSP28 synthesis during incubation at 37 degrees C for 12 h following heat shock was not detected by [3H]-leucine labeling followed by two-dimensional polyacrylamide gel electrophoresis. In addition, Northern blots failed to demonstrate expression of the HSP28 gene. Unlike HSP28, the expression of constitutive and inducible HSP70 genes, along with the synthesis of their proteins, was observed during incubation at 37 degrees C after heat shock. These results demonstrate that HSP28 synthesis and its phosphorylation are not required to develop acute thermotolerance in L929 cells.