Increased heat shock protein 70 expression following toxicant-mediated cytotoxicity: a ubiquitous marker of toxicant exposure?

The up-regulation of heat shock protein (HSP) expression has been proposed as a general biomarker of cellular protection against various environmental stresses and chemicals. The present study investigated the possibility of using HSP70 up-regulation as a biomarker of toxicant exposure in vitro. Cells of a rat hepatoma cell line (FGC4) were exposed to concentrations of 1,3-dichloroacetone, duroquinone, diquat dibromide, menadione, hydrogen peroxide, cadmium chloride (CdCl2) and sodium (meta)arsenite (NaAsO2) that elicited 20-50% cytotoxicity over a 24-hour period, and HSP70 levels were measured by ELISA. Up-regulation of HSP70 expression was demonstrated following treatment with menadione, CdCl2 and NaAsO2, but not with the other chemicals tested. A shorter exposure time (6 hours) and/or the use of non-toxic concentrations reduced the level of HSP70 up-regulation with menadione, CdCl2 and NaAsO2, but did not uncover any up-regulation with the other chemicals. Although the toxicity of the majority of the chemicals tested is believed to involve an oxidative stress component, the results of this study clearly demonstrate that up-regulation of HSP70 expression cannot be used as a general biomarker of toxicant exposure in vitro.     

Inhibition of the ubiquitin-proteasome pathway induces differential heat-shock protein response in cardiomyocytes and renders early cardiac protection

The effects of proteasome inhibition (PI) on heat-shock protein (HSP) expression in cardiomyocytes were investigated. Neonatal rat cardiomyocytes were incubated with MG132 (0.1-10 microM) for 1 h. Induction of various HSPs was determined by real-time PCR and Western blotting. PI induced a 2- to 3-fold increase in HSP27, HSP60, and HSP90, and a 18-fold increase in HSP70 mRNA expression, whereas HSP40 levels were unaffected. Western blotting revealed increased protein expression for HSP70 after PI. Similar results were obtained with MG262. HSP induction correlated with enhanced survival of neonatal cardiomyocytes after sublethal heat stress in XTT testing. In papillary muscles, pretreatment with MG132 (10 microM, 90 min) was associated with enhanced recovery of the contractile parameters after a 40-min hypoxia. In these proof-of-principle experiments, we show that PI induces differential heat-shock response in cardiomyocytes, accompanied by enhanced cell survival and functional recovery after various forms of stress.     

Effects of a 2450 MHz high-frequency electromagnetic field with a wide range of SARs on the induction of heat-shock proteins in A172 cells

In this study, we investigated whether exposure to 2450 MHz high-frequency electromagnetic fields (HFEMFs) could act as an environmental insult to evoke a stress response in A172 cells, using HSP70 and HSP27 as stress markers. The cells were exposed to a 2450 MHz HFEMF with a wide range of specific absorption rates (SARs: 5-200 W/kg) or sham conditions. Because exposure to 2450 MHz HFEMF at 50-200 W/kg SAR causes temperature increases in culture medium, appropriate heat control groups (38-44 degrees C) were also included. The expression of HSP 70 and HSP 27, as well as the level of phosphorylated HSP 27 ((78)Ser) (p-HSP27), was determined by Western blotting. Our results showed that the expression of HSP 70 increased in a time and dose-dependent manner at >50 W/kg SAR for 1-3 h. A similar effect was also observed in corresponding heat controls. There was no significant change in HSP 27 expression caused by HFEMF at 5-200 W/kg or by comparable heating for 1-3 h. However, HSP 27 phosphorylation increased transiently at 100 and 200 W/kg to a greater extent than at 40-44 degrees C. Phosphorylation of HSP 27 reached a maximum after 1 h exposure at 100 W/kg HFEMF. Our results suggest that exposure to a 2450 MHz HFEMF has little or no apparent effect on HSP70 and HSP27 expression, but it may induce a transient increase in HSP27 Phosphorylation in A172 cells at very high SAR (>100 W/kg).     

HSP70 induction by bleomycin metal core analogs

Induction of heat shock protein 70 (HSP70) is known to be effective against various diseases. We are interested in HSP70 induction capability of an antitumor antibiotic bleomycin which produces oxidative stress by iron chelate formation and oxygen activation in a cell. The HSP70 induction activity of bleomycin and its six metal core analogs was examined, and a compound HPH-1Trt of 10 μM was found to induce this protein in a pheochromocytoma cell line and some T cell and monocytic cell lines. Its mechanism is increase of HSP70 mRNA, but higher concentration of this compound showed toxicity. Two new derivatives were then synthesized, and one of them named DHPH-1Trt was shown to have less toxicity and higher HSP70 induction activity. This study would lead to a clue for new HSP70 inducer clinically used in near future.     

Stress proteins in oligodendrocytes: differential effects of heat shock and oxidative stress

Heat shock proteins (HSP) or stress proteins serve as biomarkers to identify the contribution of stress situations underlying the pathogenesis of degenerative diseases of the CNS. We have analyzed by immunoblot technique the constitutive and inducible occurrence of stress proteins in cultured rat brain oligodendrocytes subjected to heat shock or oxidative stress exerted by hydrogen peroxide, or a combination of both. The data demonstrate that oligodendrocytes constitutively express HSP32, HSP60 and the cognate form of the HSP70 family of proteins, HSC70. After heat shock, HSP25, alpha B-crystallin and HSP70 were up-regulated, while after oxidative stress the specific induction of HSP32 and alpha B-crystallin was observed. HSP32 represents heme oxygenase 1 (HO-1), a small stress protein with enzymatic activity involved in the oxidative degradation of heme which participates in iron metabolism. The presence of the iron chelators phenanthroline or deferoxamine (DFO), which previously has been shown to protect oligodendrocytes from oxidative stress-induced onset of apoptosis, caused a marked stimulation of HSP32 without affecting HSP70. This indicates that DFO possibly exerts its protective role by directly influencing the antioxidant capacity of HO-1. In summary, HSP in oligodendrocytes are differentially stimulated by heat stress and oxidative stress. Heme oxygenase-1 has been linked to inflammatory processes and oxidative stress, its specific up-regulation after oxidative stress in oligodendrocytes suggests that it is an ideal candidate to investigate the involvement of oxidative stress in demyelinating diseases.     

Expression of the HSP 70 gene family in rat hepatoma cell lines of different growth rates

We have studied the expression of different members of the HSP 70 gene family in MH1C1, FAO, and 3924A hepatoma cell lines, which possess different growth rates and show different levels of histone H3 gene expression. The cells have been subjected to mild (42 degrees C/1 h) or severe (45 degrees C/25 min) heat shock that causes a decrease in cell proliferation and histone H3 gene expression correlated to the severity of stress: previous mild heat shock protects against the effects of the subsequent severe exposure. All cell lines, irrespective of their growth rate, show a high constitutive expression of the HSC 73 gene, which is barely detectable in normal liver, and a good induction of the heat-inducible HSP 70 gene, which, however, seems to be induced less than in the normal tissue. The relative amount of grp 78 mRNA is high in all hepatoma cells lines, but only FAO cells maintain a significant expression of the albumin gene. The basic diversity in HSP 70 family gene expression between normal and tumors is still maintained in hepatoma cell lines, but the growth-related, quantitative differences among the transplantable hepatomas that we previously found in the animal (Bardella et al., Br. J. Cancer 55, 642-645, 1987; Cairo et al., Hepatology 9, 740-746, 1989), seem to be lost, or at least strongly blunted, in vitro.     

The heat shock protein 70 inhibitor VER155008 suppresses the expression of HSP27, HOP and HSP90β and the androgen receptor,induces apoptosis,and attenuates prostate cancer cell growth

Heat shock proteins (HSPs) are molecular chaperones that play a pivotal role in correct folding, stabilization and intracellular transport of many client proteins including those involved in oncogenesis. HSP70, which is frequently overexpressed in prostate cancer (PCa), has been shown to critically contribute to tumor cell survival, and might therefore represent a potential therapeutic target. We treated both the androgen receptor (AR)-positive LNCaP and the AR-negative PC-3 cell lines with the pharmacologic HSP70 inhibitor VER155008. Although we observed antiproliferative effects and induction of apoptosis upon HSP70 inhibition, the apoptotic effect was more pronounced in AR-positive LNCaP cells. In addition, VER155008 treatment induced G1 cell cycle arrest in LNCaP cells and decreased AR expression. Further analysis of the HSP system by Western blot analysis revealed that expression of HSP27, HOP and HSP90β was significantly inhibited by VER155008 treatment, whereas the HSP40, HSP60, and HSP90α expression remained unchanged. Taken together, VER155008 might serve as a novel therapeutic option in PCa patients independent of the AR expression status.     

过表达热休克蛋白70提高CHO细胞表达抗体的能力

通过在中国仓鼠卵巢细胞(CHO)中过表达热休克蛋白70以提高其表达抗体的能力。首先从中国仓鼠基因组DNA中扩取HSP70基因,构建真核表达质粒pcDNA3.1-HSP70,再将重组质粒稳定转染到CHO/dhfr-细胞中,筛选获得稳定的细胞系,运用RT-qPCR检测和Western blot分析HSP70基因的过表达。在过表达HSP70的CHO细胞组和对照细胞组(转染空载体pcDNA3.1的CHO细胞组)中分别转染表达抗-HBs的质粒,应用ELISA检测两组细胞表达抗-HBs的能力。RT-qPCR结果显示实验组CHO细胞中HSP70基因的表达量明显高于对照组细胞;ELISA检测结果表明过表达HSP70的CHO细胞组抗-HBs表达量高于对照组细胞(P<0.05)。研究揭示HSP70能有效促进细胞内分泌性蛋白的表达。     

The maximal cytoprotective function of the heat shock protein 27 is dependent on heat shock protein 70

Endogenous heat shock proteins (HSPs) 70 and 25/27 are induced in renal cells by injury from energy depletion. Transfected over-expression of HSPs 70 or 27 (human analogue of HSP25), provide protection against renal cell injury from ATP deprivation. This study examines whether over-expressed HSP27 depends on induction of endogenous HSPs, in particular HSP70, to afford protection against cell injury. LLC-PK1 cells transfected with HSP27 (27OE cells) were injured by ATP depletion for 2 h and recovered for 4 h in the presence of HSF decoy, HSP70 specific siRNA (siRNA-70) and their respective controls. Injury in the presence of HSF decoy, a synthetic oligonucleotide identical to the heat shock element, the nuclear binding site of HSF, decreased HSP70 induction by 80% without affecting the over-expression of transfected HSP27. The HSP70 stress response was completely ablated in the presence of siRNA-70. Protection against injury, provided by over-expression of HSP27, was reduced by treatment with HSF decoy and abolished by treatment with siRNA-70. Immunoprecipitation studies demonstrated association of HSP27 with actin that was not affected by either treatment with HSF decoy or siRNA. Therefore, HSP27 is dependent on HSP70 to provide its maximal cytoprotective effect, but not for its interaction with actin. This study suggests that, while it has specific action on the cytoskeleton, HSP 25/27 must have coordinated activity with other HSP classes, especially HSP70, to provide the full extent of resistance to injury from energy depletion.     

汉滩病毒感染诱导热休克蛋白70表达

为了解汉滩病毒感染后细胞的应激反应及ＨＳＰ７０的表达与病毒复制的关系,在汉滩病毒Ａ９株感染Ｖｅｒｏ－Ｅ６细胞后,用免疫组织化学及核酸分子原位杂交法,对细胞ＨＳＰ７０基因的表达进行了检测。结果表明,汉滩病毒感染细胞４ｈｙ后即可诱导Ｖｅｒｐ－Ｅ６细胞表达ＨＳＰ７０,表达可持续至感染后５ｄ且ＨＳＰ７０在细胞内的分布也有改变。提示汉滩病毒可直接诱导ＨＳＰ７０的高表达。     

Expression and induction of the stress protein alpha-B-crystallin in vascular endothelial cells

Stress-induced development of enhanced tolerance against various kinds of stresses has been observed in vascular endothelial cells as well as in several other cell types. Stress proteins are thought to play a key role in the development of stress tolerance. In this study we show that endothelial cells of various sources contain the major stress protein of the eye lens, alphaB-crystallin. In the mouse myocardial microvascular cell line, MyEnd, alphaB-crystallin as well as the heat shock proteins HSP 70i and HSP 25 display a low constitutive expression but can be significantly upregulated by sodium arsenite stress. Osmotic stress also resulted in strong upregulation of alphaB-crystallin and HSP 70i but not of HSP 25. Both osmotic and arsenite stress resulted in significant stress tolerance of MyEnd cells against glucose deprivation as assayed by lactate dehydrogenase release and overall cellular morphology. Development of stress tolerance without induction of HSP 25 indicates that HSP 25 is not essential for the protective effect. MyEnd cells from alphaB-crystallin-/- mice displayed a similar degree of stress tolerance showing that alphaB-crystallin is dispensable for protection of cells against energy depletion. The functional role of alphaB-crystallin in endothelial cells needs to be further elucidated. In our experiments HSP 70i turned out to be the only potential candidate of the stress proteins assayed to be involved in the development of tolerance against energy depletion.     

Heat Shock Proteins 27 and 70: Anti-Apoptotic Proteins with Tumorigenic Properties

Heat shock proteins (HSP) HSP27 and HSP70 are expressed in response to a wide variety of physiological and environmental insults including anticancer chemotherapy, thus allowing the cell to survive to lethal conditions. Several mechanisms account for the cytoprotective effect of HSP27 and HSP70. 1) Both proteins are powerful chaperones. 2) They both inhibit key effectors of the apoptotic machinery at the pre- and post-mitochondrial level. 3) They participate in the proteasome-mediated degradation of proteins under stress conditions, thereby contributing to the so called “protein triage”. In cancer cells, the expression of HSP27 and/or HSP70 is abnormally high, and both HSP27 and HSP70 may participate in oncogenesis and in resistance to chemotherapy. In rodent models, HSP27 or HSP70 over-expression increases tumor growth and metastatic potential. The depletion or inhibition of HSP27 and HS70 frequently reduces the size of the tumors and even can cause their complete involution (for HSP70). Therefore, the inhibition of HSP70 and HSP27 has become a novel strategy of cancer therapy.     

Expression of HSP27 results in increased sensitivity to tumor necrosis factor, etoposide, and H2O2 in an oxidative stress-resistant cell line

The role of HSP27 in cell growth and resistance to stress was investigated using murine fibrosarcoma L929 cells (normally devoid of constitutively expressed small HSPs) and human osteoblast-like SaOS-2 cells stably transfected with a human hsp27 expression vector. Our data showed that our L929 cells were more resistant to oxidative stress than generally observed for this line. Production of HSP27 in these cells led to a marked decrease in growth rate associated with a series of phenotypical changes, including cell spreading, cellular and nuclear hypertrophy, development of an irregular outline, and a tremendous accumulation of actin stress fibers. By contrast, none of these changes was observable in SaOS-2/hsp27 transfectants overexpressing the protein product. Together, these observations are consistent with a cause-to-effect cascade relationship between increased (or induced) HSP27 expression, changes in cytoskeletal organization, and decreased growth. On the other hand, whereas the transfection of the hsp27 gene increased the cell resistance to heat in both cell lines, only in SaOS-2 cells was this associated with protection to the cytotoxic action of tumor necrosis factor-alpha (TNF-alpha) and etoposide. Unexpectedly, L929/hsp27 transfectants exhibited an increased sensitivity to both agents and also to H2O2. These data thus imply that different mechanisms are involved in the cell resistance to heat shock and to the cytotoxic action of TNF-alpha, etoposide, and H2O2. They also plead against the simple view that overexpression of a phosphorylatable HSP27 would necessarily be beneficial in terms of increased cell resistance to any type of stress. Our data further indicate that the role of HSP27 in cellular resistance to stress and in cell proliferation involves different targets and that the ultimate result of its interference with these processes depends on the intracellular context in which the protein is expressed.     

Activation of stress response by ionomycin in rat hepatoma cells

All living systems respond to a variety of stress conditions by inducing the synthesis of stress or heat shock proteins (HSPs), which transiently protect cells. HSP synthesis was preceded by an increase in intracellular free calcium concentration [(Ca(2+))i]. In this study, we show that Ca(2+) ionophore, ionomycin, induced an immediate increase in intracellular free Ca(2+) and examined how this increase affects heat shock response in rat hepatoma cell line H4II-E-C3. Results indicate that incubating H4II-E-C3 cells with 0.3 microM ionomycin at 37 degrees C for 15 min results in the induction of HSP 70 in both Ca(2+)-containing and Ca(2+)-free medium. Associated with this increase in free Ca(2+) is an in vivo change in membrane organization and activation of signaling molecules like ERKS and SAPKs/JNK. In Ca(2+) containing medium HSP 70 induction mediated by HSF-HSE interaction was faster upon ionomycin treatment as compared to heat shock. Our results show that ionomycin, at sub lethal concentration, increases intracellular free Ca(2+) concentration, activates SAPK/JNK and HSF-HSE interaction, and induces HSP 70 synthesis.     

The endogenous danger signals HSP70 and MICA cooperate in the activation of cytotoxic effector functions of NK cells

Although natural killer (NK) cells are often described as first line defence against infected or malignant cells which act without the need of prior activation, it is known now that the NK cell activity is tightly regulated by other cells and soluble factors. We show here that the stress‐inducible heat shock protein (HSP) 70 activates human NK cells to kill target cells expressing major histocompatibility complex class I chain‐related molecule A (MICA) in a natural killer group 2 member D (NKG2D‐) dependent manner. The HSP70‐derived peptide TKD (TKDNNLLGRFELSG) was able to replace the full‐length HSP70 and to exert the same function. Interestingly, the expression of the cytotoxic effector protease granzyme B in NK cells was increased after TKD stimulation. When MICA and MICB expression was induced in human tumour cells by a histone deacetylase inhibitor and NK cells were activated by HSP70 or TKD, both treatments jointly improved the killing of the tumour cells. Thus, the synergistic activity of two stress‐inducible immunological danger signals, HSP70 and MICA/B, leads to activation and enhanced cytotoxicity of human NK cells against tumour cells.     

Heat shock protein 25 or inducible heat shock protein 70 activates heat shock factor 1: dephosphorylation on serine 307 through inhibition of ERK1/2 phosphorylation

The expression of heat shock proteins (HSPs) is known to be increased via activation of heat shock factor 1 (HSF1), and excess expression of HSPs exerts feedback inhibition of HSF1. However, the molecular mechanism to modulate such relationships between HSPs and HSF1 is not clear. In the present study, we show that stable transfection of either Hsp25 or inducible Hsp70 (Hsp70i) increased expression of endogenous HSPs such as HSP25 and HSP70i through HSF1 activation. However, these phenomena were abolished when the dominant negative Hsf1 mutant was transfected to HSP25 or HSP70i overexpressed cells. Moreover, the increased HSF1 activity by either HSP25 or HSP70i was found to result from dephosphorylation of HSF1 on serine 307 that increased the stability of HSF1. Either HSP25 or HSP70i inhibited ERK1/2 phosphorylation because of increased MKP1 phosphorylation by direct interaction of these HSPs with MKP1. Treatment of HOS and NCI-H358 cells, which showed high expressions of endogenous HSF1, with small interfering RNA (siRNA) of either HSP27 (siHSP27)or HSP70i (siHSP70i) inhibited both HSP27 and HSP70i proteins; this was because of increased ERK1/2 phosphorylation and serine phosphorylation of HSF1. The results, therefore, suggested that when the HSF1 protein level was high in cancer cells, excess expression of HSP27 or HSP70i strongly facilitates the expression of HSP proteins through HSF1 activation, resulting in severe radio- or chemoresistance.