Glutamine is a powerful effector of heat shock protein expression in Drosophila Kc cells.

We have investigated the effects of extracellular anions on the regulation of expression of the heat shock response in Drosophila Kc cells incubated in defined balanced salt solutions. Widely varying chloride concentrations had no effect on normal or heat shock protein (hsp) expression. Increasing glutamate concentrations from zero to 15 mM increased hsp expression more than 100-fold while affecting expression of non-heat-shock proteins minimally. Glutamine was 20-100-fold more potent than glutamate in supporting hsp expression, while other amino acids were less effective or supported no detectable hsp synthesis in heat shock. Inhibition of glutamine synthetase with methionine-sulfoximine resulted in very low hsp expression with glutamate and normal high level expression with glutamine, confirming the importance of glutamine. The absence of glucose and treatment with 2-deoxyglucose did not change the requirement for adequate glutamine for hsp expression. Cells heat shocked under conditions which gave very low hsp expression resumed growth when returned to normal medium as well as cells which expressed normal levels of hsps. Measurements of free amino acid levels in cells heat shocked in the presence and absence of glutamine showed a correlation between glutamine levels and amount of hsp expression. We conclude that a physiological process regulated by glutamine or a glutamine metabolite is important for normal hsp expression in heat shock conditions in Drosophila.     

Cellular localization of Drosophila 83-kilodalton heat shock protein in normal, heat-shocked, and recovering cultured cells with a specific antibody

To gain insight on the possible functions of heat shock proteins (hsp's) in Drosophila, we have purified the 83-kilodalton hsp (hsp 83) from cultured cells and studied its intracellular localization by immunofluorescence in normal, heat-shocked, and recovering cells. The specificity of the antibody was assessed by one- and two-dimensional gel immunoblotting and by partial proteolytic digestion. The anti-hsp 83 antibody does not show any significant cross-reactivity with hsp's of different avian or mammalian cell lines, but cross-reacts with hsp's of similar molecular masses in other dipteran insects. The partial proteolytic peptide maps of Drosophila hsp 83 differ from those of mouse hsp 89 and chicken hsp 84. Immunoblotting of Drosophila Kc cells heat shocked at different temperatures indicates a maximal expression of hsp 83 at 33 degrees C. By immunofluorescence, hsp 83 is shown to have a strictly cytoplasmic localization. In unstressed cells, it is distributed in the entire cytoplasm with a slight enrichment in the perinuclear region. After heat shock, it seems to concentrate at the cell periphery close to the plasma membrane and it gradually redistributes to the whole cytoplasm during cellular recovery at normal temperatures.     

Analysis of the expression of the two major proteins of the 70 kilodalton mammalian heat shock family

This study compares the expression after heat shock of the two major variants of the mammalian 70 kilodalton heat shock family in three separate systems. The ability of wild type and temperature sensitive mutant (ts85) FM3A cells to elicit a heat shock response following a 45 degrees C, 12 min exposure was examined. The ts85 cells were found to be both significantly more thermosensitive than parent FM3A cells and to induce a 66kDa heat shock protein (hsp66) not visibly synthesized in the parent line by this exposure. However, a constitutive (synthesized at 37 degrees C) 68kDa heat shock protein (hsp68) is comparably induced in both cell lines after heat. A relationship between the severity of the heat exposure as seen by the cell and hsp66 expression is suggested and tested in Chinese hamster ovary cells. In CHO cells a brief 45 degrees C heat shock induces the constitutive hsp68 (but not hsp66), while longer and more severe exposures are required for the expression of hsp66. The induction of these two proteins is also examined in situ in mouse skeletal muscle. In this case both hsp66 and hsp68 are induced following comparatively mild heat treatments, and the 'threshold' for hsp66 induction observed in cultured cells either does not occur or is greatly reduced. However, once again, hsp68 is naturally synthesized at 37 degrees C while hsp66 appears to be de novo synthesized after heat shock.     

Effects of cycloheximide on thermotolerance expression, heat shock protein synthesis, and heat shock protein mRNA accumulation in rat fibroblasts.

A single hyperthermic exposure can render cells transiently resistant to subsequent high temperature stresses. Treatment of rat embryonic fibroblasts with cycloheximide for 6 h after a 20-min interval at 45 degrees C inhibits protein synthesis, including heat shock protein (hsp) synthesis, and results in an accumulation of hsp 70 mRNA, but has no effect on subsequent survival responses to 45 degrees C hyperthermia. hsp 70 mRNA levels decreased within 1 h after removal of cycloheximide but then appeared to stabilize during the next 2 h (3 h after drug removal and 9 h after heat shock). hsp 70 mRNA accumulation could be further increased by a second heat shock at 45 degrees C for 20 min 6 h after the first hyperthermic exposure in cycloheximide-treated cells. Both normal protein and hsp synthesis appeared increased during the 6-h interval after hyperthermia in cultures which received two exposures to 45 degrees C for 20 min compared with those which received only one treatment. No increased hsp synthesis was observed in cultures treated with cycloheximide, even though hsp 70 mRNA levels appeared elevated. These data indicate that, although heat shock induces the accumulation of hsp 70 mRNA in both normal and thermotolerant cells, neither general protein synthesis nor hsp synthesis is required during the interval between two hyperthermic stresses for Rat-1 cells to express either thermotolerance (survival resistance) or resistance to heat shock-induced inhibition of protein synthesis.     

Induction of heat shock protein and thermal sensitivity of mammalian cells maintained at low culture temperature.

Effects of low culture temperature on the induction of heat shock proteins in FM3A cells by a heat shock and on the thermal sensitivity of the cells were examined. FM3A cells maintained at 33 degrees C could not induce hsp70 during continuous heating or after a short heat shock at either 39, 42, or 45 degrees C, although FM3A cells maintained at a normal culture temperature of 37 degrees C can induce the synthesis of hsp70. Furthermore, the cells maintained at 33 degrees C were more sensitive to the subsequent heat shock than the cells maintained at 37 degrees C. Thus, the culture temperature of the mammalian cells may be an important factor for the induction of hsp70, and hsp70 may play an important role to protect or repair the thermal damage of cells.     

Expression of heat shock genes during differentiation of mammalian osteoblasts and promyelocytic leukemia cells.

The progressive differentiation of both normal rat osteoblasts and HL-60 promyelocytic leukemia cells involves the sequential expression of specific genes encoding proteins that are characteristic of their respective developing cellular phenotypes. In addition to the selective expression of various phenotype marker genes, several members of the heat shock gene family exhibit differential expression throughout the developmental sequence of these two cell types. As determined by steady state mRNA levels, in both osteoblasts and HL-60 cells expression of hsp27, hsp60, hsp70, hsp89 alpha, and hsp89 beta may be associated with the modifications in gene expression and cellular architecture that occur during differentiation. In both differentiation systems, the expression of hsp27 mRNA shows a 2.5-fold increase with the down-regulation of proliferation while hsp60 mRNA levels are maximal during active proliferation and subsequently decline post-proliferatively. mRNA expression of two members of the hsp90 family decreases with the shutdown of proliferation, with a parallel relationship between hsp89 alpha mRNA levels and proliferation in osteoblasts and a delay in down-regulation of hsp89 alpha mRNA levels in HL-60 cells and of hsp89 beta mRNA in both systems. Hsp70 mRNA rapidly increases, almost twofold, as proliferation decreases in HL-60 cells but during osteoblast growth and differentiation was only minimally detectable and showed no significant changes. Although the presence of the various hsp mRNA species is maintained at some level throughout the developmental sequence of both osteoblasts and HL-60 cells, changes in the extent to which the heat shock genes are expressed occur primarily in association with the decline of proliferative activity. The observed differences in patterns of expression for the various heat shock genes are consistent with involvement in mediating a series of regulatory events functionally related to the control of both cell growth and differentiation.     

Comparison of the effect of heat shock factor inhibitor, KNK437, on heat shock- and chemical stress-induced hsp30 gene expression in Xenopus laevis A6 cells

In this study, we compared the effect of KNK437 (N-formyl-3, 4-methylenedioxy-benzylidene-gamma-butyrolactam), a benzylidene lactam compound, on heat shock and chemical stressor-induced hsp30 gene expression in Xenopus laevis A6 kidney epithelial cells. Previously, KNK437 was shown to inhibit HSE-HSF1 binding activity and heat-induced hsp gene expression. In the present study, Northern and Western blot analysis revealed that pretreatment of A6 cells with KNK437 inhibited hsp30 mRNA and HSP30 and HSP70 protein accumulation induced by chemical stressors including sodium arsenite, cadmium chloride and herbimycin A. In A6 cells subjected to sodium arsenite, cadmium chloride, herbimycin A or a 33 degrees C heat shock treatment, immunocytochemistry and confocal microscopy revealed that HSP30 accumulated primarily in the cytoplasm. However, incubation of A6 cells at 35 degrees C resulted in enhanced HSP30 accumulation in the nucleus. Pre-treatment with 100 microM KNK437 completely inhibited HSP30 accumulation in A6 cells heat shocked at 33 or 35 degrees C as well as cells treated with 10 microM sodium arsenite, 100 microM cadmium chloride or 1 microg/mL herbimycin A. These results show that KNK437 is effective at inhibiting both heat shock- and chemical stress-induced hsp gene expression in amphibian cells.     

Dissociation of 68,000 Mr heat shock protein synthesis from thermotolerance expression in rat fibroblasts

Rat embryonic fibroblasts growing exponentially at either 35, 37, or 39 degrees C were exposed to 42 degrees C for times up to 6 hr. Cell survival was unaffected by this heat shock in cultures growing at 39 degrees C but survival was decreased in a temperature dependent manner in cells growing at 37 or 35 degrees C. Exposure to 42 degrees C of cells previously adapted to 35 or 37 degrees C resulted in the induction of heat shock proteins (hsps) with apparent molecular weights of 68,000 (hsp 68), 70,000 (hsp 70), and 89,000 (hsp 89); cells previously adapted to 39 degrees C expressed all hsps except hsp 68. Inasmuch as the synthesis of certain hsps may function to protect cells from thermal damage, these data indicate that hsp 68 may not be required for this adaptation-related thermotolerant survival response. Hsp 68 may only be expressed in cells destined to die.     

Patterns of protein synthesis in various cells after extreme heat shock

The analysis of proteins synthesized in rat thymocytes and mouse teratocarcinoma PCC-4 Aza 1 and myeloma Sp2/0 cells after 1 h of treatment at 42 or 44 degrees C was carried out. Shock at 42 degrees C reduced the total synthetic rate of proteins in all three cell lines and induced "classical" heat-shock protein with a mass of 70 kDa (hsp 70). Heat shock at 44 degrees C resulted in almost complete inhibition of protein synthesis; only a small amount of hsp 70 was synthesized. Meanwhile a new 48-kDa polypeptide (pI = 7.5) was found in the cells exposed to severe heat shock. This protein was compared by peptide mapping with other known polypeptides of the same size: heat-shock protein from chicken embryo cells and mitogen-stimulated polypeptide from human lymphoid cells. The peptide maps were not identical. It was also shown that after a shock at 44 degrees C teratocarcinoma cells were able to accumulate anomalous amounts of hsp 70 despite hsp 70 synthesis inhibition. The data show that reaction of various cells to extreme heat shock depends heavily on cell type.     

Expression of the small heat shock protein gene, hsp30, in Rana catesbeiana fibroblasts

In the present study, we examined the expression of the Rana catesbeiana small heat shock protein gene, hsp30, in an FT fibroblast cell line. Northern and western blot analyses revealed that hsp30 mRNA or HSP30 protein was not present constitutively but was strongly induced at a heat shock temperature of 35 degrees C. However, treatment of FT cells with sodium arsenite at concentrations that induced hsp gene expression in other amphibian systems caused cell death. Non-lethal concentrations of sodium arsenite (10 microM) induced only minimal accumulation of hsp30 mRNA or protein after 12 h. Immunocytochemical analyses employing laser scanning confocal microscopy detected the presence of heat-inducible HSP30, in a granular or punctate pattern. HSP30 was enriched in the nucleus with more diffuse localization in the cytoplasm. The nuclear localization of HSP30 was more prominent with continuous heat shock. These heat treatments did not alter FT cell shape or disrupt actin cytoskeletal organization. Also, HSP30 did not co-localize with the actin cytoskeleton.     

Calmodulin is involved in heat shock signal transduction in wheat

The involvement of calcium and calcium-activated calmodulin (Ca(2+)-CaM) in heat shock (HS) signal transduction in wheat (Triticum aestivum) was investigated. Using Fluo-3/acetoxymethyl esters and laser scanning confocal microscopy, it was found that the increase of intracellular free calcium ion concentration started within 1 min after a 37 degrees C HS. The levels of CaM mRNA and protein increased during HS at 37 degrees C in the presence of Ca(2+). The expression of hsp26 and hsp70 genes was up-regulated by the addition of CaCl(2) and down-regulated by the calcium ion chelator EGTA, the calcium ion channel blockers LaCl(3) and verapamil, or the CaM antagonists N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide and chlorpromazine. Treatment with Ca(2+) also increased, and with EGTA, verapamil, chlorpromazine, or trifluoperazine decreased, synthesis of HS proteins. The temporal expression of the CaM1-2 gene and the hsp26 and hsp70 genes demonstrated that up-regulation of the CaM1-2 gene occurred at 10 min after HS at 37 degrees C, whereas that of hsp26 and hsp70 appeared at 20 min after HS. A 5-min HS induced expression of hsp26 after a period of recovery at 22 degrees C after HS at 37 degrees C. Taken together, these results indicate that Ca(2+)-CaM is directly involved in the HS signal transduction pathway. A working hypothesis about the relationship between upstream and downstream of HS signal transduction is presented.