The effect of temperature and protein synthesis on the renaturation of firefly luciferase in intact H9c2 cells.

A mild increase in temperature that does not exert an effect on tolerance development or synthesis of heat shock proteins (Hsps) in control cells can stimulate these processes when applied to cells that have previously been heat shocked. To study the underlying mechanism of this effect, H9c2 cells were stably transfected with the gene encoding firefly luciferase (Luc). Heat-shock-induced inactivation of Luc and its subsequent reactivation is frequently used as a model for cellular protein denaturation and renaturation. Luc reactivation was determined following a damaging heat shock (43 or 44 degrees C for 30 min) in cells that were subsequently exposed to either control temperatures (37 degrees C) or various mild hyperthermic conditions (from 38.5 to 41.5 degrees C for 1 h). To prevent changes in Luc activity consequent to new synthesis of Luc, Luc reactivation was monitored in the presence of cycloheximide, an inhibitor of protein synthesis. The results showed that reactivation of Luc was inhibited when heat-treated cells were post-treated under mild hyperthermic conditions. The observed increase in Hsp synthesis under mild hyperthermic post-heat shock conditions therefore appears to be the result of an increase in the period during which denatured proteins are present. In addition, we studied Luc reactivation in the absence of protein synthesis inhibitors. This condition led to much higher Luc activity. By estimating half-life times of Luc, the contribution of new Luc synthesis in this recovery could be determined, and only partially explained the observed increase in Luc reactivation after heat shock. Thus the synthesis of other proteins must be important for the renaturation of heat-damaged proteins.

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

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

The role of hsp70 in protection and repair of luciferase activity in vivo; experimental data and mathematical modelling.

The stably transfected rat cell line HR24 expressing high levels of the inducible human hsp70 and its parental cell line Rat-1 were used for in vivo studies to analyse the role of hsp70 during thermal protein denaturation and the subsequent renaturation. In order to monitor denaturation and renaturation of a cellular protein in vivo, both cell lines were transiently transfected with firefly luciferase (Luc). The continuous monitoring of Luc activity during and after heat stress allowed a detailed analysis of the inactivation and reactivation kinetics in cells grown in monolayers. The aim of these studies was to distinguish a protective effect of increased hsp70 levels during heat shock-induced protein inactivation from a stimulation of reactivation. In this paper we show that in cells that are stably transfected with hsp70, thermal Luc inactivation decreased, and subsequent reactivation yielded higher activity levels, compared with the parental cells. The difference in early inactivation kinetics observed in the two cell lines suggests an immediate effect of the presence of an extra amount of hsp70 on enzyme inactivation. Using different mathematical models, the heat-induced inactivation and reactivation kinetics was compared with simulations of denaturation and renaturation. It is concluded that the model in which it is assumed that hsp70 is able to interact with partially denatured proteins, which did not yet lose their enzymatic activity, most optimally explains the experimental observations.

A mathematical model of the hsp70 regulation in the cell.

A mathematical model of the regulation process of the heat shock protein hsp70 in the cell is presented. The model describes the damaging effect of elevated temperature on proteins; the interaction of free hsp70 with injured proteins and its chaperone role in nascent protein translation; the relation between the amount of free hsp70 and the formation of the activated trimer form of the heat shock factor protein (HSF); the binding of activated HSF with the heat shock elements on the DNA; the transcription of mRNA of hsp70 and the synthesis of hsp70. The reaction of the model to a temporal rise in temperature shows an initial decline and a subsequent sharp rise to an ultimately increased level of free hsp70 in the cell. The response of the model to both a single and two consecutive heat shocks appears to closely resemble experimental data on hsp70 synthesis. This general agreement demonstrates the structure of the model to be sound and suitable as a basis for further modelling the complex tolerance mechanism of the cell.

NADPH-oxidase-dependent superoxide production by myocyte-derived H9c2 cells: influence of ischemia, heat shock, cycloheximide and cytochalasin D.

Extracellular oxygen radicals produced by H9c2 rat heart cells in monolayer cultures during ischemia and subsequent reoxygenation were monitored using the luminol-horseradish peroxidase-enhanced chemiluminescence technique. As expected, the photon count diminishes during ischemia but again rapidly attains normal values following reoxygenation. In the presence of superoxide dismutase, this photon emission is repressed, as is also the case in the presence of diphenylene iodonium, a specific inhibitor of NADPH-oxidase activity. Thus, the conclusion seems justified that H9c2 rat heart cells in monolayer cultures produce superoxide radicals extracellularly due to an NADPH oxidase-like action. In order to characterize this extracellular superoxide-generating system, we determined its sensitivity to increased temperatures, inhibition of protein synthesis and perturbations of cytoskeletal structures. Heat shocks result in a delayed inactivation of the NADPH oxidase activity followed by recovery, the kinetics of which depend on the imposed heat shock temperature. This inactivation is independent of protein synthesis and actin cytoskeletal structures, but the recovery of the enzyme's activity is dependent on these entities.

Enhancement of superoxide production and protection against heat shock by HSP27 in fibroblasts.

Extracellular superoxide production in the Chinese hamster CCL39 fibroblast cell line was detected by direct counting of luminol-enhanced chemiluminescence in the presence of horseradish peroxidase (HRP). This superoxide production, which is directly related to cell density, can be inhibited by diphenylene iodonium, a specific inhibitor of NADPH-oxidase. The O2- generation is transiently inhibited following exposure of the cells to elevated temperatures. To study the possible relationship between HSP27 and its modulation of NADPH-oxidase activity we measured the generation of O2- activity in CCL39 cells containing either the gene coding for human HSP27 or a mutant form of this gene leading to HSP27 unable to be phosphorylated. Cells with the enhanced expression of normal human HSP27 as well as the non-phosphorylatable protein exhibited a 20-fold higher superoxide production than control CCL39 cells. Furthermore, we demonstrate that accumulation of normal HSP27 appears to play a role in prevention of NADPH-oxidase inhibition by heat.

Hepatoma cells adapted to proliferate under normally lethal hyperthermic stress conditions show rapid decay of thermoresistance and heat shock protein synthesis when returned to 37 degrees C.

H35 hepatoma cultures were adapted to sustained growth at 41.3 degrees C. In these variant cells the 'basic' levels of various heat shock proteins (hsps), especially those of hsp60, 70 and 100, are significantly raised. These cells exhibit a thermoresistance comparable with the induced thermotolerance in normal hepatoma cells heat shocked at 42.5 degrees C for 30 min. However, this resistance of variant cells shows a rapid, exponential decay with a half-time of 2.2 h when the temperature is lowered to 37 degrees C, with a concomitant decrease of the synthesis of hsp60 and 70. Heat shock experiments with variant cells grown at 41.3 degrees C lead to increased thermoresistance and synthesis of hsps when further incubation was performed at the original temperature but not at 37 degrees C. In the latter case, only a 3-h delay in the onset of decay of thermoresistance is observed. However, when the variant cells were incubated at 37 degrees C prior to heat stress normal induction of thermoresistance and hsp synthesis return inversely proportional to the progression of thermoresistance decay. Thermoresistant cells thus seem to be valuable tools in the study of the down-regulation of thermoresistance as well as of hsp synthesis.

Relationship between cadmium-induced expression of heatshock genes, inhibition of protein synthesis and cell death.

Stress proteins (heat shock proteins, HSPs) have been proposed as markers for toxicity. This study has focussed on the pattern of HSP synthesis in relation to cytotoxicity and their dependence on doses of cadmium chloride. We investigated the relationship between cadmium-induced expression of heatshock genes, inhibition of protein synthesis and cell death in a well-differentiated hepatoma cell line, Reuber H35, under exposure conditions ranging to full (> 98%) lethality. We find a non-linearity in the responses of these cells when the duration of cadmium exposure is varied. The results indicate that sublethal concentrations of cadmium can inhibit protein synthesis and also increase the synthesis of certain HSPs. The pattern of heat shock protein induction changes when exposure conditions become more severe. The most strongly inducible heat shock protein, HSP68, is, surprisingly, only synthesized under conditions which lead to severe inhibition of protein synthesis. The comparison of HSP68 mRNA levels and HSP68 synthesis showed that HSP68 mRNA is already induced under conditions where the synthesis of HSP68 protein cannot yet be traced. From these data we conclude that a differential HSP expression takes place. The translational control of HSP synthesis might be explained by the preferential translation of this mRNA under conditions of severe shut-off of general protein synthesis.

Enhancement of the stress response by low concentrations of arsenite in arsenite-pretreated Reuber H35 hepatoma cells.

The present study is aimed at determining whether the induction of heat-shock protein (hsp) synthesis, heat-shock mRNAs, and tolerance development after arsenite application has been sensitized to low concentrations of arsenite in Reuber H35 rat hepatoma cells. Using a step-down arsenite treatment, consisting of a 1-hr pretreatment with 100 or 300 microM followed by an incubation with a lower concentration (1-10 microM), H35 cells were shown to exhibit increased sensitivity to low concentrations of sodium arsenite shortly after exposure to the high arsenite concentration, but not any longer when the low concentration was applied 4 hr after pretreatment. In this paper it is shown that exposure of H35 cells to sodium arsenite concentrations of 100 or 300 microM for 1 hr rapidly changes the sensitivity toward a second arsenite treatment with respect to the induction of the heat-shock response. It was observed that under conditions of enhanced sensitivity, an additional increase occurred in hsp synthesis as well as in hsp mRNA (as exemplified by hsp68 mRNA behavior) when low concentrations of arsenite were applied to arsenite pretreated cells. Since no effect of these low concentrations was observed in nonpretreated cells, the effect of step-down treatment results in a higher effect than could be expected based on summation. Furthermore, in sensitized cultures, cells are able to develop a higher level of tolerance in the presence of low concentrations of arsenite. It can be concluded that during a transient period of enhanced sensitivity, low concentrations of the original stressor are able to enhance hsp synthesis and to induce a higher level of tolerance in comparison with control cultures that are sensitized but not incubated in the presence of low concentrations of the original stressor.

HSP synthesis of neonatal rat heart myocytes is regulated by a collagen environment.

Studies on the stress response of isolated myocytes have gained great importance in the understanding of the response of the heart as an organ after, for instance, ischemia. However, the possible role of the extracellular matrix on these effects has thereby been neglected. The recently developed model system of neonatal heart cells cultured on a collagen gel, characterized by a high coherence of contractions, has been used to study the effects of this more in vivo-like collagen environment on the heat shock response of the myocytes as compared to 'normally used' monolayer cultures. After four days differences were found in the heat-induced synthesis of HSPs of cells grown by the two culturing procedures. The degree of induction of different HSPs appeared to be directly related to the basic level of synthesis of these HSPs under the used culturing conditions. In collagen gel-grown cultures the basic level of synthesis as well as the heat-induced synthesis of HSP84 and HSP100 was decreased, for HSP60 both were increased, and for HSP70 no differences were found compared to the monolayer cultures. Our results suggests that the collagen matrix has a regulatory role in the synthesis of HSPs.

Stressor-specific induction of heat shock proteins in rat hepatoma cells.

In order to determine whether induction of specific stress proteins is dependent on a given stressor and whether induction of these proteins is linked to survival, Reuber H35 rat hepatoma cells were exposed to five different environmental stressors (heat shock, arsenite, cadmium, dinitrophenol and ethanol). The effect of these stressors was studied on cell survival as well as on inhibition and recovery of protein synthesis and on induction of heat shock proteins (hsps). In this article, we present evidence that several well-known hsp-inducers fail to stimulate specific hsps in a degree that is comparable to the induction of these hsps by heat shock. Most evidently, hsp60 is not induced by cadmium-treatment, whereas hsp100 is hardly induced by sodium arsenite. Treatment with DNP only slightly induces hsp68 and hsp84, whereas no detectable induction of hsps is observed after treatment with ethanol. In contrast, treatment with cadmium raises the amount of hsp28 to a higher level as compared to heat shock. A comparison of the stressor-specific induction of major hsps was also made under conditions of similar impact on cellular physiology: (a) stressor conditions up to the critical point that cell death starts to occur, and (b) conditions of iso-survival (50%). We conclude that hsps cannot be simply used as a general risk-assessment tool, and that the validation of stressor-specific risk-assessment warrants further research with larger groups of proteins.

Collagen gels populated with rat neonatal heart cells can be used for optical recording of rhythmic contractions which also show ECG-like potentials.

Neonatal heart cells populated collagen gels show rhythmic contractions which can be recorded optically and electrically. Optical recordings revealed two populations of rhythmically contracting gels: 1) highly coherent contracting collagen gels with normally distributed contraction interval times and contraction amplitudes, and 2) irregularly contracting gels with a multi-modal distribution of contraction interval times and amplitudes. The irregularly contracting gels were shown to be 'semi regular', which means that a short contraction interval was preferentially followed by another short interval. The volume of the collagen gel during the contraction decreased, and our calculations indicate that the myocytes expel 3-10 times their own volume from the gel. Changes in electrical potential were observed depending on the location of the electrodes. These electrical, ECG-like changes in potential were maximal when one electrode was placed in the centre and the other at the edge of the gel. The results of this study indicate that myocyte-populated collagen gels are a very promising system for studies of electrophysiology and coherent contractions.

Serum-stimulated cell cycle progression and stress protein synthesis in C3H10T1/2 fibroblasts treated with sodium arsenite.

In this work, we demonstrated that a nonlethal dose of arsenite administered to quiescent C3H10T1/2 fibroblasts can enhance the mitogenic effect of suboptimal concentrations of serum. The mitogenic effect was dependent on the serum concentration and on the time interval between the administration of arsenite and that of serum. This suggests that mitogen sensitivity changes in time after arsenite treatment. It is shown that the concentrations of arsenite that enhance the mitogenic effect of serum also increase the mRNA levels of c-fos, HSP68, and HSP84 and induce the specific synthesis of Heat Shock Proteins (HSPs). The physiological significance of this phenomenon is most likely to counteract the long-term toxic effect of arsenite by early induction of compensation for cell loss.

Arsenite induced sensitization and self-tolerance of Reuber H35 hepatoma cells.

Our data show that a short incubation with arsenite (30-300 microM) induces a biphasic change in cellular sensitivity towards a second exposure to arsenite. A transient sensitization was followed by the development of self-tolerance. Sensitization was measured using the step-down protocol; i.e., application of a high dose of arsenite pretreatment (100 or 300 microM) followed immediately by incubation in a low dose of arsenite (1-30 microM), with extensive rinsing in between. Whereas no effect of 1 and 3 microM on cellular survival is observed without pretreatment, a large decrease in cell survival can be established when these low doses of arsenite are applied immediately after a 1 hr pretreatment with 100 or 300 microM arsenite. According to the step-down protocol, a high dose of toxic compounds is applied and is followed by prolonged incubation in a lower concentration of the initial toxic compound. This might be a more accurate model for studying the effects of toxic insults on cells and organisms in the manner in which they occur in their natural environment. The level of tolerance was determined by a 1 hr test treatment with 300 microM arsenite applied at different times after pretreatment. Using this fractionated treatment protocol, it was established that tolerance increases with the increasing time intervals between the sodium arsenite treatments, during the 6 hr studied. These observations suggest that sensitization gradually decreases, whereas tolerance develops. Furthermore, our data indicate that the condition of pretreatment determines the extent to which the early sensitivity increases, as well as the development of tolerance later on. A relatively high arsenite concentration leads to more sensitized cells, which are transformed into more tolerant cells in comparison with the effect of a lower arsenite concentration.

Fibroblastic cell cycling in collagen gels.

Quiescent C3H10T1/2 mouse fibroblasts resume DNA synthesis and proliferation following incubation in medium containing fresh serum both when grown in monolayer and when grown in a collagen matrix. We observed that the rate of DNA synthesis is reduced at high initial cell densities and low initial collagen concentrations. In a collagen matrix, fibroblasts contract the matrix causing an increase in cell density and collagen concentration. We studied the chronological relationship between the kinetics of DNA synthesis and the collagen matrix contraction. The rate of collagen collection per cell changes in time, dependent on initial cell and collagen concentration. The kinetics of the collagen collection showed a positive correlation with the kinetics of DNA synthesis, 16 h later.

Factors controlling the rhythmic contraction of collagen gels by neonatal heart cells.

A floating collagen matrix culture of neonatal rat heart myocardial cells shows rhythmic contractions which are dependent on localization of cells, cell density, and collagen concentration. The rhythmic contractions of the collagen matrix can be registered by a device scanning the optical density at the edge of the gel and have been observed over a temperature range from 9 degrees to 40 degrees C. The results of the present study underline the usefulness of myocardial cell populated collagen matrixes for studies on coherent contractions of heart cell cultures.

Thermotolerance induced by heat and ethanol.

The effect of ethanol and heat on the thermosensitivity of 3-day-old larvae of the fresh water snail Lymnaea stagnalis is investigated, especially with regard to the kinetics of thermotolerance, the effect on protein synthesis and the pattern of proteins synthesized. Both stress factors, in a mild dose, induce a state of thermotolerance with the following characteristics: (i) it is not accompanied by an enhanced synthesis of HSPs, (ii) it needs only 10-30 min to develop maximally, and (iii) it decays within 60-90 min after it has been triggered. At a higher dose both factors induce an enhanced synthesis of the HSP 65, HSP 70, and HSP 87, and also a more stable state of thermotolerance beside the thermotolerant state present shortly after the trigger. It appears that the synthesis of HSPs is enhanced only when the overall protein synthesis is depressed. The data are discussed in relation to the putative functions of heat shock proteins. It is suggested that the constitutive levels of the HSPs provide the rapid protection against heat. An induced level of HSPs is necessary for the extension of the thermotolerant state.

Thermotolerance induced by 2,4-dinitrophenol.

The capacity of 2,4-dinitrophenol (DNP) to induce thermotolerance in 3-day-old larvae of Lymnaea has been investigated. Induction of thermotolerance is dependent on the DNP concentration, the duration of treatment, and on the pH of the medium. Optimal conditions for the induction of thermotolerance are 10 min, 2.5 mM DNP at pH 4.7. Thermotolerance has been expressed in percentages of survival. Thermotolerance is maximal at 15 min after DNP treatment and decreases subsequently. DNP suppresses protein synthesis. Recovery starts 20 min after treatment, when thermotolerance is decreasing already. DNP does not induce the synthesis of the typical heat shock proteins (HSPs). It is concluded that DNP induces a state of thermotolerance of short duration, which is not accompanied by HSP synthesis.