Modification of thermosensitivity and chemosensitivity induced by combined treatments with hyperthermia and adriamycin

Both adriamycin (ADM) and hyperthermia show thermal chemo-enhancement. Tolerance induction against ADM in heated cells has been reported resulting in clinical difficulty of cancer therapy. We investigated thermo-enhancement induced with ADM (0.2 microg/ml) treatment alone or combined with ADM and 42 degrees C hyperthermia in Chinese hamster V79 cells in vitro. Intracellular accumulation of hsc70 and hsp72 proteins after hyperthermia or ADM was observed to examine the possible relationship between cell killing effect and their accumulations. Thermosensitivity of V79 cells at 42 degrees C after the simultaneous treatments with ADM showed marked thermo-enhancement within the short-term treatments for less than 1 h, while the combined treatments for longer than 1 h, the cells showed reduced thermosensitivity. Survival from the simultaneous treatments for less than 1 h was reduced markedly less than the single treatment both with ADM or 42 degrees C hyperthermia alone. Thermotolerance was markedly induced in a step-up hyperthermia (42 degrees C 2 h-44 degrees C). The combined treatments with ADM and 44 degrees C hyperthermia following the 42 degrees C preheating alone does not inhibit thermotolerance development. The combined treatments with ADM and 42 degrees C preheating showed markedly interactive cell killing, but no thermo-enhancement to the following 44 degrees C hyperthermia was shown. The leveling slope of the 44 degrees C heating period-survival curve was drawn. In the Western blot analyses, hsc70 existed constitutively in the V79 cells. Following the 42 or 44 degrees C hyperthermia alone, intracellular accumulation of hsp72 was determined. ADM treatment alone did not induce any accumulation of hsp72. In the simultaneous treatments with ADM and hyperthermia, the accumulation of hsp72 was markedly reduced. The accumulation of hsp72 after the combined treatment with ADM and hyperthermia was not observed as markedly as that after hyperthermia alone.     

Contribution of heat shock proteins to cell protection from complement-mediated lysis

The possible participation of hsc70 and hsp70 in cellular protection from complement damage was studied. Human erythroleukemia K562 cells were pretreated with reagents affecting hsc70 or hsp70, and cell sensitivity to lysis by antibody and human complement was examined. Treatment with deoxyspergualin, an hsc70 inhibitor, sensitized K562 cells to complement lysis, whereas treatment with ethanol, butanol or hemin, inducers of hsc70 synthesis, protected the cells from complement-mediated lysis. Incubation of K562 at either 42 degrees C or with the amino acid analogue L-azetidine-2-carboxylic acid induced synthesis of hsp70, but not of hsc70. The latter treatment also conferred elevated resistance to complement lysis on K562 cells. Pretreatment of K562 cells with sub-lethal doses of complement desensitizes them to lethal complement doses. No effect of sublytic complement on synthesis of hsc70 and hsp70 was found. However, the results demonstrated that complement stress causes translocation of hsc70 from the cytoplasm to the K562 cell surface. Two monoclonal and two polyclonal antibodies identified hsc70 on the surface of intact, viable complement-stressed cells, while antibodies directed to hsp70 did not bind to these cells. Altogether, the results suggest that the heat shock proteins hsc70 and hsp70 play a role in cell defense against complement.     

Purification of multiple heat shock proteins from a single tumor sample

Heat shock protein-based vaccines have been shown to immunize against cancer and infectious diseases in both prophylactic and therapeutic protocols. So far, four classes of heat shock proteins (HSPs) preparation: gp96, HSP90 (hsp86, hsp84), HSP70 (hsc70, hsp70) and calreticulin have been used successfully. The methods for purifying them individually are now readily available. However, since tumors are not always available in large quantity, a major challenge remains the development of a procedure to simultaneously isolate these HSPs from the same sample. We report here that hsp40, hsp60, hsc70, hsp70, hsp84, hsp86, and gp96 (grp94) but not BiP (grp78) and calreticulin can be separated from a single tumor sample in one step using heparin-agarose chromatography. Interestingly this procedure separates the HSP70 isoforms hsp70 from hsc70, but not the HSP90 isoforms hsp84 and hsp86. The three main immunogenic HSPs, gp96, hsp86/84, and hsc70 can be further isolated to homogeneity using additional purification methods. In addition, we have shown that the interaction of the chaperoned peptides with hsc70 and gp96 is not compromised during heparin chromatography. These observations provide a new method for preparation of multiple HSP-based vaccines, circumventing the sample size limitation, as well as providing the possibility to study how multiple HSPs can synergize in eliciting immunity.     

Differential expression of heat shock protein mRNAs under in vivo glutathione depletion in the mouse retina

Heat shock proteins (HSPs) are highly conserved proteins playing a protective role under deleterious conditions caused by a wide variety of pathophysiological, including environmental stresses. Glutathione (GSH) is known to play a critical role in the cellular defense against unregulated oxidative stress in mammalian cells including neurons. We previously demonstrated that GSH depletion induced cell death in the retina, but the mechanism(s) of cellular protection were not clear. Unregulated oxidative stress was induced by depletion of intracellular GSH by systematic administration of buthionine sulphoximine (BSO), an inhibitor of gamma-glutamylcysteine synthetase. After 0, 1, 4 and 7 days of BSO administration, we examined expression of both large and small HSP mRNAs (hsp90alpha, hsp90beta, hsp70, hsp60 and hsp25) in oxidative-stressed mouse retina. Of large HSPs, only hsp70 expression was significantly decreased from 1 day after BSO injection, whereas expression of other large hsps was not changed on day 1. Expression of hsp60 decreased on 4 days, whereas expression of hsp90 decreased on 7 days after BSO administration. Different from large HSPs, a small HSP, hsp25 increased its expression to a great extent from 1 day after BSO administration. Taken together, our results show that unregulated oxidative stress could induce differential expression of HSPs, which, in turn, may play distinct roles in the cellular defense. Targeting HSPs, therefore, may provide novel tools for treatment of retinal degenerative diseases such as glaucoma, retinopathy or age-related macular degeneration.     

Hsp110 and Grp170, members of the Hsp70 superfamily, bind to scavenger receptor-A and scavenger receptor expressed by endothelial cells-I

Heat shock protein 110 (hsp110) and glucose-regulated protein (grp170) act as anti-cancer vaccines when complexed to tumor antigens by heat shock. It has been proposed that receptors on antigen-presenting cells contribute to HSP-mediated immune responses. Here, we show that hsp110 binds in a receptor-mediated manner to RAW264.7 macrophages, as does grp170. This hsp110/grp170 binding is inhibited by scavenger receptor ligands, suggesting a role for scavenger receptors as binding structures. We examined scavenger receptor class A (SR-A) and scavenger receptor expressed by endothelial cells-I (SREC-I). We show that hsp110/grp170 binds to both SR-A- and SREC-I-expressing CHO cells in a saturable manner and scavenger receptor ligands inhibit binding. Hsp110 also saturably binds mouse bone marrow-derived dendritic cells (bmDC) and is inhibited by scavenger receptor ligands. When an hsp110-rat neu (intracellular domain) heat shock complex vaccine is used to pulse mouse bmDC in vitro, an induction of IFN-gamma secretion is observed by CD8+ T lymphocytes isolated from vaccine-immunized mice. This immune response is inhibited by the application of scavenger receptor ligands to bmDC. Thus, SR-A and SREC-I appear to contribute to the binding of hsp110 and grp170 on APC. Scavenger receptors, in general, contribute to the cross-presentation of hsp110-chaperoned protein antigen.     

吐温80合并温热对B16黑色素瘤细胞hsp 70、c-fos、泛蛋白及S-100蛋白表达的影响

目的:研究吐温80与温热合并的协同抗肿瘤机理及与凋亡的关系.方法:用免疫组化方法.观察吐温80合并41℃温热作用后即时、4小时和72小时,B16肿瘤细胞热休克蛋白70(hsp 70)、c-fos、泛蛋白以及S-100蛋白表达的改变,并进行定量分析.结果:hsp70和C-fos蛋白呈中等阳性反应,分别位于B16细胞的胞质和胞核;泛蛋白为弱阳性、S-100蛋白呈强阳性,分布于胞质;41℃ 60分钟作用可增强hsp70表达.以4小时为显著.核亦呈阳性反应;对其它蛋白表达无明显影响;吐温80单独可轻度抑制hsp70表达;温热与吐温80合并作用能显著抑制hsp70表达,c-fos和泛蛋白表达则明显增加.S-100蛋白变化各组无显著意义.结论:吐温80增强温热抗肿瘤的效应可能与其抑制hsp70合成.影响细胞热耐受性的产生和诱导细胞调亡有关.     

Heat shock proteins are present in mallory bodies (cytokeratin aggresomes) in human liver biopsy specimens

Mallory bodies (MBs) are aggresomes, composed of cytokeratin and various other proteins, which form in diseased liver because of disruption in the ubiquitin-proteasome protein degradation pathway. Heat shock proteins (hsp's) are thought to be involved in this process because it was discovered that MB formation is induced by heat shock in drug-primed mice. It has been reported that ubiquitin and a mutant form of ubiquitin (UBB(+1)) are found in aggresomes formed in the neurons in Alzheimer's disease and in the liver MBs in various liver diseases. In addition, hsp 70 has been found in aggresomes in Alzheimer's and in MBs in drug-primed mice. Therefore, we hypothesized that hsp's might be involved in MB formation in human liver diseases. Liver biopsy sections were double-stained using ubiquitin and hsp 70 or 90b antibodies. Both hsps 70 and 90b were found in MBs in all liver diseases investigated including primary billiary cirrhosis, nonalcoholic steatohepatitis, hepatitis B and C, idiopathic cirrhosis, alcoholic hepatitis, and hepatocellular carcinoma. Ubiquitin and the hsp's colocalized in all MBs in the diseased liver sections. These results indicate that hsp involvement in MB formation is similar to that seen in aggresome formation in other conformational diseases.     

Heat Shock and Proinflammatory Stressors Induce Differential Localization of Heat Shock Proteins in Human Monocytes

Heat shock (HS) proteins (HSP) are a family of molecular chaperones induced by environmental stresses such as oxidative injury, and contribute to protection from and adaptation to cellular stress. We investigated in human monocytes the expression and subcellular distribution of hsp70 and hsc70 after HS and inflammation-related stresses leading to generation of reactive oxygen species by these cells, such as the phorbol ester PMA and erythrophagocytosis (Eø). By combining immunofluorescent staining and Western blot on subcellular fractions, we found that all three stress factors resulted in an increased hsp70 expression, however the subcellular distribution pattern was different depending on the type of stress. While HS induced a rapid translocation of hsp70 into the nucleus, no nuclear translocation of hsp70 was observed after PMA or Eø. Neither of the examined stresses induced membrane expression of hsp70. The observed differences in subcellular distribution pattern might relate to distinct regulation and specific functions of hsp70 in inflammation.     

Heat shock protein 70 associations with myelin basic protein and proteolipid protein in multiple sclerosis brains

Heat shock proteins (hsp) are known to facilitate the generation of specific immune responses by chaperoning proteins and peptides involved in T cell activation. Hsp have been shown to be strikingly elevated in multiple sclerosis (MS) lesions. The unique chaperonin properties of hsp70 have allowed identification of immunogenic proteins bound to it by the ex vivo demonstration of hsp associations with proteins implicated in the immune response. We have investigated the association of hsp70 with myelin basic protein (MBP), myelin proteolipid protein (PLP) and myelin oligodendrocyte protein (MOG) in MS and control brain tissue. In co-immunoprecipitation experiments, in all samples of MS brains examined (n = 3), but not control brain tissue (n = 3), direct association of MBP with hsp70, but not with hsp90, was found. In some MS brain samples, association between PLP and hsp70 was also seen. In similar co-immunoprecipitation experiments on brain tissue obtained from mice with experimental autoimmune encephalomyelitis (n = 5) induced by immunization with PLP peptide, specific association of hsp70 with PLP and MBP was found. Using surface plasmon resonance we demonstrated specific binding of hsp70 with MBP in vitro. Analysis of the amounts of MBP bound to hsp70 yielded a molecular ratio of MBP binding to hsp70 at 6.5:1. MBP complexed with hsp70 was taken up at significantly higher rates by antigen-presenting cells than MBP alone and enhanced MBP-specific immune responses. These results indicate that hsp70 specifically associates with MBP in MS brain tissue. This association might be relevant to the enhanced immune recognition of MBP in MS.     

The role of heat shock protein (hsp70) in dendritic cell maturation: hsp70 induces the maturation of immature dendritic cells but reduces DC differentiation from monocyte precursors

Members of the heat shock protein (hsp70) family are either constitutively expressed (hsc70) or can be induced by hyperthermic stress (hsp70). Recombinant hsp70 (rhsp70) stimulates cytokine production from monocytes and enhances NK cell proliferation and cytotoxicity. Here we demonstrate that rhsp70 binds to immature dendritic cells (DC) derived from monocyte precursors and induces their maturation as evidenced by an increase in CD40, CD86 and CD83 expression. Immature DC stimulated to mature with rhsp70 show an enhanced ability to present tyrosinase peptide to specific CTL. Mature DC did not bind rhsp70, suggesting a down-regulation in the expression of its receptor. When rhsp70 was added to monocyte precursors at the same time as GM-CSF and IL-4 it reduced the differentiation of monocytes into DC as shown by a decrease in the level of CD40, CD83, CD86 and HLA-DR expression and an increase in CD14 expression. The constitutively expressed hsc70 had neither a stimulatory effect on the maturation of immature DC nor did it reduce the differentiation of monocytes into DC. These findings demonstrate the specific ability of rhsp70 to induce the maturation of immature DC. Therefore rhsp70 may be useful for its adjuvant like properties in DC based immunotherapy of certain tumors.     

Differential expression of heat shock protein 70 (hsp70) in human monocytes rendered apoptotic by IL-4 or serum deprivation

Apoptosis of monocytes is regulated by the balance between pro- and antiapoptotic triggers and pathways and may strongly influence inflammatory disorders. The major heat shock protein, hsp70, is an effective inhibitor of apoptosis in lymphocytic and monocytic tumor cell lines, but the implications in the regulation of apoptosis of freshly isolated human monocytes have not been elucidated. In this study, we examined whether two different triggers of monocyte apoptosis, serum deprivation and IL-4, respectively, altered hsp70 expression and whether expression levels correlated with monocyte survival. Monocyte apoptosis was determined quantitatively by flow cytometry detecting annexin V binding or nuclear stainability with propidium iodide (PI). Hsp70 expression was analyzed by semiquantitative RT-PCR and immunoblotting. Exposing monocytes to heat shock (47 degrees C, 20 min) induced a rapid and marked upregulation of hsp70 without evoking injury or apoptosis, suggesting that hsp70 conferred protection and survival. In accordance, when monocytes were rendered apoptotic by serum deprivation, a drastic downregulation of hsp70 occurred, which was accompanied by a reduced synthesis of the constitutive family member hsc70. However, induction of monocyte apoptosis by IL-4 increased hsp70 expression in a concentration and time-dependent fashion. A neutralizing antibody against IL-4 abolished hsp70 expression and apoptosis induction after IL-4 treatment and so excluded indirect effects. LPS rescued monocytes from apoptosis but did not alter hsp70 formation significantly. These findings suggest that, in monocytes, distinct apoptotic triggers induce different responses of hsp70 so that this molecule does not exert protection against cell death directly or in general.     

Age-related attenuation in the expression of the major heat shock proteins in human peripheral lymphocytes

A defining characteristic of human ageing is the reduced ability to maintain homeostasis in the face of adverse environmental stresses. This progressive impairment may be a major cause for the increased incidence of infections, and general morbidity and mortality in the elderly. Heat shock proteins (hsps) or stress proteins, induced in response to hyperthermia and to various other physical, chemical and biological stressors, are often also expressed constitutively at a lower level and perform many essential functions in the cell. Here we investigate age-related changes in the heat induced expression of a comprehensive range of hsps at the translational level using primary human peripheral lymphocytes in short term culture. Our study reveals age-related attenuation in the response of the well characterised up-regulated molecular chaperone system hsp 70, the steroid-receptor binding hsp 90 and the chaperonin hsp 60. A diminution with age is also demonstrated in the heat induced response of hsps 105, 56, 47, 40, 27, and 16. Differentially down-regulated proteins at 100, 38, and 18 kDa were also noted.     

Heat shock-enhanced T cell apoptosis with heat shock protein 70 on T cell surface in multicentric Castleman's disease

We report here that T cells from patients with multicentric Castleman's disease (MCD) are sensitive to hyperthermia. T cells from two of three patients with MCD revealed DNA ladder formation and chromatin condensation following heat shock (30 min at 41.5°C). Peripheral blood mononuclear cells (PBMC) from the same MCD patients exhibited high levels of spontaneous apoptosis after 72 h in culture and elevated apoptosis after heat shock, as evaluated by a quantitative flow cytometric assay. Heat shock protein 70 (hsp70) was detected on the cell surface of T cells in all three patients after heat shock. Furthermore, hsp70 was detected on T cells in the two MCD patients with apoptosis even in the absence of heat shock. T cells from normal samples did not show either heat-shock-induced expression of cell-surface hsp70 or apoptosis. Thus, heat shock treatment augmented hsp70 expression on the cell surface of T cells and enhanced apoptosis. Our studies suggest that hyperthermia may influence the clinical course of MCD.     

Comparison of the stress response to cryopreservation in monolayer and three-dimensional human fibroblast cultures: stress proteins, MAP kinases, and growth factor gene expression

Stress responses induced in fibroblasts by cryopreservation were compared in suspension or three-dimensional cultures at various times up to 5 days of recovery. Cryopreservation caused an 86% inhibition in [(35)S]methionine incorporation, with recovery over 2 days to 45% ±: 14% of its original value. Stress proteins, including heat shock protein (hsp) and glucose-regulated proteins (GRP), detected by immunoblotting, responded with transient increases in cellular content (hsp27 and hsp90 in suspension and three-dimensional culture, and hsp70 only in three-dimensional culture), decreases at 24 h (hsp56, hsp70, hsp90, and GRP78 in three-dimensional culture and hsp90 in suspension), or little change (hsp70 in suspension). Polyacrylamide gel electrophoresis of [(35)S]methionine-labeled proteins showed transient induction of hsp47 within 4 h, and increased synthesis of hsp90 and GRP78 and other unidentified proteins at 24 h, but no change in hsp70. The mitogen-activated protein (MAP) kinase, p38, showed a transient increase after thawing, followed by a peak in extracellular signal-regulated kinase at 24 h. The stress-activated protein kinase (JNK) was not activated. In both stress protein and MAP kinase responses, the three-dimensional cultures showed a more intense response than fibroblasts in suspension. Although some responses were related to osmotic and cold stress during freezing, others were unique. Cryopreservation induced mRNA for selected growth factors, including vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) A chain, which increased 5- to 20- fold at 48 h returning to basal levels by 120 h. Our results indicate the novel finding that cryopreservation of fibroblasts grown in three-dimensional culture induced a specific cellular stress response including growth factors.     

Elevated levels of inducible heat shock 70 proteins in human brain

Differential expression of heat shock genes can modulate protein folding and stress-related cell death. There have been no comparisons of their levels of expression in animals and humans. Levels of expression of heat shock 70 genes in human brain were compared to levels in non-stressed and heat-stressed brain of rat. Levels of hsp70 proteins in human brain were 43-fold higher than in non-stressed rat brain and 14-fold higher than highest induced levels in brains of heat-shocked rats. Levels of constitutively synthesized hsc70 proteins were approximately 1.5-fold higher in human than in rat. Higher levels of hsp70 proteins in human brain may serve to protect brain cells against stress-related death or dysfunction throughout the lifespan.     

Dengue virus entry into liver (HepG2) cells is independent of hsp90 and hsp70

Recently, several stress-related proteins including GRP78, hsp70, and hsp90 have been implicated as dengue virus receptors in various cell types, with hsp90/70 being implicated as a receptor complex in monocytes and macrophages, while GRP78 has been implicated as a liver cell expressed dengue virus receptor. To assess whether the hsp90/70 complex plays a role in the internalization of the dengue viruses into liver cells, we undertook infection inhibition studies with lipopolysaccharide and antibodies directed against both hsp70 and hsp90, individually and in combination. No inhibition of any dengue serotype was seen in the presence of lipopolysaccharide or antibodies directed against either hsp70 or hsp90 either singly or in combination. A moderate inhibition of dengue virus serotype 2 entry into liver cells was observed in the presence of antibodies directed against GRP78. These results confirm a proposed role for GRP78 as a dengue virus serotype 2 receptor protein and suggest that the recently identified hsp90/70 complex does not play a role in dengue virus internalization into liver cells.     

A heat shock protein gene (Hsp70.1) is critically involved in the generation of the immune response to myelin antigen

Protracted inflammation has been associated with the generation of autoimmune responses. In this respect, increase in the chaperonin, heat shock protein 70 (hsp70) is an outcome of prolonged inflammatory stress. Previous experiments have shown that overexpression of inducible hsp70 in vitro enhanced myelin autoantigen recognition. To prove the role of hsp70 in myelin-directed responses in vivo, we applied a mouse deficient in the major gene encoding inducible hsp70, hsp70.1. Hsp70.1(-/-) mice sensitized for experimental autoimmune encephalomyelitis (EAE) with myelin oligodendrocyte glycoprotein (MOG) peptide 35-55, displayed almost complete resistance to the disease. This correlated with the loss of T cell proliferation and IFN-gamma production in response to MOG(35-55). T cell transfer experiments as well as antigen presentation assays in vitro demonstrated that hsp70 deficiency was associated with dysfunction in the activation of autoreactive T cells. Moreover, T cell responses to ovalbumin (OVA) peptide 323-339 were altered and CD4(+) T cells were more prone to TCR-induced apoptosis, suggesting broader spectrum of T cell defect in hsp70.1(-/-) mice. These results provide compelling evidence for generalized effect mediated by inducible hsp70 in the recognition of myelin self and non-self antigens that influences the cytokine profile of the immune response affecting autoimmune demyelination.     

Reverse transcriptase mutation K65N confers a decreased replication capacity to HIV-1 in comparison to K65R due to a decreased RT processivity

Eight members of the HSP/HSC70 family were identified in Spodoptera frugiperda Sf9 cells infected with Autographa californica multiple nucleopolyhedrovirus (AcMNPV) by 2D electrophoresis followed by mass spectrometry (MALDI/TOF) and a Mascot search. The family includes five HSP70s induced by AcMNPV-infection and three constitutive cognate HSC70s that remained abundant in infected cells. Confocal microscopy revealed dynamic changes in subcellular localization of HSP/HSC70s in the course of infection. At the early stages (4 to 10 hpi), a fraction of HSPs is localized in distinct speckles in cytoplasm. The speckles contained ubiquitinylated proteins suggesting that they may be aggresomes where proteins targeted by ubiquitin are sequestered or processed for proteolysis. S. frugiperda HSP90 was identified in the 2D gels by Western blotting. Its amount was unchanged during infection. A selective inhibitor of HSP90, 17-AAG, decreased the rate of viral DNA synthesis in infected cells suggesting a supportive role of HSP90 in virus replication.     

Characterization and cellular distribution of human spermatozoal heat shock proteins.

Heat shock proteins (hsp) are ubiquitous components of all living systems. They are up-regulated in response to adverse changes in the cellular environment and at least one highly conserved group (hsp 70) is associated with the development of tolerance to various physico-chemical stress inducers. Spermatozoa have highly condensed chromatin and unlike somatic cells, are consequently unable to mount a stress response. However, using a combination of gel electrophoresis and immunoblotting with hsp-specific monoclonal antibodies, we report that proteins of M(r) 95 kDa and 70-75 kDa corresponding to hsp 90, and multiple forms of hsp 70 are present in human spermatozoa. Immunohistochemistry localized hsp 90 to the neck and tail of unfixed, acrosome-intact spermatozoa. In contrast, an equatorial ring surrounding the nucleus was observed in unfixed spermatozoa, acrosome-reacted with the calcium ionophore A23187. The ring was stained in cells fixed and permeabilized with ethanol, regardless of acrosomal status. Hsp 70 was an abundant surface antigen and as this protein was also abundant in seminal plasma, we believe that it may have been directly adsorbed onto the cell surface. More specific midpiece, equatorial and nuclear staining was also observed. Possible functions for spermatozoal heat shock proteins are discussed.