Role In Thermotolerance Research Articles

熱ショック蛋白 : 温熱耐性及び癌治療のための細胞内分子標的作用

熱ショック蛋白 : 温熱耐性及び癌治療のための細胞内分子標的作用

An Hsp70 antisense gene affects the expression of HSP70/HSC70, the regulation of HSF, and the acquisition of thermotolerance in transgenic Arabidopsis thaliana.

The genes and proteins of the HSP70 family, are involved in important processes in cells and organelles at normal temperature and after heat stress. Constitutive Hsc70 and heat-inducible Hsp70 genes are known in all organisms including plants. The goal of our present investigation was to generate an Hsp70 mutation in Arabidopsis thaliana. In a transgenic approach a heat-inducible antisense Hsp70 gene was constructed, plants were transformed and screened for lack of heat-inducible HSP70 mRNA; two such lines were further investigated. In these plants the Hsp70 gene was not induced by heat shock, and the level of HSC70 RNA was also greatly reduced. This negative antisense effect was specific for genes of the HSP70 family and the induction of mRNAs encoding the small HSP18 class of heat shock protein (HSP) was not affected. The level of HSP70/HSC70 proteins was significantly reduced in transgenic plants, but HSP18 was induced to the same level in different transgenic lines and in untransformed plants. The acquisition of thermotolerance was negatively affected in artisense plants, the survival temperature being 2 degrees C below the survival temperature of the wild type and other transgenic lines. Another major effect concerning the regulation of the endogenous heat shock transcription factor HSF was detected by testing the ability to form heterotrimers between authentic HSF and recombinant HSF-GUS (beta-glucuronidase) proteins. The shut-off time, required to turn of HSF activity during recovery from heat stress, was significantly prolonged in antisense plants compared with wild-type and other transgenic lines. Our results imply a dual role of HSP70 in plants, a protective role in thermotolerance and a regulatory effect on HSF activity and hence the autoregulation of the heat shock response.

Heat Shock Protein Induction in Montastraea faveolata and Aiptasia pallida Exposed to Elevated Temperatures.

Frequent widespread episodes of coral bleaching have made researchers aware of the sensitivity of reef corals to moderately elevated temperatures and led us to investigate mechanisms of temperature stress tolerance in this group. One such mechanism may be the induced synthesis of heat shock proteins (hsps), which have been shown to play a role in thermotolerance in other organisms. However, induced synthesis of hsps in scleractinian corals was not reported until recently. Experiments were conducted in which Montastraea faveolata was exposed to high temperatures (up to 35{deg}C) for short periods (2 h). Under the conditions tested, the corals produced seven different hsps with approximate molecular weights of 95, 90, 78, 74, 33, 28, and 27 kDa. Another zooxanthellate species, the sea anemone Aiptasia pallida, also synthesized hsps during temperature stress, but fewer and with different molecular weights (82, 72, 68, and 48 kDa) than those produced by Montastraea. It now remains to be determined whether hsps are involved in differences in thermotolerance and susceptibility to bleaching within and between the various species of Montastraea, and between species of reef cnidarians.

Expression and Function of the Trehalase Genes NTH1 and YBR0106 in Saccharomyces cerevisiae

The biological function of the trehalose-degrading yeast enzyme neutral trehalase consists of the control of the concentration of trehalose, which is assumed to play a role in thermotolerance, in germination of spores, and in other life functions of yeast. Resequencing of the neutral trehalase gene NTH1 on chromosome IV resulted in the observation of two possible start codons (Kopp, M., Nwaka, S., and Holzer, H. (1994) Gene (Amst.) 150, 403-404). We show here that only the most upstream start codon which initiates translation of the longest possible ORF is used for expression of NTH1 in vivo. A gene with 77% identity with NTH1, YBR0106, which was discovered during sequencing of chromosome II (Wolfe, K. H., and Lohan, A. J. E. (1994) Yeast 10, S41-S46), is shown here to be expressed into mRNA. Experiments with a mutant disrupted in the YBR0106 ORF showed, in contrast to a NTH1 deletion mutant, no changes in trehalase activity and in trehalose concentration. However, similar to the NTH1 gene a requirement of the intact YBR0106 gene for thermotolerance is demonstrated in experiments with the respective mutants. This indicates that the products of the likely duplicated YBR0106 gene and the NTH1 gene serve a heat shock protein function. In case of the YBR0106 gene, this is the only phenotypic feature found at present.

An Arabidopsis heat shock protein complements a thermotolerance defect in yeast.

The heat shock protein Hsp104 of the yeast Saccharomyces cerevisiae plays a key role in promoting survival at extreme temperatures. We found that when diverse higher plant species are exposed to high temperatures they accumulate proteins that are antigenically related to Hsp104. We isolated a cDNA corresponding to one of these proteins from Arabidopsis. The protein, AtHSP101, is 43% identical to yeast Hsp104. DNA gel blot analysis indicated that AtHSP101 is encoded by a single- or low-copy number gene. AtHsp101 mRNA was undetectable in the absence of stress but accumulated to high levels during exposure to high temperatures. When AtHSP101 was expressed in yeast, it complemented the thermotolerance defect caused by a deletion of the HSP104 gene. The ability of AtHSP101 to protect yeast from severe heat stress strongly suggests that this HSP plays an important role in thermotolerance in higher plants.

Specific binding of the transglutaminase, platelet factor XIII, to HSP27.

HSP27, the unique mammalian low molecular weight heat shock protein, is prominently phosphorylated upon activation of a wide variety of cells and has a role in thermotolerance, growth events, and regulation of actin cytoskeletal dynamics. In thrombin-stimulated platelets, HSP27 is rapidly and prominently phosphorylated in a manner highly correlated with platelet secretion. However, the function of HSP27 and the identity of proteins that interact with HSP27 remain unknown. To identify specific HSP27-protein interactions, a recombinant fusion protein affinity reagent was constructed and used to identify proteins associating with HSP27 from human platelet lysates and erythroleukemia cells. An 84-kDa protein was found to associate specifically with HSP27 and was isolated from platelet lysates, resolved on preparative gels, transferred to nitrocellulose, subjected to enzymatic digestion, and microsequenced. A 20-amino acid sequence derived from p84 proved identical to amino acids 484-503 of the transglutaminase, platelet Factor XIII. Immunoblotting studies were used to confirm the binding of FXIII from fresh platelet lysates to the HSP27 fusion protein. FXIII also was shown to coprecipitate with HSP27 in immunoprecipitation studies and to colocalize with HSP27 in immunofluorescence studies of intact glass-activated platelets. The data thus demonstrate specific binding of platelet FXIII to HSP27 and suggest that HSP27 may participate in the cellular localization and/or enzymatic regulation of platelet FXIII.

レーザーサイトメーターを用いたheat shock protein 72の加温および抗癌剤添加による動態の検討

Heat shock proteins (HSPs) are regarded as the proteins most related to the development of thermotolerance. Recently, not only their role in thermotolerance, but also their role in resistance to anticancer agents is gathering concern. In this study, the kinetics of hsp 72 in HeLa cells treated with heating and/or anticancer agents were studied. Hsp 72 was immunostained by the indirect fluorescent technique using a monoclonal anti-hsp 72 antibody (Amersham). The staining pattern was observed and analyzed using a laser cytometer, ACAS 570 (Meridian). Hsp 72 was normally found in the cytoplasm at 37°C and moved rapidly into the nucleus with heating at 43°C for 2 hours. It then returned to the cytoplasm 4 to 6 hours after heating. The hsp 72 content reached a peak at 8 hours after heating. Hsp 72 was induced in all cells treated with cisplatin, adriamycin, peplomycin, or etoposide for 48 hours. In the cells treated with both heating at 43°C for 2 hours and these anticancer agents, hsp 72 induction was most suppressed by adriamycin. However, translocation of hsp 72 to the nucleus was specific for heating and was not affected by the anticancer agents. By laser cytometry the intracellular localization of hsp 72 and the changes of its content were simultaneously detected. Moreover, the change pattern of hsp 72 content measured by laser cytometry coincided with that measured by the Western blotting procedure.

Biological and clinical implications of heat shock protein 27,000 (Hsp27): a review.

Heat shock and other environmental and pathophysiologic stresses stimulate synthesis of heat shock proteins (Hsps). These proteins enable the cell to survive and recover from stressful conditions by as yet uncompletely understood mechanisms. Hsp27 is an important small Hsp (molecular weight, 27,000) found in human cells--both cancer cells and normal cells. This protein, besides its putative role in thermotolerance, is of special clinical interest because of recent data suggesting it may also play a role in drug resistance. In adults, Hsp27 is found particularly in several cell types such as breast, uterus, cervix, placenta, skin, and platelets. Although low-molecular-weight (small) Hsps have been found to be involved in embryogenesis of Xenopus and Drosophila, they have not been detected in human fetal organs. Regulation of expression of the Hsp gene (also known as HSPB1) has been considered a paradigm of gene regulation and is actively being studied in both prokaryotes and eukaryotes. In prokaryotes, the major Hsp genes are transcriptionally regulated by positively and negatively acting transcription factors. In eukaryotes, the genes encoding Hsps contain a regulatory DNA motif (inverted repeats of the pentameric sequence nGAAn) known as the heat shock element. Hsp27 may function as a molecular chaperone and in signal transduction pathways of different cell regulators, and Hsp27 and other Hsps may be active in development of resistance to stressful conditions and agents including cytotoxic drugs. Study findings indicate that some but not all estrogen-positive breast cancers express Hsp27, and overexpression of Hsp27 has been associated with both good and poor prognosis. In endometrial carcinomas, the presence of Hsp27 is correlated with the degree of tumor differentiation as well as with the presence of estrogen and progesterone receptors. Studies suggest, however, that detection of Hsp27 should not be considered to be a method for identifying hormone-responsive tumors or detecting estrogen receptors. Hsp27 seems to be a biochemical marker of estrogenic endometrial response. In patients with cervical cancer, Hsp27 is predominantly expressed in well-differentiated and moderately differentiated squamous cell carcinomas. In addition, expression of Hsp27 seems to be a negative prognostic factor for gastric cancer. Different isoforms of Hsp27 have been found in lymphoid tissue of patients with acute lymphoblastic leukemia, and the protein has also been associated with viral infections. These aspects are summarized and discussed in the present review.

Genetic evidence for a functional relationship between Hsp104 and Hsp70

The phenotypes of single Hsp104 and Hsp70 mutants of the budding yeast Saccharomyces cerevisiae provide no clue that these proteins are functionally related. Mutation of the HSP104 gene severely reduces the ability of cells to survive short exposures to extreme temperatures (thermotolerance) but has no effect on growth rates. On the other hand, mutations in the genes that encode Hsp70 proteins have significant effects on growth rates but do not reduce thermotolerance. The absence of a thermotolerance defect in S. cerevisiae Hsp70 mutants is puzzling, since the protein clearly plays an important role in thermotolerance in a variety of other organisms. In this report, examination of the phenotypes of combined Hsp104 and Hsp70 mutants uncovers similarities in the functions of Hsp104 and Hsp70 not previously apparent. In the absence of the Hsp104 protein, Hsp70 is very important for thermotolerance in S. cerevisiae, particularly at very early times after a temperature upshift. Similarly, Hsp104 plays a substantial role in vegetative growth under conditions of decreased Hsp70 protein levels. These results suggest a close functional relationship between Hsp104 and Hsp70.

Methionine-mediated lethality in yeast cells at elevated temperature.

Saccharomyces cerevisiae cells grown at 30 degrees C in minimal medium containing methionine lose viability upon transfer to 45 degrees C, whereas cells grown in the absence of methionine survive. Cellular levels of two intermediates in the sulfate assimilation pathway, adenosine 5'-phosphosulfate (APS) and adenosine 5'-phosphosulfate 3'-phosphate, are increased by a posttranslational mechanism after sudden elevation of temperature in yeast cultures grown in the absence of methionine. Yeast cells unable to synthesize APS because of repression by methionine or mutation of the MET3 gene do not survive the temperature shift. Thus, methionine-mediated lethality at elevated temperature is linked to the inability to synthesize APS. The results demonstrate that APS plays an important role in thermotolerance.

Changes in erythrocyte membranes after fractioned hyperthermia

The structural changes in erythrocytes membranes were examined before and after the second heat shock of erythrocytes. Electrophoretic separation of protein erythrocyte membranes for cells incubated at 48.5°C was different from control i.e. from erythrocytes incubated at 37°C. No quantitative or qualitative changes were spotted in comparison with protein membranes isolated from the erythrocytes following single or double heat shock. Fluidity of erythrocytes membranes was determined by using spin labels, 5-doxylstearic acid and 16-doxylstearic acid. The membranes were more rigid in their hydrophobic regions after incubation of cells at 44°C. It can be suggested that erythrocyte membranes play some role in thermotolerance and heat damage of enuclate cells.

Heat shock proteins and thermotolerance in a cultured cell line from the Mediterranean fruit fly, Ceratitis capitata.

Heat shock proteins (hsps) were identified in a cell line from the Mediterranean fruit fly, Ceratitis capitata Wiedemann (Diptera: Tephritidae) exposed to elevated temperatures. Cells produced three hsps (Mr 87,000, 69,000, and 34,000) in response to a temperature shift from 26 degrees C to 37 degrees C (30-60 min) with a concomitant decrease in synthesis of most other cellular proteins. Synthesis of low Mr hsps was not evident. The heat shock response is triggered within 30 min at temperatures from 33 degrees C to 41 degrees C. At temperatures greater than 41 degrees C protein synthesis was shut down. Within 2-3 h after return to 26 degrees C, synthesis of proteins repressed at the higher temperatures resumed production while the major hsps disappear. Heat shock proteins were not produced in the presence of actinomycin D. Evaluations on the role of hsps in conferring thermotolerance to the cells showed an increase in cell viability in heat-shocked cells over non-heat-shocked cells (after 3 and 10 days) when subsequently placed at 45 degrees C for 1 h, a normally lethal temperature. Heat shock alone had little effect on subsequent cell viability or growth at 26 degrees C. These results suggest that hsps produced by these cells may aid in the maintenance of cell integrity and thus play a transitory role in thermotolerance.

Effect of hyperthermia on selective expression of HL-60 heat shock proteins

Hyperthermia is used experimentally to treat human malignancy. The effect of heat delivery rate and thermotolerance on the expression of heat shock proteins (hsp) by the human HL-60 cell line before and after differentiation was studied. This leukemia cell synthesized multiple hsp in response to elevated temperatures. The most obvious and consistent proteins were within the highly conserved stress-inducible family of polypeptides hsp70 which resolved as a hsp69/72 doublet. Cells which were made thermotolerant by gradual heating selectively failed to express the hsp70 doublet even though other hsp were synthesized. Mature HL-60 cells induced to differentiate by incubation in retinoic acid expressed a full complement of hsp when exposed to immediate heat, but there was selective deletion of hsp70 with gradual hyperthermia. This model for selective induction of hsp confirms that synthesis of hsp and thermotolerance can be dissociated in the HL-60. It suggests that the hsp70 does not play an obligatory role in thermotolerance of this human leukemia cell.

Involvement of rRNA synthesis in the enhanced survival and recovery of protein synthesis seen in thermotolerance.

Although acquired thermotolerance has been linked to the induction of heat shock proteins, the molecular mechanism(s) by which cells become resistant to heat is unknown. The present study shows a strong correlation between the survival of cells following heat shock and the rate of recovery of protein, total RNA, and rRNA synthesis. Increasing exposure of CHO cells to 45 degrees C was found to decrease survival and cause a lengthening delay in these synthetic processes. The same reciprocal correlation was seen in thermotolerant cells. As thermotolerance develops, more cells survive a heat challenge and the delay in synthesis decreases. These data argue that enhanced recovery of protein and RNA synthesis is one factor which plays a key role in thermotolerance. The involvement of rRNA synthesis was further investigated by using actinomycin D at 0.1 microgram m1(-1), a concentration at which rRNA synthesis is selectively inhibited. When the drug was present during the recovery from a challenge heat treatment, the survival of thermotolerant cells was approximately 3-fold lower than expected from the mild toxicity of the drug. As this could not be accounted for by an interaction of the drug with the response of cells to single heat treatments, it is concluded that the drug inhibits the expression of thermotolerance in cells which would otherwise express a full degree of thermotolerance. The time and concentration dependence of this effect indicates that the drug acts though inhibition of rRNA synthesis. Therefore, enhanced recovery of RNA synthesis, presumably rRNA synthesis, is identified as one of the mechanisms responsible for enhanced survival of thermotolerant cells following heat shock.