When prokaryotic or eukaryotic cells are submitted to a transient rise in temperature or to other proteotoxic treatments, the synthesis of a set of proteins called the heat shock proteins (hsp) is induced. The structure of these proteins has been highly conserved during evolution. The signal leading to the transcriptional activation of the corresponding genes is the accumulation of denatured and/or aggregated proteins inside the cells after stressful treatment. The expression of a subset of hsp is also induced during early embryogenesis and many differentiation processes. Two different functions have been ascribed to hsp: a molecular chaperone function: chaperones mediate the folding, assembly or translocation across the intracellular membranes of other polypeptides, and a role in protein degradation: some of the essential components of the cytoplasmic ubiquitin-dependent degradative pathway are hsp. These functions of hsp are essential in every living cell. They are required for repairing the damage resulting from stress.

In contrast to differentiated somatic cells, mouse embryonal carcinoma (EC) cell lines spontaneously express high levels of major members of the heat shock protein (HSP) family. In addition, some EC cell lines (noninducible) are not able to induce HSP gene transcription and HSP synthesis after a stress. However, after in vitro differentiation, constitutive HSP expression decreases and the differentiated derivatives become able to induce HSP gene transcription after a stress. These cells were tested by gel shift assays for the presence of an activity able to bind the heat shock element (HSE) before and after a stress. Control fibroblasts grown at 37°C did not contain significant levels of HSE-binding activity, but heat shock dramatically increased the level of HSE-binding activity. In contrast to control fibroblasts, all EC cells contained significant levels of HSE-binding activity at 37°C. In the inducible EC cell line F9, as in fibroblasts, heat shock strongly increased the level of HSE-binding activity. In the noninducible EC cells, however, HSE-binding activity markedly decreased upon heat shock. During in vitro differentiation of the noninducible cell line PCC7-S-1009, the constitutive HSE-binding activity found at 37°C disappeared and heat induction of the HSE-binding activity appeared. Therefore, a good correlation exists between the high spontaneous expression of some members of the HSP family and the constitutive level of HSE-binding activity in EC cells at 37°C. Heat induction of HSP gene transcription correlates with a strong increase in HSE-binding activity, whereas a deficiency in heat induction of HSP gene transcription is associated with a loss of HSE-binding activity upon heat shock.

Interferon (IFN) is not able to induce heat-shock protein (HSP) synthesis. However IFN pretreatment of mouse L cells has been shown to enhance the decrease of overall protein synthesis which follows a heat shock, and to stimulate the accumulation of HSPs. We show here that the synthesis of a protein (the hepatitis B virus surface antigen) under the control of a Drosophila HSP 70 promoter is also stimulated in IFN-pretreated cells. The regulation by IFN takes place at two levels: first, the rate of HSP gene transcription is increased in nuclei isolated from IFN-treated cells; second, the synthesis of HSPs is prolonged after pretreatment with IFN. Experiments performed in the presence of actinomycin D show that this effect is due to a stabilization by IFN of mRNAs coding for HSPs.

The synthesis of heat-shock proteins via activation of heat-shock genes occurs in response to heat and various physical or chemical stressing agents. Transcriptional activation of heat-shock genes requires a heat-shock regulatory element in their promoter, to which a heat-shock specific transcription factor binds. In Drosophilu cells, the heat-shock factor already exists in unstressed cells in an inactive form and acquires the capacity to bind to the heat-shock element following stress. The mechanism of this activation is not known ; neither is it known whether the different stressing agents induce the heat-shock response through a common mechanism. We previously proposed that many agents known to induce the heat-shock response (substances interfering with respiratory metabolism, agents reacting with sulphydryl groups, metals, recovery from anaerobiosis and ischemia) might act via accumulation of reactive oxygen species, i.e. superoxide ion or H202. We show here that H202, introduced either in Drosophilu cell cultures or in cell extracts, was able to activate heat-shock-element binding. Activation was rapid and H 2 0 2 concentration dependent, with a threshold of 1 pM. These results were confirmed with mouse fibroblast cells. This very rapid activation, in vivo or in vitro, suggests a direct effect of H 2 0 2 either on the heatshock factor itself or on its activator.The synthesis of heat-shock or more generally termed stress proteins in response to elevation of temperature or to a large variety of physical and chemical stressing agents seems to be common to all living organisms, from bacteria to man [l -41. A puzzling question is the mechanism by which the numerous stressing agents lead to the transcriptional activation of the same set of heat-shock genes and to what extent they might act through a common pathway.We previously proposed a hypothesis based on the following observation : reoxygenation after a period of anoxia without elevation of temperature, is sufficient to induce the heatshock proteins in Drosophila Kc cells [5] and other biological systems [6 -lo]. At the time of reoxygenation, O2 consumption is increased twofold over the control [5]. It is known that this situation, deprivation of oxygen followed by an excess, generates byproducts of the partial reduction of oxygen, i.e. free radicals or reactive oxygen species [l 11, among which two have been particularly studied, the superoxide ion (0;) and H 2 0 2 . These reactive oxygen species are involved in many diseases, in the inflammation process, and in carcinogenesis [12-141. We hypothesized that the reactive oxygen species might also be involved in the mechanism of induction of heatshock proteins [5]. Indeed, heat-shock is accompanied by an elevation of O2 consumption [5, 101 and many factors which induce the heat-shock protein response could act via accumulation of reactive oxygen species or more generally via modification of the redox equilibrium of the cell [I, 3, 15, 161. This is the case, for example, for substances interfering with the [19]...

In contrast to differentiated somatic cells, mouse embryonal carcinoma (EC) cell lines spontaneously express high levels of major members of the heat shock protein (HSP) family. In addition, some EC cell lines (noninducible) are not able to induce HSP gene transcription and HSP synthesis after a stress. However, after in vitro differentiation, constitutive HSP expression decreases and the differentiated derivatives become able to induce HSP gene transcription after a stress. These cells were tested by gel shift assays for the presence of an activity able to bind the heat shock element (HSE) before and after a stress. Control fibroblasts grown at 37 degrees C did not contain significant levels of HSE-binding activity, but heat shock dramatically increased the level of HSE-binding activity. In contrast to control fibroblasts, all EC cells contained significant levels of HSE-binding activity at 37 degrees C. In the inducible EC cell line F9, as in fibroblasts, heat shock strongly increased the level of HSE-binding activity. In the noninducible EC cells, however, HSE-binding activity markedly decreased upon heat shock. During in vitro differentiation of the noninducible cell line PCC7-S-1009, the constitutive HSE-binding activity found at 37 degrees C disappeared and heat induction of the HSE-binding activity appeared. Therefore, a good correlation exists between the high spontaneous expression of some members of the HSP family and the constitutive level of HSE-binding activity in EC cells at 37 degrees C. Heat induction of HSP gene transcription correlates with a strong increase in HSE-binding activity, whereas a deficiency in heat induction of HSP gene transcription is associated with a loss of HSE-binding activity upon heat shock.