Differential Sensitivity of Telomerase from Human Hematopoietic Stem Cells and Leukemic Cell Lines to Mild Hyperthermia

We have investigated the effects of hyperthermia (HT) on cell proliferation and telomerase activity of human hematopoietic stem cells (HSCs) and compared with human leukemic cell lines (TF-1, K562 and HL-60). The cells were exposed to HT at 42 and 43 °C up to 120 min. The cells were incubated at 37 °C for 96 h. Then the cells were collected and assayed for cell proliferation, viability, telomerase activity, and terminal restriction fragment (TRF) lengths. The enzyme activity from HSCs was decreased up to 68.6 at 42 and 85.1 % at 43 °C for 120 min. This inhibition in leukemic cells was up to 28.9 and 53.6 % in TF-1; 53 and 63.9 % in K562; 45.2 and 61.1 % in HL-60 cells. The treated cells showed TRF lengths about 5.3 kb for control HL-60 cells, 5.0 kb for HL-60 cells treated at 42 and 4.5 kb at 43 °C for 120 min. In HSCs, the TRF length was about 4.5 kb for untreated cells and 4.0–4.5 kb for treated cells at 42 and 43 °C for 120 min. The time response curves indicated that, inhibition of the enzyme activity in leukemic cells was dependent to the time of exposure to HT. But in HSCs, the inhibition was reached to steady state at 15 min exposure to 43 °C heat stress. TRF length was constant at treated two types of cells, which implies that in cells subjected to mild HT no telomere shortening was observed.     

Expression analysis of nine small heat shock protein genes from Tamarix hispida in response to different abiotic stresses and abscisic acid treatment

Heat shock proteins (HSPs) play important roles in protecting plants against environmental stresses. Furthermore, small heat shock proteins (sHSPs) are the most ubiquitous HSP subgroup with molecular weights ranging from 15 to 42 kDa. In this study, nine sHSP genes (designated as ThsHSP1–9) were cloned from Tamarix hispida. Their expression patterns in response to cold, heat shock, NaCl, PEG and abscisic acid (ABA) treatments were investigated in the roots and leaves of T. hispida by real-time RT-PCR analysis. The results showed that most of the nine ThsHSP genes were expressed at higher levels in roots than in leaves under normal growth condition. All of ThsHSP genes were highly induced under conditions of cold (4 °C) and different heat shocks (36, 40, 44, 48 and 52 °C). Under NaCl stress, all nine ThsHSPs genes were up-regulated at least one stress time-point in both roots and leaves. Under PEG and ABA treatments, the nine ThsHSPs showed various expression patterns, indicating a complex regulation pathway among these genes. This study represents an important basis for the elucidation of ThsHSP gene function and provides essential information that can be used for stress tolerance genetic engineering in future studies.     

Heat shock increases levels of reactive oxygen species,autophagy and apoptosis

Hyperthermia is a promising anticancer treatment used in combination with radiotherapy and chemotherapy. Temperatures above 41.5 °C are cytotoxic and hyperthermia treatments can target a localized area of the body that has been invaded by a tumor. However, non-lethal temperatures (39–41 °C) can increase cellular defenses, such as heat shock proteins. This adaptive survival response, thermotolerance, can protect cells against subsequent cytotoxic stress such as anticancer treatments and heat shock (>41.5 °C). Autophagy is another survival process that is activated by stress. This study aims to determine whether autophagy can be activated by heat shock at 42 °C, and if this response is mediated by reactive oxygen species (ROS). Autophagy was increased during shorter heating times (<60 min) at 42 °C in cells. Levels of acidic vesicular organelles (AVO) and autophagy proteins Beclin-1, LC3-II/LC-3I, Atg7 and Atg12-Atg5 were increased. Heat shock at 42 °C increased levels of ROS. Increased levels of LC3 and AVOs at 42 °C were inhibited by antioxidants. Therefore, increased autophagy during heat shock at 42 °C (<60 min) was mediated by ROS. Conversely, heat shock at 42 °C for longer times (1?3 h) caused apoptosis and activation of caspases in the mitochondrial, death receptor and endoplasmic reticulum (ER) pathways. Thermotolerant cells, which were developed at 40 °C, were resistant to activation of apoptosis at 42 °C. Autophagy inhibitors 3-methyladenine and bafilomycin sensitized cells to activation of apoptosis by heat shock (42 °C). Improved understanding of autophagy in cellular responses to heat shock could be useful for optimizing the efficacy of hyperthermia in the clinic.     

Effect of heat shock on protein synthesis in Locusta migratoria epidermis

Heat shock induced by an increase in temperature from 30°C to 47°C led to changes in protein synthesis in wing pads of the fifth larval instar of Locusta migratoria. Synthesis of heat shock proteins in the molecular weight range of 85,000, 70,000 and 18,000–22,000 was first detected at a threshold temperature of 45°C and was found to be highest at 47°C. A marked decline in the synthesis of many other proteins was also evident at 47°C. Recovery of general protein synthesis was observed when wing pads were shifted back to 30°C after a 2-h heat shock at 47°C. Heat shock protein patterns in Locusta and Drosophila were compared.     

Heat Shock Preconditioning Reduces the Formation of 8-hydroxy-2′-deoxyguanosine and 4-hydroxy-2-nonenal Modified Proteins in Ischemia-reperfused Liver of Rats

Heat shock preconditioning (HSPC) is a promising strategy for providing ischemic tolerance. The objective of this study is to investigate the effectiveness of HSPC in preventing oxidative damage of cellular proteins and DNA during ischemia-reperfusion of the liver. Male Wistar rats were divided into a heat shock group (group HS) and control (group C). Forty-eight hours prior to ischemia, rats in group HS received HSPC at 42°C for 15 &#117 min. All rats received hepatic warm ischemia for 30 &#117 min and subsequent reperfusion. The formation of 8-hydroxy-2'-deoxyguanosine (8-OHdG), 4-hydroxy-2-nonenal (HNE) modified proteins in liver tissue, survival rate of the animals, and changes in biochemical and histological parameters were compared between groups. Heat shock protein 72 was produced only in group HS. The 7-day survival of rats was significantly better in group HS (10/10) than in group C (5/10) ( p <0.01). The serum release of alanine aminotransferase ( n =10, p <0.01) and the concentration of adenosine triphosphate in liver tissue ( n =10, p <0.01) 40 &#117 min after reperfusion was significantly better in group HS than in group C. The formation of 8-OHdG in liver tissue measured by high-performance liquid chromatography was suppressed in group HS ( p <0.01). The production of HNE-modified proteins as determined by Western-blot analysis was also decreased in group HS. These results were also confirmed by immunohistochemical analysis. As determined by levels of 8-OHdG and HNE-modified proteins produced during ischemia-reperfusion of the liver, HSPC reduced the oxidative injury of cellular proteins and DNA in the liver tissue.     

Analysis of Proteins Expressed by an Abiotic Stress Tolerant Pseudomonas putida (NBAII-RPF9) Isolate Under Saline and High Temperature Conditions

Pseudomonas putida (NBAII-RPF9) was identified as an abiotic stress tolerant bacterium capable of growing at 45 °C as well as in 1 M NaCl. The proteins expressed by this bacterium when subjected to these two stresses were analyzed by 2D gel and MALDI-TOF/MS. Two parameters viz., heat/saline shock (20 min at 45 °C/1 M solid NaCl added at mid log phase and incubated for 1 h) and heat/saline tolerance (24 h growth at 45 °C/in 1 M NaCl) were studied. Under heat shock 13 upregulated proteins and 1 downregulated protein were identified and under tolerance 6 upregulated proteins were identified. GroES and GroEL proteins were expressed under both tolerance and shock. Under saline shock 11 upregulated proteins were identified whereas under saline tolerance 6 upregulated proteins were identified and all these proteins had pI between 3 and 10 with molecular weights ranging from 14.3 to 97 kDa. Aspartate carbamoyltransferase was common under both the saline conditions studied. The analysis revealed involvement of heat stress responsive molecular chaperones and membrane proteins during heat stress. During salt stress, proteins involved in metabolic processes were found to be upregulated to favor growth and adaptation of the bacterium. Heat shock chaperones viz., DnaK and DnaJ were expressed under both saline and heat stress. This is the first report of protein profile obtained from a single bacterium under saline and heat stress and the studies reveal the complex mechanisms adapted by the organism to survive under high temperature or saline conditions.     

Mechanism of Saccharomyces cerevisiae yeast cell death induced by heat shock. Effect of cycloheximide on thermotolerance

The mechanism of yeast cell death induced by heat shock was found to be dependent on the intensity of heat exposure. Moderate (45°C) heat shock strongly increased the generation of reactive oxygen species (ROS) and cell death. Pretreatment with cycloheximide (at 30°C) suppressed cell death, but produced no effect on ROS production. The protective effect was absent if cycloheximide was added immediately before heat exposure and the cells were incubated with the drug during the heat treatment and recovery period. The rate of ROS production and protective effect of cycloheximide on viability were significantly decreased in the case of severe (50°C) heat shock. Treatment with cycloheximide at 39°C inhibited the induction of Hsp104 synthesis and suppressed the development of induced thermotolerance to severe shock (50°C), but it had no effect on induced thermotolerance to moderate (45°C) heat shock. At the same time, Hsp104 effectively protected cells from death independently of the intensity of heat exposure. These data indicate that moderate heat shock induced programmed cell death in the yeast cells, and cycloheximide suppressed this process by inhibiting general synthesis of proteins.     

Heat shock and cold shock in Deinococcus radiodurans

On the basis of acquired thermotolerance and cryotolerance, the optimal heat shock and cold shock temperatures have been determined for Deinococcus radiodurans. A heat shock at 42°C maximized survival at the lethal temperature of 52°C and a cold shock at 20°C maximized survival after repeated freeze-thawing. Enhanced survival from heat shock was found to be strongly dependent on growth stage, with its greatest effect shortly after phase. Increased synthesis of a total of 67 proteins during heat shock and 42 proteins during cold shock were observed by two-dimensional polyacrylamide gel electrophoresis (2D PAGE) and autoradiography. Eight of the most highly induced heat shock proteins shown by 2D PAGE were identified by MALDI-MS as Hsp20, GroEL, DnaK, SodA, Csp, Protease I and two proteins of unknown function.     

Changes in ultrastructure and transcription induced by elevated temperature in Zea mays embryonic root cells

A study has been made of the changes occurring in Zea mays kernels exposed to thermal shock at the 48th h of soaking at 16°C, i.e., just before the protrusion of the root. Heat shock of 5 h at 46°C temporarily inhibits the resumption of root growth and consequently retards the protrusion of the root. On the ultrastructural level, it is the nucleolus which undergoes the most dramatic changes. Total loss of the granular component occurs and new electron opaque corpuscles with diameters ranging from 80 to 140 nm appear. These corpuscles contain RNA and proteins. Microstereology shows that the vacuolation of the nucleolus is increased whereas its volume is decreased. Autoradiographical and biochemical studies of RNA synthesis show that the heat shock induces an inhibition of pre-rRNA synthesis. Only RNAs of low molecular weight are still synthesized. After 19 h of a return to 16°C, the ultrastructural changes of the nucleolus are reversed. Nevertheless, increased nucleolar vacuolation persists up to that time. Exposure of maize kernels to 46°C also produces the appearance of corpuscles in some areas of the cytoplasm and in the matrix of mitochondria. Disappearance of these structures is complete within 19 h, except in some mitochondria.     

Effects of heat shock on survival and predation of an important whitefly predator,Serangium japonicum

Climate change is expected to increase the frequency of periods of extreme weather events, including heat waves that are harmful to arthropod natural enemies. We studied the thermotolerance of the ladybeetle Serangium japonicum Chapin (Coleoptera: Coccinellidae), an important native predator of the whitefly Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) in China. Serangium japonicum eggs, first‐ and fourth‐instar larvae, pupae, and adults were subjected to a range of high temperatures (36, 39, 42, 43.5, and 45 °C) at intervals between 15 and 720 min. Survival was compromised and declined sharply for all life stages at temperatures of 42 °C and higher. First instars were the least heat tolerant, and eggs were typically the most resistant. Egg predation by S. japonicum did not differ whether the adult beetles were subjected to either heat shock or starvation first. Heat shock treatments at 36 °C did not impede adult egg consumption, treatments at 39 °C crippled ladybeetle's egg consumption for 8 h following treatment but recovered predatory capacity within 24 h after treatment. However, treatments at 42 °C greatly impaired ladybeetle's predatory capacity. After experiencing heat shock at 39 and 42 °C, adults significantly increased the amount of time they spent resting, which is the probable cause of predation decline after heat exposure. These results demonstrate that short periods of extreme heat exposures have a detrimental impact on S. japonicum survival and predation, but the effect was dependent on duration and life stage. Overall, these findings can help predict population dynamics and success of biological control of whitefly by S. japonicum in a warming world.     

Cortisol Effect on Heat Shock Proteins in the C2C12 and 3T3-L1 Cells

The present study was carried out to understand the effect of cortisol on heat shock protein system (Hsps) in the C2C12 and 3T3-L1 cells under co-culture system. Cells were co-cultured by using Transwell inserts with a 0.4-μm porous membrane to separate C2C12 and 3T3-L1 cells. Each cell type was grown independently on the Transwell plates. After cell differentiation, inserts containing 3T3-L1 cells were transferred to C2C12 plates and inserts containing C2C12 cells transferred to 3T3-L1 plates. Ten micrograms per microliter of cortisol was added to the medium. Following 72 h of treatment, the cells in the lower wells were harvested for analysis. Heat shock proteins (Hsps) such as Hsp27, Hsp70, and Hsp90 were selected for the analysis. The qRT-PCR results showed the significant increase in the mRNA expression of as Hsp27, Hsp70, and Hsp90. In addition, confocal microscopical investigation showed the cortisol treatment increases Hsps expressions in the mono and co-cultured C2C12 and 3T3-L1 cells. From the results, we concluded that the cortisol increases Hsps expression in the co-cultured C2C12 and 3T3-L1 cells, which is differed from one-dimensional mono-cultured C2C12 and 3T3-L1 cells.     

Characteristics and expression of genes encoding two small heat shock protein genes lacking introns from <Emphasis Type="Italic">Chilo suppressalis</Emphasis>

Small heat shock proteins (sHSPs) constitute a large, diverse, and functionally uncharacterized family of heat shock proteins. To gain insight regarding the function of sHSPs in insects, we identified genes encoding two sHSPs, Cshsp22.9b and Cshsp24.3, from the rice pest Chilo suppressalis. The cDNAs of Cshsp22.9b and Cshsp24.3 encoded proteins of 206 and 216 amino acids with isoelectric points of 5.79 and 9.28, respectively. Further characterization indicated that both Cshsp22.9b and Cshsp24.3 lacked introns. Real-time quantitative PCR indicated that Cshsp22.9b and Cshsp24.3 were expressed at higher levels within the fat body as compared to other tissues (head, epidermis, foregut, midgut, hindgut, Malpighian tubules, and hemocytes). Expression of Cshsp22.9b and Cshsp24.3 was lowest in the hindgut and Malpighian tubules, respectively. Cshsp22.9b and Cshsp24.3 showed identical patterns in response to thermal stress from ?11 to 43 °C, and both genes were up-regulated by hot and cold temperatures. The mRNA (messenger ribonucleic acid) expression levels of Cshsp22.9b (KY701308) and Cshsp24.3 (KY701309) were highest after a 2-h exposure at 39 °C and started to decline at 42 °C. In response to cold temperatures, both Cshsp22.9b and Cshsp24.3 showed maximal expression after a 2-h exposure to ?3 °C. The two Cshsps were more responsive to hot than cold temperature stress and were not induced by mildly cold or warm temperatures. In conclusion, Cshsp22.9b and Cshsp24.3 could play a very important role in the regulation of physiological activities in C. suppressalis that are impacted by environmental stimuli.     

Evaluation of DNA methylation and mRNA expression of heat shock proteins in thermal manipulated chicken

Thermal manipulation during embryogenesis has been demonstrated to enhance the thermotolerance capacity of broilers through epigenetic modifications. Heat shock proteins (HSPs) are induced in response to stress for guarding cells against damage. The present study investigates the effect of thermal conditioning during embryogenesis and thermal challenge at 42 days of age on HSP gene and protein expression, DNA methylation and in vitro luciferase assay in brain tissue of Naked Neck (NN) and Punjab Broiler-2 (PB-2) chicken. On the 15th day of incubation, fertile eggs from two breeds, NN and PB-2, were randomly divided in to two groups: control (C)—eggs were incubated under standard incubation conditions, and thermal conditioning (TC)—eggs were exposed to higher incubation temperature (40.5°C) for 3 h on the 15th, 16th, and 17th days of incubation. The chicks obtained from each group were further subdivided and reared under different environmental conditions from the 15th to the 42nd day as normal [N; 25 ± 1 °C, 70% relative humidity (RH)] and heat exposed (HE; 35 ± 1 °C, 50% RH) resulting in four treatment groups (CN, CHE, TCN, and TCHE). The results revealed that HSP promoter activity was stronger in CHE, which had lesser methylation and higher gene expression. The activity of promoter region was lesser in TCHE birds that were thermally manipulated at the embryonic stage, thus reflecting their stress-free condition. This was confirmed by the lower level of mRNA expression of all the HSP genes. In conclusion, thermal conditioning during embryogenesis has a positive impact and improves chicken thermotolerance capacity in postnatal life.     

Heat-shock gene expression and cell cycle changes during mammalian embryonic development

Synchronized regulation of cell division during gastrulation is essential for the regional proliferation of cells and pattern formation of the early CNS. The neural plate and neuroectoderm cells are a rapidly dividing and differentiating population of cells with a unique and rapid heat-shock response. Heat shock and the heat-shock genes were studied during neural plate development in a whole rat embryo culture system at 9.5-11.5 days. A lethal heat shock can cause cell death and severe developmental defects to the forebrain and eye during organogenesis. Heat shock can also result in acquired thermotolerance whereby cell progression is delayed at the G1/S and S/G2 boundaries of the cell cycle. This delay in cell cycle progression caused an overall lengthening of the cell cycle time of at least 2 hr. The heat shock genes may therefore function as cell cycle regulators in neuroectoderm induction and differentiation. The kinetics and expression of the hsp genes were examined in neuroectodermal cells by flow cytometry and Northern analysis. The levels of hsp mRNA 27, 71, 73, and 88 were identified following exposure at 42°C (nonlethal), 43deg;C (lethal) and 42deg;/43deg;C (thermotolerant) heat shock. Examination of hsp gene expression in the neural plate showed tight regulation in the cell cycle phases. Hsp 88 expression was enhanced at Go and hsp71 induction at G2 + M of the cell cycle. Cells exposed to a thermotolerant heat shock of 42deg;C induced hsp71 mRNA expression in all phases of the cell cycle with the mRNA levels of hsp27, 73, and 88 increased but relatively constant. Following a lethal heat shock, dramatic changes in hsp expression were seen especially enhanced hsp71 induction in late S phase. The regulated expression of hsps during the cell cycle at various phases could play a unique and important role in the fate and recovery of neuroectoderm cells during early mammalian embryo development. © 1993Wiley-Liss, Inc.