Kinetics of thermally induced heat shock protein 27 and 70 expression by bone marrow-derived mesenchymal stem cells

Although bone marrow-derived mesenchymal stem cells (MSCs) are an attractive cell therapy candidate, their potential is limited by poor survival following transplantation. Over-expression of anti-apoptotic heat shock proteins using viral vectors can improve the survival of these cells under stressful conditions in vitro and in vivo. It is also possible to induce heat shock protein expression in many cell types by simply exposing them to a transient, nonlethal elevation in temperature. The response profile of MSCs to such a thermal stress has not yet been reported. Therefore, this study sought to determine the kinetics of thermally induced heat shock protein expression by MSCs in vitro. To determine if heat shock protein expression was a function of thermal stress exposure time, MSCs were exposed to 42°C for 15, 30, 45, and 60 min and were harvested 24 h later. To establish the time-course of heat shock protein expression, MSCs were heat shocked for 60 min and harvested 2, 24, 48, 72, 96, and 120 h later. The cells were then analyzed for Hsp27 and Hsp70 expression by Western blot. Densitometric analysis revealed that exposure to a thermal stress induced expression of both Hsp27 and Hsp70 and that the level of expression was dependant on stress exposure time. Following 60 min of heat stress, both Hsp27 and Hsp70 accumulated maximal expression after 48 h with both proteins returning to constitutive expression levels by 120 h. This study demonstrates that heat shock protein expression can be induced in MSCs by a simple thermal stress.     

Chronic electromagnetic field exposure decreases HSP70 levels and lowers cytoprotection

Electromagnetic field (EMF) exposures have been shown to induce heat shock proteins (HSPs), which help to maintain the conformation of cellular proteins during periods of stress. We have previously reported that short-term exposure of chick embryos to either 60 Hz (extremely low frequency: ELF), or radio-frequency (RF: 915 MHz) EMFs induce protection against hypoxia. Experiments presented in the current report are based on a study in which long-term (4 days), continuous exposure to ELF-EMFs decreased protection against ultraviolet radiation. Based on this result, it was hypothesized that de-protection against hypoxia should also occur following long-term, continuous, or daily, repeated exposures to EMFs. To test this hypothesis, chick embryos were exposed to ELF-EMFs (8 microT) continuously for 4 days, or to ELF or RF (3.5 mW incident power)-EMFs repeated daily (20, 30, or 60 min once or twice daily for 4 days). Several of the exposure protocols yielded embryos that had statistically significant decreases in protection against hypoxic stress (continuous and 30 or 60 min ELF twice daily; or 30 or 60 min once daily RF). This is consistent with our finding that following 4 days of ELF-EMF exposure, HSP70 levels decline by 27% as compared to controls. In addition, the superposition of ELF-EMF noise, previously shown to minimize ELF-EMF induced hypoxia protection, inhibited hypoxia de-protection caused by long term, continuous ELF or daily, repeated RF exposures. This EMF-induced decrease in HSP70 levels and resulting decline in cytoprotection suggests a mechanism by which daily exposure (such as might be experienced by mobile phone users) could enhance the probability of cancer and other diseases.     

Thermal biology of horseshoe crab embryos and larvae: A role for heat shock proteins

Eggs of the American horseshoe crab, Limulus polyphemus L., develop on sandy estuarine beaches during the spring and summer, and are potentially vulnerable to thermal stress during the 3-4 weeks of development to the first instar (trilobite) larval stage. In many marine taxa, heat shock (stress) proteins (Hsp's) help individuals acclimate to stresses by restoring the proper folding of cellular proteins whose shape has been altered by temperature shock or other forms of environmental stress. We examined the survival of embryos and first instar (trilobite) larvae following heat shock, and compared the levels of Hsp70 in heat shocked and control animals. Animals acclimated to 13 or 22 °C had close to 100% survival when heat shocked for 3 h at 35 or 40 °C, but exposure to 45 °C for 3 h was lethal. To study the effect of heat shock on Hsp70 production under environmentally realistic conditions, animals were acclimated to either 13 or 22 °C, heat-shocked at 35 °C for 3 h, and soluble proteins were extracted following 0, 2, 4, or 6 h recovery at 22 °C. The relative amounts of Hsp70 in horseshoe crab embryos and larvae were examined using SDS-PAGE and Western blotting. Relative to controls animals held at a constant temperature, there was a slight elevation of Hsp70 only among heat shocked trilobite larvae in the 6 h recovery treatment. Hsp70 levels did not differ significantly between control and heat shocked embryos. Horseshoe crabs have adapted to living in a thermally stressful environment by maintaining a high baseline (constitutive) level of cellular stress proteins such as Hsp70, rather than by synthesizing inducible Hsp's when stressful temperatures are encountered. This may be an effective strategy given that the heat shocks encountered by intertidal embryos and larvae occur regularly as a function of diurnal and tidal temperature changes.     

Thermal and metabolic responsiveness of Japanese quail embryos following periodic exposure to 2,450 MHz microwaves

Two studies were performed to determine if repeated exposure of the avian egg to microwaves can alter metabolism, temperature, and growth rate of embryos. Another aim was to supplement conventional heating with microwave heating and provide an optimal temperature for growth. Japanese quail (Coturnix coturnix japonica) eggs were exposed from day 1 through 15 of incubation (8 h/day) to sham or microwave (2,450 MHz) irradiation. Microwave exposures were at two power densities, 5 or 20 mW/cm2, and at three ambient temperatures (Tas), 30.0, 33.1, or 35.4 degrees C. Specific absorption rates for unincubated and 15-day-old incubated eggs were, respectively, 0.76 and 0.66 W kg-1 mW-1 cm-2 (i.e., 3.8 and 3.3 W/kg at 5 mW/cm2 and 15.2 and 13.2 W/kg at 20 mW/cm2). Eggs were concurrently sham exposed at each of five Tas, ranging from 27.9 to 37.5 degrees C. Tests were conducted during the 16th day of incubation (i.e., 1 day post-treatment), in the absence of microwaves, to determine metabolic rate of embryos and internal and external egg temperatures at different Tas. Repeated exposures to microwaves at 5 and 20 mW/cm2 at the same Ta (30 degrees C) increased wet-embryo mass on the 16th day by an average, respectively, of 9% and 61% when compared with predicted masses for embryos exposed at the same Ta in the absence of microwave radiation. There was no reliable indication, from post-treatment tests and comparisons with control embryos of similar mass, that repeated exposure to microwave radiation resulted in abnormal physiological development. Microwave radiation can be used to increase egg temperature and embryonic growth rate at Tas below normal incubation level without altering basic metabolic and thermal characteristics of the developing bird.     

Mild heat stress at a young age in<Emphasis Type="Italic">Drosophila melanogaster</Emphasis> leads to increased Hsp70 synthesis after stress exposure later in life

In a number of animal species it has been shown that exposure to low levels of stress at a young age has a positive effect on stress resistance later in life, and on longevity. The positive effects have been attributed to the activation of defence/cleaning systems (heat shock proteins (Hsps), antioxidases, DNA repair) or to effects of a changed metabolic rate, or both. We investigated the effect of mild stress exposures early in life on Hsp70 synthesis after a harder stress exposure later in life in five isofemale lines ofDrosophila melanogaster. Female flies were either exposed to repeated bouts of mild heat stress (3 h at 34‡C) at a young age (days 2, 4 and 6 post-eclosion) or held under standard laboratory conditions. At 16 and 32 days of adult age, respectively, flies were exposed to a high temperature treatment known to induce Hsp70 in the investigated species (1 h at 37‡C). Thereafter, the inducible Hsp70 levels were measured. Our data show a tendency towards increased Hsp70 synthesis with increased age for both ’mild stress’ and ’no stress’ flies. Moreover, the results show that flies exposed to mild stress at a young age synthesized more Hsp70 upon induction, compared to control flies, and that this difference was accentuated at 32 days compared to 16 days of age. Thus, bouts of mild heat stress at a young age impact on the physiological stress response system later in life. This may be caused by an increased ability to react to future stresses. Alternatively, the mild stress exposure at a young age may actually have caused cellular damages increasing the need for Hsp70 levels after stress exposure later in life. The importance of an Hsp70 upregulation (throughout life) in explaining the phenomenon of hormesis is discussed, together with alternative hypotheses, and suggestions for further studies.     

Effects of exposure to a 1950 MHz radio frequency field on expression of Hsp70 and Hsp27 in human glioma cells

Human glioma MO54 cells were used to investigate whether radio frequency (RF) field exposure could activate stress response genes. Cells were exposed to continuous wave 1950 MHz or sham conditions for up to 2 h. Specific absorption rates (SARs) were 1, 2, and 10 W/kg. For the cell growth experiment, cell numbers were counted at 0-4 days after exposure. Expression of Hsp27 and Hsp70, as well as the level of phosphorylated Hsp27 (78Ser) protein, was determined by Western blotting. It was found that sham exposed and RF exposed cells demonstrated a similar growth pattern up to 4 days after RF field exposure. RF field exposure at both 2 and 10 W/kg did not affect the growth of MO54 cells. In addition, there were no significant differences in protein expression of Hsp27 and Hsp70 between sham exposed and RF exposed cells at a SAR of 1, 2, or 10 W/kg for 1 and 2 h. However, exposure to RF field at a SAR of 10 W/kg for 1 and 2 h decreased the protein level of phosphorylated Hsp27 (78Ser) significantly. Our results suggest that although exposure to a 1950 MHz RF field has no effect on cell proliferation and expression of Hsp 27 and Hsp70, it may inhibit the phosphorylation of Hsp27 at Serine 78 in MO54 cells.     

Stress proteins are not induced in mammalian cells exposed to radiofrequency or microwave radiation

The induction of stress proteins in HeLa and CHO cells was investigated following a 2 h exposure to radiofrequency (RF) or microwave radiation. Cells were exposed or sham exposed in vitro under isothermal (37 ± 0.2 °C) conditions. HeLa cells were exposed to 27- or 2450 MHz continuous wave (CW) radiation at a specific absorption rate (SAR) of 25 W/kg. CHO cells were exposed to CW 27 MHz radiation at a SAR of 100 W/kg. Parallel positive control studies included 2 h exposure of HeLa or CHO cells to 40 °C or to 45 μM cadmium sulfate. Stress protein induction was assayed 24 h after treatment by electrophoresis of whole-cell extracted protein labeled with [³⁵S]-methionine. Both cell types exhibited well-characterized responses to the positive control stresses. Under these exposure conditions, neither microwave nor RF radiation had a detectable effect on stress protein induction as determined by either comparison of RF-exposed cells with sham-exposed cells or comparison with heat-stressed or Cd⁺⁺ positive control cells. Bioelectromagnetics 18:499–505, 1997. © 1997 Wiley-Liss, Inc.     

Effect of microwave radiation on the biophysical properties of liposomes

Steadily growing use of electromagnetic fields, especially in conjunction with wireless communication systems, has led to increasing public concern about possible health effects of electromagnetic radiation. However, besides the well-known thermal effect of electromagnetic fields on biological tissue, there is no clear evidence of further athermal interaction mechanisms with biological systems. The present study was designed to determine the changes in bilayer permeability in egg lecithin multilamellar vesicles after exposure to 900 MHz microwave radiation for a period of 5 h. Specific absorption rate (SAR) of the radiation for the investigated liposome sample was found to be 12 +/- 1 W/kg. Liposomal changes in permeability were monitored using a light scattering technique. Optical anisotropy of the liposome sample decreased dramatically upon exposure to microwave radiation, indicating structural changes in acyl chain packing. IR and NMR ((1)H NMR) studies, which have been employed to reveal structural alterations in microwave, exposed vesicles showed an increased damage upon exposure to microwave. The changes observed in the (1)H NMR spectrum of the microwave exposed sample indicated hydrolysis of carboxylic and phosphoric esters. IR study showed conformational changes in the acyl chains of the lipids upon microwave exposure. However, both IR and (31)P NMR did not show any appreciable changes in the head group part of the lipids.     

Lack of effect of 2.45-GHz microwave radiation on the development of preimplantation embryos of mice

The development of preimplantation embryos after exposure to microwave radiation was studied. Female CD-1 mice were induced to superovulate, mated, and exposed to 2.45-GHz microwave or sham radiation for 3 h at power densities of 9 mW/cm² and 19 mW/cm² on either day 2 or 3 of pregnancy (plug day was considered day 1). Another group of mice was exposed to heat stress by placing the dams in an environmental room at an ambient temperature of 38 °C and relative humidity at 62% for 3 h on day 2 of pregnancy. All groups were euthanized on day 4 of pregnancy and embryos were recovered by flushing excised uterine horns. Embryos were examined for abnormalities and classified by the developmental stages. They were then treated with hypotonic solution and dissociated for counting blastomeres. Heat stress caused stunted development of embryos, but no remarkable effect of microwave radiation could be found on the development of preimplantation embryos.     

Expression of the cochaperone HspBP1 is not coordinately regulated with Hsp70 expression

Intracellular levels of the heat stress protein Hsp70 are elevated following exposure to elevated temperature. The cochaperone HspBP1 is an intracellular protein that is known to bind to and regulate Hsp70 activity. The purpose of this study was to determine if HspBP1 levels changed when Hsp70 levels were altered. Heat stress resulted in an increase in Hsp70 levels but no change in HspBP1 levels. Treatment of cells with the apoptosis inducing drug camptothecin lowered Hsp70 levels but again had no effect on HspBP1 levels. Cells treated with camptothecin plus heat stress did not exhibit an increase in Hsp70 levels. Over-expression in cells stably transfected with HspBP1 cDNA resulted in a 290% increase in HspBP1 levels without a similar change in Hsp70 levels. These results demonstrate that Hsp70 and HspBP1 are not coordinately regulated but provide evidence that an increase in the ratio of HspBP1 to Hsp70 correlates with apoptosis, in a similar way to reducing the amount of Hsp70.     

Effects of 1.8 GHz radiofrequency field on DNA damage and expression of heat shock protein 70 in human lens epithelial cells

To investigate the DNA damage, expression of heat shock protein 70 (Hsp70) and cell proliferation of human lens epithelial cells (hLEC) after exposure to the 1.8 GHz radiofrequency field (RF) of a global system for mobile communications (GSM). An Xc-1800 RF exposure system was used to employ a GSM signal at 1.8 GHz (217 Hz amplitude-modulated) with the output power in the specific absorption rate (SAR) of 1, 2 and 3 W/kg. After 2 h exposure to RF, the DNA damage of hLEC was accessed by comet assay at five different incubation times: 0, 30, 60, 120 and 240 min, respectively. Western blot and RT-PCR were used to determine the expression of Hsp70 in hLECs after RF exposure. The proliferation rate of cells was evaluated by bromodeoxyuridine incorporation on days 0, 1 and 4 after exposure. The results show that the difference of DNA-breaks between the exposed and sham-exposed (control) groups induced by 1 and 2 W/kg irradiation were not significant at any incubation time point (P > 0.05). The DNA damage caused by 3 W/kg irradiation was significantly increased at the times of 0 and 30 min after exposure (P < 0.05), a phenomenon that could not be seen at the time points of 60, 120 or 240 min (P > 0.05). Detectable mRNA as well as protein expression of Hsp70 was found in all groups. Exposure at SARs of 2 and 3 W/kg for 2 h exhibited significantly increased Hsp70 protein expression (P < 0.05), while no change in Hsp70 mRNA expression could be found in any of the groups (P > 0.05). No difference of the cell proliferation rate between the sham-exposed and exposed cells was found at any exposure dose tested (P > 0.05). The results indicate that exposure to non-thermal dosages of RF for wireless communications can induce no or repairable DNA damage and the increased Hsp70 protein expression in hLECs occurred without change in the cell proliferation rate. The non-thermal stress response of Hsp70 protein increase to RF exposure might be involved in protecting hLEC from DNA damage and maintaining the cellular capacity for proliferation.     

Increased expression of stress proteins in the surf clam Donax variabilis following hydrogen sulfide exposure

Endogenous free radical production and resulting oxidative damage may result from exposure to hypoxia, hyperoxia, or hydrogen sulfide. Previous investigations of sulfide-induced oxidative damage have produced conflicting results, perhaps because these studies utilized species presumably adapted to sulfide. We examined the effects of sulfide, hypoxia and hyperoxia on the surf clam Donax variabilis to test whether these stressors induce a cellular response to oxidative stress. These clams inhabit high-energy sandy beaches and are unlikely to have specific adaptations to these stressors. In duplicate flow-through experiments performed in fall and spring, clams were exposed to normoxia (22 kPa P(O(2))), hypoxia (10 kPa), hyperoxia (37 kPa), or sulfide with normoxia ( approximately 100 mumol L(-1), 22 kPa respectively) for 24 h. We quantified whole-animal expression of three antioxidants (Cu/Zn and Mn superoxide dismutases, glutathione peroxidase), a lipid peroxidation marker (4-hydroxy-2E-nonenol-adducted protein), a DNA repair enzyme (OGG1-m), four heat shock proteins (small Hsp, Hsp60, Hsp70, and mitochondrial Hsp70), ubiquitin, and actin. Clams exposed to sulfide showed upregulation of the greatest number of stress proteins and the pattern was consistent with a cellular response to oxidative stress. Furthermore, there was a marked seasonality, with greater stress protein expression in clams from the spring.     

Evaluation for Hsp70 as a biomarker of effect of pollutants on the earthworm Lumbricus terrestris

Induction of heat shock proteins (Hsps) is often associated with a cellular response to a harmful stress or to adverse life conditions. The main aims of the present study were (1) to assess if stress-induced Hsp70 could be used to monitor exposure of the earthworm species Lumbricus terrestris to various soil pollutants, (2) to assess the specificity of pollutants in their tissue targeting and in Hsp70 induction, and (3) to evaluate if dose-response relationships could be established and if the stress-response observed was specific. The midgut/intestinal tissues of L. terrestris are shown to express an inducible member of the Hsp70 family after heat shock treatment in vitro and exposures to different soil toxicants in vivo (re: artificial soil). Short-term (24-72 hours) and long-term (14-16 days) exposures to the chemical standards chloroacetamide and pentachlorophenol and to heavy metals (Pb++, Cd++, Cu++, and Hg++) also affected the earthworms, and Hsp70 was induced in their midgut/intestinal tissues. After a 3-day exposure to heavy metals, the level of Hsp70 induction in the midgut/intestinal tissues appears to correlate well with the reported in vivo and in vitro toxicity data. Comparatively, in proximal and midbody wall muscle tissues of animals exposed to the heavy metals, a decrease in expression of Hsp70 was sometimes detected. Thus Hsp analysis by Western blot in L. terrestris tissues and particularly in the midgut/intestine proved to be a suitable and sensitive assay for adverse effects in earthworms and showed a good level of reproducibility despite some individual variations. The use of pristine/nonexposed animals transposed into contaminated environments as in the present study should therefore be of high ecological relevance. Induction of Hsp70 in earthworms should represent not only a good wide-spectrum biomarker of exposure but also a biomarker of effect since known toxicants altered gene expression in tissues of these animals, as contrasted with a simple accumulation of Hsp. Hence, the detection of Hsp70 in earthworms can constitute an early-warning marker for the presence of potentially deleterious agents in soils, with L. terrestris in particular and earthworms in general acting as potential sentinel animal species.     

Intracellular localization of constitutive and inducible heat shock protein 70 in rat liver after in vivo heat stress

The level and intracellular redistribution of the two nucleo-cytoplasmic members of 70 kDa heat shock protein family (constitutive, Hsc70 or Hsp73, and inducible, Hsp72) were studied in rat liver during a 24-h period after exposure of the animals to 41 degrees C whole body hyperthermic stress. The examined proteins were detected in the liver cytosol and nuclei by Western blotting and immunohistochemical staining of paraffin sections, as well as by immnocytochemical staining of isolated nuclear smears. All three techniques applied were based on the use of monoclonal antibodies recognizing both constitutive and inducible Hsp70 isoforms or only the inducible isoform, and gave consistent results. The exposure of the animals to in vivo heat stress was shown to induce the synthesis of otherwise non-existing Hsp72, rendering Hsc70 level unchanged in comparison to unstressed controls. However, immediately after the stress the intracellular redistribution of Hsc70, i.e. its nuclear accumulation, was observed. The maximal level of Hsp70 both in the cytoplasm and in the nuclei was registered 5 h after the stress, which coincided with the maximal level of Hsp72 induction. The alterations in the level and intracellular distribution of examined proteins were still noticeable 24 h after the stress. The results of this study could shed some more light on, as yet uncertain, differences between cellular functions of these two proteins, as well as on the role of the constitutive form under normal and stress conditions.     

Induction of heat shock gene expression in RAT1 primary fibroblast cells by ELF electric fields

Recent studies have demonstrated that the Ku70 gene fragment can be placed in the anti‐sense orientation under the control of a heat‐inducible heat shock protein 70 (HSP70) promoter and activated through heat shock exposure. This results in attenuation of the Ku70 protein expression, inhibiting cellular repair processes, and sensitizing the transfected cells to exposures such as the ionizing radiation exposures used clinically. However, achieving the tissue temperatures necessary to thermally induce the HSP70 response presents significant limitations to the clinical application of this strategy. Previous findings suggest an alternative approach to inducing a heat shock response, specifically through the use of extremely low frequency (ELF) electrical field stimulation. To further pursue this approach, we investigated HSP70 responses in transfected rat primary fibroblast (RAT1) cells exposed to 10 Hz electric fields at intensities of 20–500 V/m. We confirmed that low frequency electric fields can induce HSP70 heat shock expression, with peak responses obtained at 8 h following a 2 h field exposure. However, the approximate threefold increase in expression is substantially lower than that obtained using thermal stimulation, raising questions of the clinical utility of the response. Bioelectromagnetics 34:405–413, 2013. © 2012 Wiley Periodicals, Inc.     

Mytilus trossulus hsp70 as a biomarker for arsenic exposure in the marine environment: laboratory and real-world results.

The highly conserved heat shock protein 70 (hsp70) is induced by heat and chemical toxins, particularly heavy metals such as arsenic (As). The use of Mytilus trossulus (bay mussel) hsp70 as a 'screening' biomarker for marine heavy metals contamination was assessed. Some studies have found high hsp70 sensitivity to heavy metals, while others have found the opposite. Few studies have realistically used low heavy metals exposures, and fewer have used real-world contamination exposures. Clean sub-tidal mussels from the Puget Sound, Washington State (WA), USA, were acclimatized for 2 weeks and exposed for 24 h to As-spiked seawater (n=9) or to contaminated seawater from an arsenical pesticide plant in Tacoma, WA (n=10) followed by a Western blot for hsp70. Hsp70 inductions were insignificant at 10 microg l(-1) As(III), but were strong at 100 microg l(-1) (p<0.05) and 1000 microg l(-1) (p<0.01), with the induction threshold estimated at 30-50 microg l(-1) As(III). Hsp70 induction roughly correlated with arsenical toxicity, with As(III) > As(V) > (CH(3))(2)As(V). Altogether, the inter-individual variability of hsp70 levels tends to mask inductions at low As concentrations, making it a crude toxicity biomarker. In addressing this problem, the following options could prove promising: (1) pre- or post-stressing specimens for greater hsp70 sensitivity, (2) use of internal protein controls such as actin, (3) use of hsp70-reporter gene constructs, and (4) detection with hsp60, heme oxygenase-1, metallothionein, CYP450, MXR or GPx.     

Inducible heat shock protein 70 and its role in preconditioning and exercise

Heat shock proteins (Hsp) are well known to be expressed in response to a range of cellular stresses. They are known to convey protection against protein denaturation and a subsequent immediate stress. Inducible heat shock protein 70 (Hsp70) is among the most studied of these stress proteins and its role and function are discussed here in terms of thermal and in particular exercise preconditioning. Preconditioning has been shown to confer cellular protection via expression Hsp, which may be of benefit in preventing protein damage following subsequent periods of exercise. Many studies have used animal models to gather data on Hsp70 and these and the most recent human studies are discussed.     

Heat shock proteins, substrate specificity and modulation of function

Hsp70 is a universally conserved essential protein chaperone. In addition to its roles in many cellular process, Hsp70 protects cells from stress by binding partially unfolded proteins. Therefore, Hsp70 prevents protein aggregation and prion formation. Prions are infectious agents and are responsible for several fatal neurodegenerative diseases. Eukaryotic cells have several cytosolic Hsp70 isoforms, some constitutively expressed (Hsc70s), and others expressed only when cells are exposed to stress (Hsp70s). To determine which factors conferred functional specificity, we constructed hybrid Hsc/Hsp chaperones. All hybrids supported growth except those that contained the ATPase domain derived from inducible Hsp70. Thus, regulation of peptide binding by ATP hydrolysis must differ significantly between Hsc- and Hsp70 isoforms. In this work, nucleotide and peptide binding domain communication of Hsp70 proteins during their interaction with nucleotides and peptide substrates were investigated in vitro by using hybrid constructs.