Effect of hydrogen peroxide on cytoskeletal proteins of Drosophila cells: comparison with heat shock and other stresses.

Hydrogen peroxide, which was shown to trigger the heat-shock response by activating the immediate binding of the heat-shock factor to DNA heat shock regulatory elements in the promoter of heat-shock genes of Drosophila cells, has also been reported to enhance the synthesis of actin. We show here that very short and transient H2O2 treatments, from 1 s to 2 min, are sufficient to induce an increase of actin synthesis. This increase becomes apparent 2 to 3 h after the short H2O2 treatment. It is inhibited if actinomycin D is present during the short H2O2 treatment. An increase of actin synthesis was also observed during the recovery period after two other stresses: reoxygenation after anoxia and ethanol treatment. The synthesis of two cytoskeletal proteins, tubulin and a 46-kDa insoluble protein of the intermediate filament fraction, was also slightly increased by H2O2 in Drosophila cells, but this increase was not actinomycin D-dependent. H2O2 does not provoke the translocation of the 46-kDa protein to the nuclear fraction as does heat shock. The very rapid stimulation of actin synthesis by H2O2 and the involvement of cytoskeletal elements in many stress situations suggest that actin may play a key role in the response to external stimuli.

On the mechanism of action of H2O2 in the cellular stress.

We propose a hypothesis according to which the reactive and reduced species of oxygen could be the intracellular inducers of the stress (or "heat-shock") response. This hypothesis is based on the following observations on Drosophila cells: a) the return to normoxia after 24 h anaerobiosis is sufficient to induce the synthesis of the "heat shock" proteins without elevation of temperature together with a rapid increase of O2 consumption; b) hydrogen peroxide introduced in the culture medium induces the early transcriptional activation of the "heat shock" genes (maximal after 5 minutes); c) hydrogen peroxide added to cellular extracts in vitro (thus acting as an intracellular metabolite) activates instantaneously the binding capacity of a "heat shock" factor to a DNA "heat shock" regulatory element. Thus, hydrogen peroxide, and possibly other reactive reduced species of oxygen, could trigger the onset of the stress (or "heat shock") response.

The response of the centrosome to heat shock and related stresses in a Drosophila cell line.

The centrosome of Drosophila melanogaster cells cultured in vitro has been followed by immunofluorescence techniques with the Bx63 antibody of Frasch and Saumweber. After a heat shock, the centrosome labelling becomes very small and finally disappears after 30 min. Other heat-shock protein (hsp) inducers such as ethanol, arsenite and ecdysterone lead to the same disappearance. Moreover, the functional ability of centrosomes to nucleate microtubule assembly is inhibited by these treatments, particularly by heat shock, ethanol and ecdysterone. Two other hsp inducers, cadmium chloride and hydrogen peroxide, do not affect the centrosome seriously. With the exception of cadmium, the rapidity and the intensity of hsp induction are in good agreement with the kinetics of alteration of the organelle. We propose that a close link exists between the heat-shock response and the centrosome and that the physiological induction of hsps could be reinterpreted in terms of cell division control.

Hydrogen peroxide activates immediate binding of a Drosophila factor to DNA heat-shock regulatory element in vivo and in vitro.

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 Drosophila 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 H2O2. We show here that H2O2, introduced either in Drosophila cell cultures or in cell extracts, was able to activate heat-shock-element binding. Activation was rapid and H2O2 concentration dependent, with a threshold of 1 microM. These results were confirmed with mouse fibroblast cells. This very rapid activation, in vivo or in vitro, suggests a direct effect of H2O2 either on the heat-shock factor itself or on its activator.

Early activation of heat shock genes in H2O2-treated Drosophila cells.

Drosophila cells of a diploid clone derived from line Kc were treated with 1 mM H2O2 for 1 to 20 minutes. Dot blot and Northern blot analysis of RNAs extracted from control and treated cells showed that the transcriptional activation of the 6 heat-shock genes tested was early, and maximal within 5 minutes of H2O2 treatment. Analysis of the kinetics of induction of the heat-shock proteins (hsps) after an exposure to H2O2 of 2 or 5 minutes, followed by removal, suggests that this brief treatment was sufficient to trigger the synthesis of all the hsps, which was maximal 1.5 to 3h after this short H2O2 treatment.

Hydrogen peroxide (H2O2) induces actin and some heat-shock proteins in Drosophila cells.

Drosophila cells of a clone derived from line Kc were treated with various concentrations of hydrogen peroxide (H2O2). The concentration of 10 mM was lethal, whereas concentrations of 1-100 microM did not affect cell viability, rate of multiplication or protein synthesis. The intermediate concentration of 1 mM H2O2 was used to study the response of the cells to an oxidative stress. We observed a transitory decrease of the global protein synthesis, which was accompanied by changes in the polypeptide pattern. There was a 2.5-fold increase of the synthesis of the heat-shock proteins 70-68 and 23. The most prominent response was a 6.5-fold increase of actin synthesis 3 h after a 1 mM H2O2 treatment. When aminotriazole (an inhibitor of catalase) was added in association with H2O2, the increase of actin synthesis became 8.5-fold. Experiments in which catalase was added at various times after H2O2 showed that a 10-min treatment with H2O2 was sufficient to induce actin and heat-shock protein synthesis 3 h later. H2O2 was shown to induce the transcriptional activation of an actin gene and of the heat-shock protein genes 70 and 23 within minutes. These results are coherent with the hypothesis that the byproducts of O2 reduction (the superoxide ion and hydrogen peroxide) could be inducers of the heat-shock response. Whether the increase of actin synthesis is a stress-related response, and the mode of action of H2O2 are discussed.

Heat shock proteins are induced by cadmium in Drosophila cells.

Drosophila cells were treated with increasing concentrations of CdCl2 (10 microM-1 mM). The toxicity of cadmium, as observed by cellular death and the ability of the cells to survive after removal of CdCl2, depended on concentration and duration of treatment. The overall synthesis of protein, measured by incorporation of [35S]methionine, decreased. It fell to 66% of the controls after 24 h of exposition to 50 microM CdCl2 and to 29% after 48 h. We showed that cadmium induced the synthesis of 'heat shock proteins' (hsps), which started after 6 h and was maximal after 24 h of 50-100 microM CdCl2 treatment.

The possible role of the superoxide ion in the induction of heat-shock and specific proteins in aerobic Drosophila cells during return to normoxia after a period of anaerobiosis.

In vitro cultured Drosophila melanogaster cells were shown to be aerobic and several kinetic parameters of their respiration were measured. This allowed us to define experimental conditions for a transient period of anaerobiosis followed by a reexposure to normal oxygenation. This treatment, applied without any change of temperature, induced not only the heat-shock proteins, but also a new specific peptide of 27 000 daltons and a twofold increase of the maximal rate of O2 uptake. This evokes a common molecular mechanism activated either by heat or by O2, which could involve the increase of the products of oxygen reduction such as the superoxide ion.

A critical period of ecdysterone action on sensitive clones of Drosophila cultured in vitro: the maturation of the cells.

Ecdysterone-sensitive clones cultured in vitro were isolated from established cell lines of Drosophila melanogaster. The clones FC and 89K are ecdysterone-inducible for two enzymatic activities: acetylcholinesterase and beta-galactosidase. No activity could be detected in untreated cells, whereas after treatment with 50-250 nM ecdysterone, the activity appeared after one day and increased during 3-4 days. We wanted to modulate the response of the cells by varying the conditions of the hormonal stimulus. Mimicking the physiological situation of Drosophila (the ecdysterone peak corresponding to the molts is preceded by low levels) we pretreated the cells with a subthreshold concentration (1-5 nM) for 2 days and then we added the stimulating concentration of 50-250 nM ecdysterone. The enzymatic activities were then detectable within the following hours and the final level of induction was about twice the one of cells without pretreatment. Thus, the continuous presence of a subthreshold concentration of ecdysterone provokes the maturation of the cells which become able to respond to the hormonal stimulus by a quicker and higher enzymatic induction. The cellular maturation seems to be a critical period. It is altosid-sensitive. Altosid (a juvenile hormone analog) abolishes the effects of the ecdysterone-induced maturation.

beta-Galactosidase is induced by hormone in Drosophila melanogaster cell cultures.

Drosophila melanogaster cell lines Kc and Ca and clones FC and RF6, cultured in vitro, have no detectable beta-galactosidase (beta-galactoside galactohydrolase, EC 3.2.1.23) activity (as measured by hydrolysis of o-nitrophenyl-beta-D-galoctoside). Ecdysterone, a hormonal steroid of critical importance in insect physiology, clearly induces beta-galactosidase activity in D. melanogaster cells cultured in vitro. Induction occurs in cell lines or clones known to be sensitive to ecdysterone (K, Ca, and Fc) and does not occur in clones known to be resistant to the hormone (RF6). Some properties of the hormone-induced beta-galactosidase activity were studied. The Km for o-nitrophenyl galactoside is 0.35 mM and the Ki for lactose is 12 mM (similar to those of Escherichia coli beta-galactosidase); the activity can be recovered after sodium dodecyl sulfate treatment; the enzyme is a tetramer (Mr of the monomer is 64,000).

[Comparison of the effects of alpha and beta ecdysone on a diploid clone of cultured Drosophila melanogaster cells in vitro: induction of proteins and morphological changes]. / Etude comparative des effets de l'alpha et de la beta-ecdysone sur un clone de cellules diploïdes de Drosophila melanogaster en culture in vitro: induction protéique et modifications morphologiques

The specific induction of a similar (class of) protein(s) by alpha or beta-ecdysone in the cells of a "sensitive" clone of Drosophila melanogaster cultured in vitro was shown by polyacrylamide gel electrophoresis. However, differences can be seen in the action of the two hormones. (1) The threshold concentration is about 10 times higher for alpha-ecdysone (10 nM) than for beta-ecdysone (1 nM). (2) Beyond this threshold (i.e. 100 nM) the rate of induction is greater for beta-ecdysone (less than or equal to 24 h) than for alpha-ecdysone (larger than or equal to 48 h). The morphological modifications and the protein induction are seen simultaneously after 1 day of 100 nM beta-ecdysone treatment. On the contrary, a dissociation of these two phenomena is noted with the same concentration of alpha-ecdysone.

[Combined action of juvenile hormone and ecdysterone on Drosophila cell lines in vitro]. / Action conjuguée de l'hormone juvénile et de l'ecdystérone sur des lignées cellulaires de Drosophile in vitro

The action of juvenile hormone used alone or combined with ecdysterone was studied on in vitro diploid cell lines or clones of Drosophila. The solubility of juvenile hormone in the culture medium was estimated to be 10(-8) M. At this concentration, which can be considered as physiological, juvenile hormone slows down the process of ecdysone-induced morphological modifications of the cells. This action is reversible. Concentrations of about 10(-4) M inhibit all reactions to ecdysterone and bring about the death of the cells. This effect is probably non-specific.