Heat induced expression of CD95 and its correlation with the activation of apoptosis upon heat shock in rat histiocytic tumor cells

The heat shock response is a universal phenomenon and is among the most highly conserved cellular responses. However, BC-8, a rat histiocytoma, fails to mount a heat shock response unlike all other eukaryotic cells. In the absence of induction of heat shock proteins, apoptotic cell death is activated in BC-8 tumor cells upon heat shock. We demonstrate here that stable transformants of BC-8 tumor cells transfected with hsp70 cDNA constitutively express hsp70 protein and are transiently protected from heat induced apoptosis for 6-8 h. In addition heat stress induces CD95 gene expression in these tumor cells. There is a delay in CD95 expression in hsp70 transfected cells suggesting a correlation between the cell surface expression of CD95 and the time of induction of apoptosis in this tumor cell line. Also expression of CD95 antigen appears to inhibit the interaction between heat shock factors and heat shock elements in these cells resulting in the lack of heat shock response.     

Hsp105 reduces the protein aggregation and cytotoxicity by expanded-polyglutamine proteins through the induction of Hsp70

Hsp105α and Hsp105β are major heat shock proteins in mammalian cells and belong to the HSP105/110 family. Hsp105α is expressed constitutively in the cytoplasm of cells, while Hsp105β, an alternatively spliced form of Hsp105α, is expressed specifically in the nucleus of cells during mild heat shock. Here, we show that not only Hsp105β but also Hsp105α accumulated in the nucleus of cells following the expression of enhanced green fluorescent protein with a pathological length polyQ tract (EGFP-polyQ97) and suppressed the intranuclear aggregation of polyQ proteins and apoptosis induced by EGFP-polyQ97. Mutants of Hsp105α and Hsp105β with changes in the nuclear localization signal sequences, which localized exclusively in the cytoplasm with or without the expression of EGFP-polyQ97, did not suppress the intranuclear aggregation of polyQ proteins and apoptosis induced by EGFP-polyQ97. Furthermore, Hsp70 was induced by the co-expression of Hsp105α and EGFP-polyQ97, and the knockdown of Hsp70 reduced the inhibitory effect of Hsp105α and Hsp105β on the intranuclear aggregation of polyQ proteins and apoptosis induced by EGFP-polyQ97. These observations suggested that Hsp105α and Hsp105β suppressed the expanded polyQ tract-induced protein aggregation and apoptosis through the induction of Hsp70.     

Production of heat shock proteins, cytokines, and nitric oxide in toxic stress

Expression of heat shock proteins Hsp27, Hsp90, and Hsp70 and production of tumor necrosis factors (TNF-alpha, TNF-beta), interferon-gamma (IFN-gamma), interleukin-2, -3, -6, and nitric oxide (NO) were studied under conditions of acute and chronic intoxication of animals with lipopolysaccharides. Injection of endotoxin increased expression of heat shock proteins Hsp70 and Hsp90-alpha in mouse cells. Acute toxic stress also provoked a sharp increase in the production of TNF-alpha, TNF-beta, and NO in mouse cells. The production of other cytokines (interleukins and IFN-gamma) was changed insignificantly. In the model of chronic toxic stress, changes in the production of Hsp70, Hsp90, TNF, and NO were followed during 11 days after the beginning of the toxin injections. The expression of Hsp70 and Hsp90 in acute stress was significantly higher than at the final stage of the chronic exposure. The changes in the TNF and NO productions, on one hand, and the production of heat shock proteins, on the other hand, were synchronous. The findings indicate that repeated injections of increasing endotoxin doses result in a decreased ability of the body cells to respond to stress by overproduction of heat shock proteins, TNF, and NO.     

Suppression of ceramide-mediated apoptosis by HSP70.

Ceramide has been known as an important second messenger in programmed cell death (apoptosis) which is induced by various stimuli such as the tumor necrosis factor-alpha (TNF-alpha), Fas ligand, and environmental stresses such as UV-irradiation and heat shock. Although the precise molecular mechanism of apoptosis is not fully understood, ceramide generated by sphingomyelinase (SMase) mediates the activation of several downstream molecules that are implicated in the regulation of apoptosis. Here, we show that stress-inducible heat shock protein 70 (Hsp70) prevents apoptosis induced by increased level of intracellular ceramide. In T-cell hybridoma DO11.10, we examined the effect of Hsp70 on apoptosis mediated by TNF-alpha, Fas ligation, SMase, and C2-ceramide, all of which elevate intracellular ceramide levels. Hsp70 not only markedly reduced internucleosomal DNA fragmentation, but also enhanced cell viability measured by the Trypan blue dye exclusion test. Similarly, the ceramide-induced c-jun amino-terminal kinase (JNK/SAPK) activation is impaired in cells overexpressing Hsp70. These data strongly suggest that hsp70 functions as a regulator of apoptosis downstream of ceramide.     

CD95-mediated alteration in Hsp70 levels is dependent on protein stabilization

Engagement of death receptors induces caspase activation and apoptosis. A recent study reported altered protein expression, including increased Hsp70 levels during CD95-mediated apoptosis. Here, we examined the mechanism underlying increased Hsp70 levels in cells challenged with a monoclonal antibody directed against the CD95 receptor. Levels of Hsp70 were found to increase in a dose-dependent manner, occurring independently of either heat shock factor 1 activation or the accumulation of Hsp70 messenger ribonucleic acid (mRNA), suggesting the involvement of posttranslational modifications. Inhibition of translation and de novo protein synthesis by cycloheximide resulted in Hsp70 protein levels diminishing over time in control cells, whereas its level remained constant during CD95 signaling. In addition, death receptor activation through exposure of cells to tumor necrosis factor-related apoptosis-inducing ligand did not alter Hsp70 levels. These findings demonstrate that receptor-specific signaling through the CD95 increases the stability of Hsp70 protein, rather than mRNA, when compared with control cells. The results describe a novel mechanism of heat shock protein accumulation, where increased protein stability and reduced turnover, is the mechanism by which Hsp70 accumulates in cells during CD95-mediated apoptosis.     

Induction and accumulation of HSP70 leads to formation of its complexes with other cell proteins

The expression of a major stress protein Hsp70 may be induced by a great number of cytotoxic agents and also by factors known to cause process of cell proliferation or apoptosis. After induction the synthesis of Hsp70 is reduced to its basic level within first 2-6 hours after inducer application, while the high content of the protein may persist up to several days. The aim of this study was to understand the reason of such a long storage of Hsp70 in U-937 cells induced with three distinct factors. The data of immunoblotting showed that the mild heat shock at 43 degrees C for 60 min, phorbol ester and tumor necrosis factor (the two latter known to induce differentiation and apoptosis, respectively) caused Hsp70 accumulation, the protein level being high within 48 hours and then slowly declined to the basal level. According to the results of chromatography on anti-Hsp70 antibody-Sepharose gel, approximately 50% of the protein was retarded by the gel, while the other part was not recognized by immunoglobulins. Among proteins bound to the Hsp70, three polypeptides were identified as actin, p65 and c-Rel, two latter being constituents of NF-kappa B regulatory complex. The data demonstrate that besides its traditional target, i.e. newly synthesized or abnormal polypeptides, Hsp70 is able to bind mature, active proteins.     

Glucoronic acid is a novel inducer of heat shock response

The elevated expression of 70 kDa heat shock protein (Hsp70) induces resistance to stress-induced apoptosis. We have screened a variety of natural products for their ability to enhance Hsp70 expression as anti-apoptotic agent. We found that glucuronic acid (GA) induced the synthesis of Hsp70 and that cells pretreated with GA were significantly tolerant to stress including heat shock and hydrogen peroxide. We also found that GA induces the production of reactive oxygen species (ROS), a process inhibited by NADPH oxidase inhibitor, diphenyleneiodonium chloride (DPI) and antioxidant N-acetylcysteine (NAC). GA-induced ROS production was also inhibited in RacN17 cell line overexpressing a dominant negative mutant of Rac1. Furthermore, GA treatment induces MAPKs activation (SAPK/JNK and p38) and Hsp70 expression in ROS dependent manner, suggesting that GA turns on the signaling pathway by generation of ROS through Rac1. We analyzed the profiles of newly synthesized proteins by GA with 2-dimensional gel electrophoresis and MALDI-TOF MS and found that two families of proteins were expressed by GA. One was similar to the protein family synthesized by heat shock (Hsp70, Hsp73, Hsp65, Hsp90, vimentin, tubulin, Ras homolog); and the other was a family of protein specific to GA (calreticulin, annexin III, thioredoxin peroxidase). These results suggest that GA-induced stress responses are mediated by ROS generation and are similar, in part, to heat shock-induced responses and GA can be possibly adopted for the protecting agent from cell death.     

Heat shock protein 70 is able to prevent heat shock-induced resistance of target cells to CTL

Heat shock or transfection with heat shock protein 70 (Hsp70) genes has been shown to protect tumor cell lines against immune mechanisms of cytotoxicity. We have reported previously that heat shock confers resistance to CTL in the rat myeloma cell line Y3 that is Hsp70 defective. Evidence is now presented that Hsp70 is able to prevent the induction of the resistant phenotype. In Con A-stimulated lymphocytes and in lymphocyte x Y3 somatic cell hybrid clones a severe, non-Hsp70-inducing heat shock elicits resistance to CTL in contrast to a heat shock that results in Hsp70 expression. Thus, Hsp70 expression appears to be negatively associated with the development of resistance. Furthermore, loading of Y3 cells with recombinant Hsp70 protein before heat shock is able to prevent resistance. Because apoptosis induced in Y3 cells by heat shock is not affected, Hsp70 appears to interfere selectively with the CTL-induced lethal pathway that is found to be calcium but not caspase dependent. It is suggested that after heat shock Hsp70 enhances the CTL-induced apoptotic pathway by chaperoning certain proteins in the target cell that are involved in the execution of cell death. Thus, although shown to confer protection against many cytotoxic mechanisms, Hsp70 does not appear to be generally cytoprotective. This observation could also be of relevance when interpreting the effectiveness of tumor immunity.     

Biological heterogeneity of the peptide-binding motif of the 70-kDa heat shock protein by surface plasmon resonance analysis

70-kDa heat shock protein family is a molecular chaperone that binds to a variety of client proteins and peptides in the cytoplasm. Several studies have revealed binding motifs between 70-kDa heat shock protein family and cytoplasmic proteins by conventional techniques such as phage display library screening. However, little is known about the binding motif based on kinetic parameters determined by surface plasmon resonance analysis. We investigated the major inducible cytosolic 70-kDa heat shock protein (Hsp70)-binding motif with the human leukocyte antigen B*2702-derived peptide Bw4 (RENLRIALRY) by using a Biacore system based on surface plasmon resonance analysis. The K(D) value of Hsp70-Bw4 interaction was 1.8 x 10(-6) m. Analyses with truncated Bw4 variant peptides showed the binding motif of Hsp70 to be seven residues, LRIALRY. To further study the characteristics of this motif, 126 peptides derived from Bw4, each with single amino acid substitution, were synthesized and analyzed for Hsp70 binding affinity. Interestingly, the Hsp70 binding affinity was abrogated when the residues were substituted for by acidic (Asp and Glu) ones at any position. In contrast, if the substitute residue was aromatic (Trp, Tyr, and Phe) or an Arg residue at any position, Hsp70 binding affinity was maintained. Thus, this study presents a new binding motif between Hsp70 and peptides derived from the natural protein human leukocyte antigen B*2702 and may also elucidate some characteristics of the Hsp70 binding characteristic, enhancing our understanding of Hsp70-binding determinants that may influence diverse cellular and physiological processes.     

In vivo chaperone activity of heat shock protein 70 and thermotolerance

Heat shock protein 70 (Hsp70) is thought to play a critical role in the thermotolerance of mammalian cells, presumably due to its chaperone activity. We examined the chaperone activity and cellular heat resistance of a clonal cell line in which overexpression of Hsp70 was transiently induced by means of the tetracycline-regulated gene expression system. This single-cell-line approach circumvents problems associated with clonal variation and indirect effects resulting from constitutive overexpression of Hsp70. The in vivo chaperone function of Hsp70 was quantitatively investigated by using firefly luciferase as a reporter protein. Chaperone activity was found to strictly correlate to the level of Hsp70 expression. In addition, we observed an Hsp70 concentration dependent increase in the cellular heat resistance. In order to study the contribution of the Hsp70 chaperone activity, heat resistance of cells that expressed tetracycline-regulated Hsp70 was compared to thermotolerant cells expressing the same level of Hsp70 plus all of the other heat shock proteins. Overexpression of Hsp70 alone was sufficient to induce a similar recovery of cytoplasmic luciferase activity, as does expression of all Hsps in thermotolerant cells. However, when the luciferase reporter protein was directed to the nucleus, expression of Hsp70 alone was not sufficient to yield the level of recovery observed in thermotolerant cells. In addition, cells expressing the same level of Hsp70 found in heat-induced thermotolerant cells containing additional Hsps showed increased resistance to thermal killing but were more sensitive than thermotolerant cells. These results suggest that the inducible form of Hsp70 contributes to the stress-tolerant state by increasing the chaperone activity in the cytoplasm. However, its expression alone is apparently insufficient for protection of other subcellular compartments to yield clonal heat resistance to the level observed in thermotolerant cells.     

Heat shock protein 70 and glycoprotein 96 are differentially expressed on the surface of malignant and nonmalignant breast cells

Heat shock proteins (HSPs), which are important for a number of different intracellular functions, are occasionally found on the surface of cells. The function of heat shock protein on the cell surface is not understood, although it has been shown to be greater in some tumor cells and some virally infected cells. Surface expression of both glycoprotein 96 (gp96) and Hsp70 occurs on tumor cells, and this expression correlates with natural killer cell killing of the cells. We examined the surface expression of gp96 and Hsp70 on human breast cell lines MCF7, MCF10A, AU565, and HS578, and in primary human mammary epithelial cells by immunofluorescence microscopy and flow cytometry. The nonmalignant cell lines HS578, MCF10A, and HMEC showed no surface expression of gp96, whereas malignant cell lines MCF7 and AU565 were positive for gp96 surface expression. All of the breast cell lines examined showed Hsp70 surface expression. These results also confirm previous studies, demonstrating that Hsp70 is on the plasma membrane of tumor cell lines. Given the involvement of heat shock proteins, gp96 and Hsp70, in innate and adaptive immunity, these observations may be important in the immune response to tumor cells.     

The effect of quercetin on the expression of heat shock proteins and apoptosis induction in monkey kidney cell line GMK

The present study was designed to investigate whether quercetin, a very common flavonoid widely distributed in many plants, can induce apoptosis in monkey kidney cells (GMK). Involvement of the expression of heat shock proteins Hsp72, Hsp73, Hsp27 and the significance of cell culture model in this process were also examined. The studies have shown that quercetin alone and in combination with the heat shock can induce apoptosis and necrosis in vitro in the studiedcells, but the percentage of affected cells did not exceed 3.9%. In the same experimental conditions, the expression of Hsp 73, Hsp72 and Hsp27 increased in cells cultured in two-dimensional system and decreased in three-dimensional model. This indicates that strong inhibition of heat shock proteins in GMK cells is not correlated with an adequate increase in the sensitivity of cells to undergo apoptosis. It also shows that the sensitivity on the manifested by Hsp expression and apoptosis induction, depends on the culture model and culture conditions.     

Apoptosis-resistant phenotype in HL-60-derived cells HCW-2 is related to changes in expression of stress-induced proteins that impact on redox status and mitochondrial metabolism

The onset of resistance to drug-induced apoptosis of tumour cells is a major problem in cancer therapy. We studied a drug-selected clone of promyelocytic HL-60 cells, called HCW-2, which display a complex resistance to a wide variety of apoptosis-inducing agents and we found that these cells show a dramatic increase in the expression of heat shock proteins (Hsps) 70 and 27, while the parental cell line does not. It is known that stress proteins such as Hsps can confer resistance to a variety of damaging agents other than heat shock, such as TNF-alpha, monocyte-induced cytotoxicity, and also play a role in resistance to chemotherapy. This elevated expression of Hsps is paralleled by an increased activity of mitochondrial metabolism and pentose phosphate pathway, this latter leading to high levels of glucose-6-phosphate dehydrogenase and, consequently, of glutathione. Thus, the apoptotic-deficient phenotype is likely because of the presence of high levels of stress response proteins and GSH, which may confer resistance to apoptotic agents, including chemotherapy drugs. Moreover, the fact that in HCW-2 cells Hsp70 are mainly localised in mitochondria may account for the increased performances of mitochondrial metabolism. These observations could have some implications for the therapy of cancer, and for the design of combined strategies that act on antioxidant defences of the neoplastic cell.     

The heat shock protein 70 family: Highly homologous proteins with overlapping and distinct functions

The human heat shock protein 70 (Hsp70) family contains at least eight homologous chaperone proteins. Endoplasmatic reticulum and mitochondria have their specific Hsp70 proteins, whereas the remaining six family members reside mainly in the cytosol and nucleus. The requirement for multiple highly homologous although different Hsp70 proteins is still far from clear, but their individual and tissue-specific expression suggests that they are assigned distinct biological tasks. This concept is supported by the fact that mice knockout for different Hsp70 genes display remarkably discrete phenotypes. Moreover, emerging data suggest that individual Hsp70 proteins can bring about non-overlapping and chaperone-independent functions essential for growth and survival of cancer cells. This review summarizes our present knowledge of the individual members of human Hsp70 family and elaborate on the functional differences between the cytosolic/nuclear representatives.     

Neuronal differentiation in PC12 cells is accompanied by diminished inducibility of Hsp 70 and Hsp60 in response to heat and ethanol

The stress response of PC12 cells was characterized by evaluating the production of heat shock proteins of the 70 kDa (Hsp70), 60 kDa (Hsp60) and 90 kDa (Hsp90) families by western blot analysis. Induction of Hsp synthesis was elicited by brief exposure to elevated temperatures or by addition of ethanol to the cultures. Normal PC12 cells responded to stress with rapid up-regulation of Hsp70 and Hsp60 production. However, fully differentiated PC12 cells (induced by nerve growth factor, NGF) failed to produce Hsp70 or Hsp60 in response to heat or ethanol treatment. The disappearance of the heat shock response of the cells was directly related to the extent of neuronal differentiation. The cellular levels of the constitutive proteins, Hsc70 and Hsp90, were not altered by differentiation of the cells. Production of Hsps was restored in the differentiated cells by removal of NGF which coincided with the loss of neurite expression and retraction of processes.