Changes in membrane lipid composition following rapid cold hardening in Drosophila melanogaster

Naturally occurring diurnal variations in temperature are sufficient to induce a rapid cold hardening (RCH) response in insects. RCH can increase cold tolerance by 1-2 degrees C and extend the temperature interval at which insects can remain active. While the benefits of RCH are well established, the underlying physiological mechanisms remain unresolved. In this study we investigated the role of RCH on expression of heat shock proteins (Hsp70) after a cold shock, and the effect of RCH on the composition of phospholipid fatty acids (PLFAs) in membranes of Drosophila melanogaster. These experiments were performed on both "control" flies and flies selected for cold resistance in order to additionally examine a possible target for selection for cold tolerance. RCH improved survival following cold shock at -4, -6 and -8 degrees C. No induction of Hsp70 was found following cold shock irrespective of the pre-treatment. In contrast, a 5h RCH treatment was sufficient to induce small, but significant, changes in the composition of PLFAs. Here, the polyunsaturated linoleic acid, 18:2(n-6), increased while monounsaturated (18:1) and saturated (14:0) PLFAs decreased in abundance. These changes were observed in both selection groups and caused a significant increase in the overall degree of unsaturation. This response is consistent with the membrane response typically found during cold acclimation in ectothermic animals and it is likely adaptive to maintain membrane function during cold. Cold selection resulted in PLFA changes (decrease of 18:0 and 18:1 and increase of 14:0 and 16:1), which may improve the ability to harden during RCH.     

Effects of cold- and heat hardening on thermal resistance in Drosophila melanogaster

The effects of cold- and heat hardening on resistance to both low and high temperature stress was examined in Drosophila melanogaster lines selected for resistance to either cold or heat. The hardening effect was positive when the hardening was of the same type as the stress in all selection regimes. The effect of cold hardening on survival after heat stress was further examined in the lines selected for cold resistance and corresponding controls. A cross-protection effect (increased heat resistance after cold hardening) was present and this effect was lower in the lines selected for resistance to cold than in the controls. The level of Hsp70 expression induced by a non-lethal cold hardening was examined, showing that cold hardening induced Hsp70 expression. The results suggest that the cross-protection effect is at least partly due to Hsp70 expression induced by cold exposure.     

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.     

Post-eclosion decline in 'knock-down' thermal resistance and reduced effect of heat hardening in Drosophila melanogaster

The dependency of knock-down resistance on age, from 0 to 12 days post eclosion, was studied in two lines of Drosophila melanogaster, one selected for knock-down resistance and one unselected control. Additionally, the inducible Hsp70 expression level was assessed at maintenance temperature and after a heat hardening treatment (1 h at 35 degrees C) at the same ages. Knock-down resistance decreased roughly linearly with age in both control and knock-down selected lines and in both sexes regardless of maintenance temperature. Hsp70 expression, however, fell only from day 0 to day 3 and was thereafter changed little. Acclimation did not result in detectable difference between lines. The effect of hardening was only significant for control flies. This suggests that the increased knock-down resistance of the selected line was reached at the expense of hardening ability.     

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.     

Sensitization of Chemo-Resistant Human Chronic Myeloid Leukemia Stem-Like Cells to Hsp90 Inhibitor by SIRT1 Inhibition

Development of effective therapeutic strategies to eliminate cancer stem-like cells (CSCs), which play a major role in drug resistance and disease recurrence, is critical to improve cancer treatment outcomes. The current investigation was undertaken to examine the effectiveness of the combination treatment of Hsp90 inhibitor and SIRT1 inhibitor in inhibiting the growth of chemo-resistant stem-like cells isolated from human chronic myeloid leukemia K562 cells. Inhibition of SIRT1 by use of SIRT1 siRNA or SIRT1 inhibitors (amurensin G and EX527) effectively potentiated sensitivity of Hsp90 inhibitors (17-AAG and AUY922) in CD44^high K562 stem-like cells expressing high levels of CSC-related molecules including Oct4, CD34, β-catenin, c-Myc, mutant p53 (mut p53), BCRP and P-glycoprotein (P-gp) as well as CD44. SIRT1 depletion caused significant down-regulation of heat shock factor 1 (HSF1)/heat shock proteins (Hsps) as well as these CSC-related molecules, which led to the sensitization of CD44^high K562 cells to Hsp90 inhibitor by SIRT1 inhibitor. Moreover, 17-AAG-mediated activation of HSF1/Hsps and P-gp-mediated efflux, major causes of Hsp90 inhibitor resistance, was suppressed by SIRT1 inhibitor in K562-CD44^high cells. Our data suggest that combined treatment with Hsp90 inhibitor and SIRT1 inhibitor could be an effective therapeutic approach to target CSCs that are resistant to current therapies.     

Resistance to stress as a function of age in transgenic Drosophila melanogaster overexpressing Hsp70

This study investigated the resistance to stress as a function of age in Drosophila melanogaster overexpressing Hsp70. The resistances to starvation, paraquat, and cold in flies from 1 to 7 week-old have been measured. The line carrying the insertion vector without the transgenes is more resistant to starvation and cold than the parental and transgenic lines. In contrast, transgenic flies carrying extra-copies of hsp70 are more resistant to paraquat, however this is due to an especially high resistance in two age groups compared to all the other groups. I showed that exposure to a mild heat shock does not increase starvation resistance, slightly increases paraquat resistance, and strongly increases cold resistance. The transgenic flies expressing Hsp70 at higher levels after the heat shock do not exhibit enhanced stress resistance compared to control lines expressing less Hsp70 after the heat shock. The lack of effect of a mild heat shock on starvation and paraquat resistance is not due to a disappearance of the effect with age, since no effect is observed at any age. In contrast, when an effect of Hsp70 induction is observed as on cold resistance, this effect is still observed in old flies.