Stressor-specific activation of heat shock genes in H35 rat hepatoma cells.

Responses of Reuber H35 hepatoma cells exposed to increasing doses of heat, arsenite ions, or cadmium ions were investigated. Doses which are capable of activating the heat shock transcription factor (HSF) were determined. The activity of this factor in the poststress period was correlated to the recovery of total protein synthesis in this same period. Subsequently, increases of mRNA levels and rates of synthesis of heat shock protein (HSP)60, HSP68, and HSP84 after exposure to the stressors were determined. We generally found that the rate of HSP synthesis correlated well with HSP mRNA levels, supporting the idea that the stress response is regulated mainly at the transcriptional level. A stressor-specific pattern of HSP mRNA induction can be observed. The most striking example is cadmium chloride which does not induce HSP60 nor its mRNA. Interestingly, under these conditions maximal activation of HSF is observed. Therefore, we conclude that more processes than just HSF activations are involved in the induction of heat shock genes.

Modulation of HSP68 gene expression after heat shock in thermosensitized and thermotolerant cells is not solely regulated by binding of HSF to HSE.

Induction of heat shock proteins (HSP) is generally regarded as a consequence of binding of the heat shock transcription factor (HSF) to heat shock elements (HSE), i.e. to be a single hit induction. The activation of HSF and the induction of HSP68 mRNA were studied in non pretreated Reuber H35 rat hepatoma cells in a thermosensitized and in a thermotolerant state. It was found that HSF in Reuber H35 hepatoma cells already acquires maximum DNA binding activity at temperatures that are too low to induce HSP68 mRNA. Directly following heat shock cells are in a transient thermosensitized state. In this state a second stress of lower impact leads to even higher production of HSP68, which corresponds with a decreased decay rate HSF-HSE binding. Directly following the thermosensitized state cells become refractory. In this period a second stress of the same impact does lead to HSF-HSE binding but the production of HSP68 mRNA is lowered, while only higher-impact stresses lead to high inductions of the said mRNA. The results indicate that regulation of HSP68 gene transcription involves at least one additional event outside the acquisition of DNA-binding activity by HSF and that this process can thus be described as a multiple-hit occurrence.

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.

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.