J. E. M. Souren

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.

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.

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.

FACTORS CONTROLLING THE RHYTHMIC CONTRACTION OF COLLAGEN GELS BY NEONATAL HEART CELLS

SummaryA 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° to 40° 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.

Contraction of collagen by human fibroblasts and keratinocytes

SummaryIn the process of wound healing keratinocytes and fibroblasts play an important role, keratinocytes in the re-epithelization process and fibroblasts in the process of wound contraction. We have studied the role of human keratinocytes and fibroblasts in the rearrangement of collagen in a collagen lattice model system. Our results revealed that keratinocytes as well as fibroblasts rearrange the collagen lattice; this occurs in a cell number and collagen concentration dependent manner. The optimal gel contraction is obtained in the presence of keratinocytes on the top of and of fibroblasts in the collagen lattice, the situation most closely approaching the in vivo situation. Between the two types of cells, differences in morphologic behavior were observed: when incorporated into the gel the keratinocytes retained their spherical shape throughout the whole culture period, but fibroblasts became elongated and formed extensions. Our data suggest that not only fibroblasts but also keratinocytes may be involved in the process of wound contraction.

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

1 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. 2 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. 3 The degree of stimulation appears to be stressorspecific, 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. 4 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. 5 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. 6 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. 7 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.

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.

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

Abstract. 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.

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

Our data show that a short incubation with arsenite (30–300 μM) induces a biphasic change in ceSlular 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 μM) followed immediately by incubation in a low dose of arsenite (1–30 μM), with extensive rinsing in between. Whereas no effect of 1 and 3 μM 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 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 pM 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.

A Molecular Basis for Understanding the Benefits from Subharmful Doses of Toxicants

In the past many scientists have published papers on hormesis, on molecular stress responses, and on the similia principle in homoeopathy. Very few, however, have stressed a common base of interdependence of these fields. Reviews the most important of these studies to demonstrate their evolution and their mutual importance. Furthermore, a multidisciplinary approach is chosen to demonstrate research into the beneficial effects of subharmful doses of toxicants administered in suboptimal conditions (such as in stressed or injured organisms and cells).