Anticancer efficacies of doxorubicin,verapamil and quercetin on FM3A cells under hyperthermic temperature

Hyperthermia (HT) in combination with anticancer drugs (ACDs) had proven to more efficacious in various cancers, although efficacies vary according to chemotherapeutic compounds and cancer types. Presently there are few data that compares anticancer efficacies among ACDs under hyperthermic conditions. Therefore, we selected three commonly used ACDs (quercetin, verapamil and doxorubicin) and compared their antitumor effects when each was treated with 43°C HT exposure. Firstly, FM3A, a murine breast cancer cell line, was treated with each ACD for 1 h followed by 43°C exposure for additional 1 h, and examined the effects of: 1) each drug, 2) 43°C HT exposure, and 3) the combination of each drug and 43°C HT exposure for 1, 6 and 24 h. The determined overall effects on FM3A cells were arrested cell proliferation, clonogenic efficiency and apoptosis. Pre-treatment of FM3A cells to each ACD followed by 43°C HT exposure produced greater antitumor effects including suppressed cell proliferation, reduced clonogenic efficiency and increased apoptotic cell death, compared to ACD treatment or HT exposure alone. Apoptotic cell death occurred in a time-dependent manner. Among the ACDs, antitumor efficacies varied in the order of doxorubicin > verapamil > quercetin. It was concluded that heat exposure during ACD treatment of caner cells may be an important factor to get a better antitumor benefit, even though this benefit may differ from one drug to another.     

Nitric oxide‐mediated inhibition of NFκB regulates hyperthermia‐induced apoptosis

Ascertaining the upstream regulatory mechanisms of hyperthermia‐induced apoptosis is important to understand the role of hyperthermia in combined modality cancer therapy. Accordingly, we investigated whether (i) hyperthermia‐induced apoptosis is mediated through the nitric oxide (NO) signaling pathway and (ii) inhibition of post‐translational modification of IκBα and down regulation of NFκB‐DNA binding activity is an intermediate step in NO‐dependent apoptosis in MCF‐7 breast cancer cells. For hyperthermia treatment, the cells were exposed to 43°C. Intracellular NO levels measured by the fluorescent intensity of DAF‐2A and iNOS expression by immunobloting revealed an increased level of iNOS dependent NO production after 43°C. Apoptosis measured by Annexin V expression and cell survival by clonogenic assay showed a 20% increase in apoptosis after 43°C treatments. EMSA analysis showed a dose‐dependent inhibition of NFκB‐DNA binding activity. The hyperthermia‐mediated inhibition of NFκB was persistent even after 48 h. Inhibition of NO by L ‐NAME rescued the NFκB‐DNA binding activity and inhibits heat‐induced apoptosis. Similarly, over‐expression of NFκB by transient transfection inhibits heat‐induced apoptosis. These results demonstrate that apoptosis upon hyperthermia exposure of MCF‐7 cells is regulated by NO‐mediated suppression of NFκB. J. Cell. Biochem. 106: 999–1009, 2009. © 2009 Wiley‐Liss, Inc.     

Induced thermotolerance to apoptosis in a human T lymphocyte cell line.

A brief exposure to elevated temperatures elicits, in all organisms, a transient state of increased heat resistance known as thermotolerance. The mechanism for this thermotolerant state is unknown primarily because it is not clear how mild hyperthermia leads to cell death. The realization that cell death can occur through an active process of self destruction, known as apoptosis, led us to consider whether thermotolerance provides protection against this mode of cell death. Apoptosis is a common and essential form of cell death that occurs under both physiological and pathological conditions. This mode of cell death requires the active participation of the dying cell and in this way differs mechanistically from the alternative mode of cell death, necrosis. Here we show that mild hyperthermia induces apoptosis in a human leukemic T cell line. This is evidenced by chromatin condensation, nuclear fragmentation and the cleavage of DNA into oligonucleosome size units. DNA fragmentation is a biochemical hallmark of apoptosis and requires the activation of an endogenous endonuclease. The extent of DNA fragmentation was proportional to the severity of heat stress for cells heated at 43 degrees C from 30 to 90 minutes. A brief conditioning heat treatment induced a resistance to apoptosis. This was evident as a resistance to DNA fragmentation and a reduction in the number of apoptotic cells after a heat challenge. Resistance to DNA fragmentation developed during a recovery period at 37 degrees C and was correlated with enhanced heat shock protein (hsp) synthesis. This heat-induced resistance to apoptosis suggests that thermotolerant cells have gained the capacity to prevent the onset of this pathway of self-destruction. An examination of this process in heated cells should provide new insights into the molecular basis of cellular thermotolerance.     

The effect of seed conditioning,short-term heat shock and salicylic,jasmonic acid or brasinolide on sunflower (Helianthus annuus L.) chilling resistance and polysome formation

The aim of this study was to develop the method for increasing resistance of sunflower seedlings ‘Wielkopolski’ to chilling. Seeds were conditioned at 25 °C for 2 days in water to 15, 20 and 25 % moisture content or in salicylic or jasmonic acid in concentration of 10^?2; 10^?3 and 10^?4 M or brassinolide in concentration of 10^?6; 10^?8 and 10^?10–15 % moisture content. After 2 days of incubation the conditioned seeds were heat shocked at 45 °C for 0, 30, 60, 120 and 240 min and 5 mm seedlings were exposed to chilling at 0 °C for 21 days. The effectiveness of the methods was assessed by evaluation of roots growth in Phytotoxkit Microbiotest, changes in the activity of dehydrogenases, the integrity of the cytoplasmic membrane and formation of polysomes after seedling were returned to 25 °C for 72 h. Seeds were conditioned at 25 °C for 2 days in water to 15 % moisture content and then heat shocked at 45 °C for 2 h decreased chilling injury of seedlings expressed by subsequent growth of the roots, electrolyte leakage, dehydrogenases activity and polysomes formation. Application of heat shock of 45 °C for 2 h during seed conditioning additionally provided seedling protection against subsequent chilling conditions. Brasinolide, salicylic acid or jasmonic acid applied during seeds conditioning exhibited further beneficial effect on seedling resistance to chilling. The most pronounced effect was obtained due to seed conditioning to 15 % moisture content in solutions of brassinolide in concentration of 10^?8 M. After 2 days of imbibition treated in this way seeds were exposed to heat shock at 45 °C for 2 h. The role of physiological events in improvement of sunflower chilling tolerance are discussed.     

The effect of timing of thermal conditioning during incubation on embryo physiological parameters and its relationship to thermotolerance in adult broiler chickens

Previously, we reported that thermal conditioning at 39°C on days 13–17 of incubation of broiler eggs enabled thermotolerance during post-hatch growth (J. Therm. Biol. 28 (2003) 133). Tolerance to a temperature of 30°C was accompanied by changes in thyroid hormones and metabolic parameters. In the current study, we determined the mechanism of epigenetic heat adaptation during embryonic age by measuring blood physiological parameters that may be associated with the ultimate effects of thermal conditioning. Hatching eggs from Ross breeders were subjected to heat treatment of 39°C at days 13, 14, 15, 16 and 17 of incubation for 2 h per day. Control eggs were incubated at 37.6°C. Samples of eggs were withdrawn on each day of thermal conditioning and at internal pipping (IP) to obtain blood samples from embryos. The remaining eggs were weighed at day 18 and transferred to hatchers. The timing of IP, external pipping (EP) and hatching were monitored every 2 h. At hatch, chicks were weighed and hatchability was determined. Blood samples were obtained from samples of day-old chicks. T3, T4, corticosterone, pCO₂, pO₂ levels were determined in the blood. Blood pH was measured and T3/T4 ratios were calculated. Heat conditioning significantly increased corticosterone and pO₂ levels and blood pH but depressed pCO₂ at day 14. These were followed by a significant depression of T4 level on day 15. Remarkably, at day 16, all these parameters were back to normal as in the control embryos. Hatching was delayed by thermal conditioning probably as a result of the depressed corticosterone levels at IP. Hatchability was also lower in the heat-treated group but 1-day old chick weights were comparable to those of the controls. The result suggests that epigenetic thermal conditioning involves changes in these physiological parameters and probably serve as a method for epigenetic temperature adaptation since the same mechanisms are employed for coping with heat during post-embryonic growth. It also suggests that days 14–15 may be the optimal and most sensitive timing for evoking this mechanism during embryonic development. The adverse effects of heat treatment observed in this study may have been due to the continued exposure to heat until day 17. Fine-tuning thermal conditioning to days 14–15 only may improve these production parameters.     

Synchronized turbo apoptosis induced by cold-shock

In our research on the role of apoptosis in the pathogenesis of the autoimmune disease systemic lupus erythematosus (SLE), we aim to evaluate the effects of early and late apoptotic cells and blebs on antigen presenting cells. This requires the in vitro generation of sufficiently large and homogeneous populations of early and late apoptotic cells. Here, we present a quick method encountered by serendipity that results in highly reproducible synchronized homogeneous apoptotic cell populations. In brief, granulocytic 32Dcl3 cells are incubated on ice for 2 h and subsequently rewarmed at 37°C. After 30–90 min at 37°C more than 80–90% of the cells become early apoptotic (Annexin V positive/propidium iodide negative). After 24 h of rewarming at 37°C 98% of the cells were late apoptotic (secondary necrotic; Annexin V positive/propidium iodide positive). Cells already formed apoptotic blebs at their cell surface after approximately 20 min at 37°C. Inter-nucleosomal chromatin cleavage and caspase activation were other characteristics of this cold-shock-induced process of apoptosis. Consequently, apoptosis could be inhibited by a caspase inhibitor. Finally, SLE-derived anti-chromatin autoantibodies showed a high affinity for apoptotic blebs generated by cold-shock. Overall, cold-shock induced apoptosis is achieved without the addition of toxic compounds or antibodies, and quickly leads to synchronized homogeneous apoptotic cell populations, which can be applied for various research questions addressing apoptosis.     

HSP16.6 Is Involved in the Development of Thermotolerance and Thylakoid Stability in the Unicellular Cyanobacterium, Synechocystis sp. PCC 6803

The low molecular weight (LMW) heat shock protein (HSP), HSP16.6, in the unicellular cyanobacterium, Synechocystis sp. PCC 6803, protects cells from elevated temperatures. A 95% reduction in the survival of mutant cells with an inactivated hsp16.6 was observed after exposure for 1 h at 47°C. Wild-type cell survival was reduced to only 41%. HSP16.6 is also involved in the development of thermotolerance. After a sublethal heat shock at 43°C for 1 h and subsequent challenge exposure at 49°C for 40 min, mutant cells did not survive, while 64% of wild-type cells survived. Ultrastructural changes in the integrity of thylakoid membranes of heat-shocked mutant cells also are discussed. These results demonstrate an important protective role for HSP16.6 in the protection of cells and, in particular, thylakoid membrane against thermal stress. Received: 14 October 1999 / Accepted: 16 November 1999     

Evaluation of thermal inactivation of Escherichia coli using microelectrode ion flux measurements with osmotic stress

Aims: To elucidate the potential use of microelectrode ion flux measurements to evaluate bacterial responses to heat treatment. Methods and Results: Escherichia coli K12 was used as a test bacterium to determine whether various heat treatments (55–70°C for 15 min) affected net ion flux across E. coli cell membranes using the MIFE? system to measure net K⁺ fluxes. No difference in K⁺ fluxes was observed before and after heat treatments regardless of the magnitude of the treatment. Applying hyperosmotic stress (3% NaCl w/v) during flux measurement led to a net K⁺ loss from the heat‐treated E. coli cells below 65°C as well as from nonheated cells. In contrast, with E. coli cells treated at and above 65°C, hyperosmotic stress disrupted the pattern of K⁺ flux observed at lower temperatures and resulted in large flux noise with random scatter. This phenomenon was particularly apparent above 70°C. Although E. coli cells lost the potential to recover and grow at and above 62°C, K⁺ flux disruption was not clearly observed until 68°C was reached. Conclusions: No changes in net K⁺ flux from heat‐stressed E. coli cells were observed directly as a result of thermal treatments. However, regardless of the magnitude of heat treatment above 55°C, loss of viability indicated by enrichment culture correlated with disrupted K⁺ fluxes when previously heated cells were further challenged by imposing hyperosmotic stress during flux measurement. This two‐stage process enabled evaluation of the lethality of heat‐treated bacterial cells within 2 h and may be an alternative and more rapid method to confirm the lethality of heat treatment. Significance and Impact of the Study: The ability to confirm the lethality of thermal treatments and to specify minimal time/temperature combinations by a nonculture‐dependent test offers an alternative system to culture‐based methods.     

Induction of heat shock protein 27 in rat embryos exposed to hyperthermia

Previously we reported that eight proteins were reproducibly induced in postimplantation rat embryos exposed to a brief heat shock (43°C, 15 min). The major heat-inducible rat embryo protein has now been identified as heat shock protein 72 (Hsp 72). In addition, the induction of Hsp 72 is temporally correlated with induction of thermotolerance. One of the other rat embryo proteins previously shown to be induced by elevated temperature is a heat shock protein of approximately 27 kilodaltons (Hsp 27). In this report we show that this protein is recognized by an antibody directed against a conserved peptide sequence of Hsp 27. Unlike Hsp 72, Hsp 27 is constitutively expressed in the rat embryo in the absence of any thermal stress; however, the level of Hsp 27 is increased approximately 2–3-fold after thermal stress (43°C, 10 min). Immunohistochemical analysis revealed that the constitutively expressed Hsp 27 is localized primarily to cells of the heart, cells that are uniquely resistant to the cytotoxic effects of hyperthermia. After thermal stress, Hsp 27 is expressed in all tissues of the embryo. Finally, our data show that Hsp 27 exists in the rat embryo as three major isoforms indicative of different phosphorylation states. Furthermore, most Hsp 27 in the heart is phosphorylated, whereas in the rest of the embryo, nonphosphorylated Hsp 27 predominates. After thermal stress, levels of phosphorylated isoforms increase dramatically in nonheart tissues of the embryo. Together, these results suggest that Hsp 27 may play a role in the development of thermotolerance in the postimplantation mammalian embryo. © 1996 Wiley-Liss, Inc.     

Apparent absence of viruses in most symptomless field-grown sweet potato in Uganda

Heat tolerance of groundnut (Arachis hypogaea L.) genotypes was evaluated by solute leakage and chlorophyll fluorescence techniques in heat-hardened and non-hardened plants. To determine the appropriate hardening treatment, 1-month-old plants of two groundnut genotypes, ICGV 86707 and Chico were conditioned at five combinations of hardening (37°C) and non-hardening (30°C) air temperatures over a 5-day period. Heat injury, was assessed through measurements of electrolyte leakage after stressing leaf discs to 55°C for 15 min. The relative injury was significantly influenced by the conditioning temperatures and by the temperature during 24 h prior to measurement if those involved non-hardening conditions. Relative injury and chlorophyll fluorescence were measured after stressing leaves of six genotypes at a range of temperatures between 49°C and 55°C. Significant genotype × hardening treatment interactions were observed in relative injury and chlorophyll fluorescence. Chico was susceptible to heat stress, the relative injury test identified ICGV 86707 as tolerant, and the chlorophyll fluorescence test identified ICGV 86707 as tolerant under hardened conditions and ICGV 87358 as tolerant when non-hardened. When expressed as percentage of control values, the relative injury and chlorophyll fluorescence measurements over the 49–53°C stress temperature range were strongly correlated. Chlorophyll concentrations were increased by hardening in all genotypes except Chico. In Chico, chl_b concentration was decreased and the chl_a/b ratio increased by hardening, and chlorophyll concentrations were correlated with chlorophyll fluorescence parameters. Chlorophyll concentration may therefore provide an alternative means of screening for heat tolerance.     

Heat shock prevents simulated ischemia-induced apoptosis in renal tubular cells via a PKC-dependent mechanism

Heat shock produces cellular tolerance to a variety of adverse conditions; however, the protective effect of heat shock on renal cell ischemic injury remains unclear. Protein kinase C (PKC) has been implicated in the signaling mechanisms of acute preconditioning, yet it remains unknown whether PKC mediates heat shock-induced delayed preconditioning in renal cells. To study this, renal tubular cells (LLC-PK1) were exposed to thermal stress (43 degrees C) for 1 h and heat shock protein (HSP) 72 induction was confirmed by Western blot analysis. Cells were subjected to simulated ischemia 24 h after thermal stress, and the effect of heat shock (delayed preconditioning) on ischemia-induced apoptosis (terminal deoxynucleotidyl transferase dUTP nick-end labeling) and B cell lymphoma 2 (Bcl(2)) expression (Western) was determined. Subsequently, the effect of PKC inhibition on HSP72 induction and heat stress-induced ischemic tolerance was evaluated. Thermal stress induced HSP72 production, increased Bcl(2) expression, and prevented simulated ischemia-induced renal tubular cell apoptosis. PKC inhibition abolished thermal induction of HSP72 and prevented heat stress-induced ischemic tolerance. These data demonstrate that thermal stress protects renal tubular cells from simulated ischemia-induced apoptosis through a PKC-dependent mechanism.     

Rapid cold-hardening protects <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> from cold-induced apoptosis

The rapid cold-hardening (RCH) response increases the cold tolerance of insects by protecting against non-freezing, cold-shock injury. Apoptosis, or programmed cell death, plays important roles in development and the elimination of sub-lethally damaged cells. Our objectives were to determine whether apoptosis plays a role in cold-shock injury and, if so, whether the RCH response protects against cold-induced apoptosis in Drosophila melanogaster. The present study confirmed that RCH increased the cold tolerance of the adults at the organismal level. No flies in the cold-shocked group survived direct exposure to ‒7°C for 2 h, whereas significantly more flies in the RCH group survived exposure to ‒7°C for 2 h after a 2-h exposure to 5°C. We used a TUNEL assay to detect and quantify apoptotic cell death in five groups of flies including control, cold-shocked, RCH, heat-shocked (37.5°C, 30 min), and frozen (‒20°C, 24 h) and found that apoptosis was induced by cold shock, heat shock, and freezing. The RCH treatment significantly improved cell viability by 38% compared to the cold-shocked group. Cold shock-induced DNA fragmentation shown by electrophoresis provided further evidence for apoptosis. SDS-PAGE analysis revealed an RCH-specific protein band with molecular mass of ∼150 kDa. Western-blotting revealed three proteins that play key roles in the apoptotic pathway: caspase-9-like (apoptotic initiator), caspase-3-like (apoptotic executioner) and Bcl-2 (anti-apoptotic protein). Consequently, the results of this study support the hypothesis that the RCH response protects against cold-shock-induced apoptosis.     

Afferent Modulation of Warmth Sensation and Heat Pain in the Human Hand

The hands of 14 normal humans were used to determine the somatotopic organization of the modulation of warmth sensation and heat pain by different forms of cutaneous stimuli. Test stimuli were 5-sec heat pulses ranging from 36° to 51°C, delivered to the fingerpads of digits 1, 2, 4, and 5 with a contact thermode. Conditioning stimuli (15 sec) bracketed the test stimuli and included vibration, noxious and innocuous heat, cold, and electrical pulses delivered to the fingerpads of digits that were adjacent or nonadjacent to the tested digits. Noxious (48° ± 1.3°C), but not innocuous (43°C), heat stimuli increased the perceived magnitude estimation of innocuous test stimuli (36–43°C) by 20–37% when delivered to adjacent, but not to nonadjacent, digits. No other conditioning stimuli had any effect on the intensity of warmth perception. In contrast, both noxious and innocuous heat or electrical conditioning reduced the magnitude estimation of noxious (50–5°C), but not innocuous, test pulses by 12–22% when delivered to adjacent digits. Conditioning of nonadjacent digits was significantly less effective. The analgesic effects of noxious and innocuous conditioning were approximately equal. Vibratory (120 Hz, 3.5 μm) and cold (15°C) conditioning stimuli were ineffective. The results are consistent with a dermatomal somatotopic organization of tactile and heat modulatory influences on warmth sensation and heat pain. The results further suggest that the neural mechanisms subserving warmth mediate a negative feedback influence on heat pain intensity.     

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.     

Characterization of a Tetrahymena thermophila mutant strain unable to develop normal thermotolerance.

For Tetrahymena thermophila cells to survive extended periods of time at 43 degrees C, they must continuously synthesize heat shock proteins. For its translational machinery to function at 43 degrees C, T. thermophila requires either prior nonlethal heat shock treatment or brief treatment with partially inhibiting doses of cycloheximide or emetine. We have identified and characterized a mutant strain of T. thermophila (MC-3) in which prior nonlethal heat shock does not prevent protein synthesis inactivation at 43 degrees C. In addition, treatment of MC-3 cells with either of the antibiotics that normally confer 43 degrees C thermoprotection on wild-type cells elicited no similar thermoprotective response in these cells. Despite these phenotypic characteristics, by other criteria MC-3 synthesized a normal, functional array of heat shock proteins at 40 degrees C, a nonlethal heat shock protein-inducing temperature. The mutation in MC-3 which prevents the thermostabilization of protein synthesis by nonlethal heat shock is, by genetic criteria, most likely the same one which prevents the induction of thermotolerance by drug treatments. We present evidence that this mutation may affect some ribosome-associated functions.     

Effects of mild heat shock on glycogenesis and its regulation by insulin in cultured fetal hepatocytes

The effects of a mild heat shock were investigated using cultured 15-day-old fetal rat hepatocytes in which an acute glucocorticoid-dependent glycogenic response to insulin was present. After exposure from 15 min to 2 h at 42.5°C, cell surface [¹²⁵I]insulin binding progressively decreased down to 60% of the value shown in cells kept at 37°C, due to a decrease in the apparent number of insulin binding sites with little change in insulin receptor affinity. In parallel cultures, protein labeling with [³⁵S]methionine exhibited stimulated synthesis of specific proteins, in particular, 73-kDa Hsc (heat shock cognate) and 72-kDa Hsp (heat shock protein). When cells were returned to 37°C after 2 h at 42.5°C, cell surface insulin binding showed a two-third restoration within 3 h (insulin receptor half-life = 13 h), with similar concomitant return of Hsps72,73 synthesis to preinduction levels. The rate of [¹⁴C]glucose incorporation into glycogen measured at 37°C after 1- to 2-h heat treatment revealed a striking yet transient increase in basal glycogenesis (up to 5-fold). At the same time, the glycogenesis stimulation by insulin was reduced (from 3.2 to 1.4—fold), whereas that induced by a glucose load was maintained. Induction of thermotolerance after a first heating was obtained for the heat shock-dependent events except for the enhanced basal glycogenesis. In insulin-unresponsive cells grown in the absence of glucocorticoids, heat shock decreased the glycogenic capacity without modifying the glucose load stimulation, supporting the hypothesis that insulin and thermal stimulation of glycogenesis share at least part of the same pathway. Inverse variations were observed between Hsps72,73 synthesis and both cell surface insulin receptor level and insulin glycogenic response in fetal hepatocytes experiencing heat stress. © 1995 Wiley-Liss, Inc.     

Changes in cell morphology and actin organization during heat shock in Dictyostelium discoideum: does HSP70 play a role in acquired thermotolerance?

In response to heat shock (34°C, 30 min), cell morphology and actin organization in Dictyostelium discoideum are drastically changed. Loss of pseudopodia and disappearance of F-actin-containing structures were observed by using fluorescence microscopy. These changes were paralleled by a rapid decrease of the F-actin content measured by a TRITC-phalloidin binding assay. The effects of heat shock on cell morphology and actin organization are transient: After heat shock (34°C) or during a long-term heat treatment (30°C), cell morphology, F-actin patterns and F-actin content recovered/adapted to a state which is characteristic for untreated cells. Because F-actin may be stabilized by increased amounts of heat shock proteins, their response and interaction with F-actin was analyzed. After a 1 h heat treatment (34°C), the major heat shock protein of D. discoideum (HSP70) showed maximally increased synthesis rates and levels. During recovery from a 34°C shock or during a continuous heat treatment at 30°C, the HSP70 content first increased and then declined slowly toward normal levels. Pre-treatment of cells with a short heat shock of 30 min at 34°C stabilized the F-actin content when the cells were exposed to a second heat shock. Furthermore, a transient colocalization of HSP70 and actin was observed at the beginning of heat treatment (30°C) using immunological detection of HSP70 in the cytoskeletal actin fraction.     

A microarray analysis of the effects of moderate hypothermia and rewarming on gene expression by human hepatocytes (HepG2)

The gene expression changes produced by moderate hypothermia are not fully known, but appear to differ in important ways from those produced by heat shock. We examined the gene expression changes produced by moderate hypothermia and tested the hypothesis that rewarming after hypothermia approximates a heat-shock response. Six sets of human HepG2 hepatocytes were subjected to moderate hypothermia (31°C for 16 h), a conventional in vitro heat shock (43°C for 30 min) or control conditions (37°C), then harvested immediately or allowed to recover for 3 h at 37°C. Expression analysis was performed with Affymetrix U133A gene chips, using analysis of variance-based techniques. Moderate hypothermia led to distinct time-dependent expression changes, as did heat shock. Hypothermia initially caused statistically significant, greater than or equal to twofold changes in expression (relative to controls) of 409 sequences (143 increased and 266 decreased), whereas heat shock affected 71 (35 increased and 36 decreased). After 3 h of recovery, 192 sequences (83 increased, 109 decreased) were affected by hypothermia and 231 (146 increased, 85 decreased) by heat shock. Expression of many heat shock proteins was decreased by hypothermia but significantly increased after rewarming. A comparison of sequences affected by thermal stress without regard to the magnitude of change revealed that the overlap between heat and cold stress was greater after 3 h of recovery than immediately following thermal stress. Thus, while some overlap occurs (particularly after rewarming), moderate hypothermia produces extensive, time-dependent gene expression changes in HepG2 cells that differ in important ways from those induced by heat shock.