Lack of heat shock response triggers programmed cell death in a rat histiocytic cell line.

Stress response is a universal phenomenon. However, a rat histiocytic cell line, BC-8, showed no heat shock response and failed to synthesize heat shock protein 70 (hsp70) upon heat shock at 42 degrees C for 30 min. BC-8 is a clone of AK-5, a rat macrophage tumor line that is adapted to grow in culture and has the same chromosome number and tumorigenic potential as AK-5. An increase in either the incubation temperature or time or both to BC-8 cells leads to loss of cell viability. In addition, heat shock conditions activated apoptotic cell death in these cells as observed by cell fragmentation, formation of nuclear comets, apoptotic bodies, DNA fragmentation and activation of ICE-like cysteine proteases. Results presented here demonstrate that BC-8 cells cannot mount a typical heat shock response unlike all other eukaryotic cells and that in the absence of induction of hsps upon stress, these cells undergo apoptosis at 42 degrees C.     

Heat shock and oxidative stress-induced exposure of hydrophobic protein domains as common signal in the induction of hsp68

The hypothesis of a common signal for heat shock (HS) and oxidative stress (OS) was analyzed in C6 cells with regard to the induction of heat shock proteins (Hsps). The synthesis rate and level of the strictly inducible Hsp68 was significantly higher after HS (44 degrees C) compared with OS (2 mm H2O2). This difference corresponded to higher and lower activation of the heat shock factor (HSF) by HS and OS, respectively. OS, on the other hand, showed stronger cytotoxicity compared with HS as indicated by drastic lipid peroxidation and inhibition of protein synthesis as well as of mitochondrial and endocytotic activity. Lactic dehydrogenase also revealed stronger inhibition of enzyme activity by OS than by HS as shown in cells and in vitro experiments. Conformational analysis of lactic dehydrogenase by the fluorophore 1-anilinonaphtalene-8-sulfonic acid, however, showed stronger exposure of hydrophobic domains after HS than after OS which correlates positively with the Hsp68 response. Treatment of cells with deoxyspergualin, which exhibits high affinity to Hsps, the putative inhibitors of HSF, strongly increased only OS-induced hsp68 expression. In conclusion, the results suggest that exposure of hydrophobic domains of cytosolic proteins represents the common first signal in the multistep activation pathway of HSF.     

Higher induction of heat shock protein 72 by heat stress in cisplatin-resistant than in cisplatin-sensitive cancer cells

Induction of the heat shock proteins (HSPs) is involved in the increased resistance to cancer therapies such as chemotherapy and hyperthermia. We used two human ovarian cancer cell lines; a cisplatin (CDDP)-sensitive line A2780 and its CDDP-resistant derivative, A2780CP. The concentration of intracellular glutathione (GSH) is higher (2.7-fold increase) in A2780CP cells than in A2780 cells. A mild treatment with a heat stress (42 degrees C for 30 min) induced synthesis of both the heat shock protein 72 (Hsp72) mRNA and the HSP72 protein in A2780CP cells, but not in A2780 cells. In contrast, a severe heat stress (45 degrees C for 30 min) increased synthesis of the HSP72 protein in the two cell lines. The induced level of the HSP72 protein by the severe treatment was higher in A2780CP than in A2780 cells. The gel mobility shift assay showed that DNA binding activities of the heat shock factor (HSF) in the two cell lines were induced similarly and significantly by the mild heat stress. Immunocytochemistry using an anti HSF1 antibody also indicated that mild heat stress activated the HSF1 translocation from the cytosol to the nucleus similarly in the both cell lines. Pretreatment of CDDP-sensitive A2780 cells with N-acetyl-L-cysteine, a precursor of GSH, effectively enhanced induction of the Hsp72 mRNA by the mild heat stress. The present findings demonstrate that induction of the Hsp72 mRNA by the mild heat stress was more extensive in CDDP-resistant A2780CP cells. It is likely that the higher GSH concentration in A2780CP cells plays an important role in promoting Hsp72 gene expression induced by the mild heat stress probably through processes downstream of activation of HSF-DNA binding.     

Differential temperature dependency of chemical stressors in HSF1-mediated stress response in mammalian cells

Expression of stress proteins is generally induced by a variety of stressors. To gain a better understanding of the sensing and induction mechanisms of stress responses, we studied the effects of culture temperature on responses to various stressors, since the induction of hsp70 in mammalian cells by heat shock is somehow modulated by culture temperature. Hsp70 was not induced by treatment with sodium arsenite, azetidine-2-carboxylic acid, or zinc sulfate at the level of heat shock factor (HSF) 1 activation in cells incubated at low temperature, although these treatments induced hsp70 in cells incubated at 37 degrees C. The repression of sodium arsenite or zinc sulfate-induced HSF1 activation by low temperature was not simply due to the inhibition of protein synthesis. On the other hand, heat shock and iodoacetamide induced HSF 1 activation in cells incubated at either temperature. Thus, there seem to be two kinds of stressors that induce HSF1 activation independently of or dependent on culture temperature. Furthermore, the reduction of glutathione level seemed to be essential for HSF1 activation by chemical stressors.     

Geldanamycin restores a defective heat shock response in vivo

Induced expression of heat shock proteins (Hsps) plays a central role in promoting cellular survival after environmental and physiological stress. We have previously shown that scrapie-infected mouse neuroblastoma (ScN2a) cells fail to induce the expression of Hsp72 and Hsp28 after various stress conditions. Here we present evidence that this impaired stress response is due to an altered regulation of HSF1 activity. Upon stress in ScN2a cells, HSF1 was converted into hyperphosphorylated trimers but failed to acquire transactivation competence. A kinetic analysis of HSF1 activation revealed that in ScN2a cells trimer formation after stress was efficient, but disassembly of trimers proceeded much faster than in the uninfected cell line. Geldanamycin, a Hsp90-binding drug, significantly delayed disassembly of HSF1 trimers after a heat shock and restored stress-induced expression of Hsp72 in ScN2a cells. Heat-induced Hsp72 expression required geldanamycin to be present; following removal of the drug ScN2a cells again lost their ability to mount a stress response. Thus, our studies show that a defective stress response can be pharmacologically restored and suggest that the HSF1 deactivation pathway may play an important role in the regulation of Hsp expression.     

Acclimation of the photosynthetic machinery to high temperature in Chlamydomonas reinhardtii requires synthesis de novo of proteins encoded by the nuclear and chloroplast genomes

The mechanism responsible for the enhancement of the thermal stability of the oxygen-evolving machinery of photosystem II during acclimation of Chlamydomonas reinhardtii to high temperatures such as 35 degrees C remains unknown. When cells that had been grown at 20 degrees C were transferred to 35 degrees C, the thermal stability of the oxygen-evolving machinery increased and within 8 h it was equivalent to that in cells grown initially at 35 degrees C. Such enhancement of thermal stability was prevented by cycloheximide and by lincomycin, suggesting that the synthesis de novo of proteins encoded by both the nuclear and the chloroplast genome was required for this process. No increase in thermal stability was observed when cells that had been grown at 35 degrees C were exposed to heat shock at 41 degrees C, optimum conditions for the induction of the synthesis of homologs of three heat shock proteins (Hsps), namely, Hsp60, Hsp70, and Hsp22. Moreover, no synthesis of these homologs of Hsps was induced at 35 degrees C. Thus it appears likely that Hsps are not involved in the enhancement of the thermal stability of the oxygen-evolving machinery.     

Thermosensitization,heat shock protein synthesis and development of thermotolerance in M-14 human tumor cells subjected to step-down heating

M-14 human tumor cells have been subjected to two regimens of step-down heating (SDH) consisting of a conditioning treatment at 42 degrees C for 1 h or at 44.5 degrees C for 20 min, immediately followed by heating at 40 degrees C. Both conditioning treatments thermosensitize the cells towards the subsequent heating at 40 degrees C; the thermosensitization ratio is 6.4 for cells conditioned at 42 degrees C for 1 h and 32.3 for cells conditioned at 44.5 degrees C for 20 min. The overall protein synthetic activity is reduced to 32.7% or 18.4% of control values following 1 h at 42 degrees C and 20 min at 44.5 degrees C, respectively; this inhibition is followed by a full recovery of the synthetic activity during the subsequent exposure at 40 degrees C. SDH-treated cells synthetize four heat shock proteins, with approximate molecular weights of 28, 64, 70 and 90 kDa. The pattern of HSPs induction observed in SDH-treated cells is similar to that found in cells subjected to single hyperthermic exposures. Cells subjected to the SDH sequence 42 degrees C/1 h-->40 degrees C/4 h develop thermotolerance, as indicated by a reduced sensitivity to further hyperthermic challenges.