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Enhancement of expression of stress proteins by agents that lower the levels of glutathione in cells
Biochimica et Biophysica Acta 1397 Ž1998. 223–230
Enhancement of expression of stress proteins by agents that lower the levels of glutathione in cells Hidenori Ito, Keiko Okamoto, Kanefusa Kato
)
Department of Biochemistry, Institute for DeÕelopmental Research, Aichi Human SerÕice Center, Kamiya, Kasugai, Aichi 480-03, Japan Received 21 November 1997; accepted 8 January 1998
Abstract The effects of diethyl maleate and buthionine sulfoximine, agents that lower cellular levels of glutathione, on expression of hsp27 and a B crystallin in response to stress were studied. When C6 rat glioma cells were treated with 100 m M arsenite for 1 h, accumulation of the two proteins, estimated by specific immunoassays, was markedly enhanced by additional exposure to 1 mM diethyl maleate or 2.5 mM buthionine sulfoximine. The latter also increased heat- and CdCl 2-induced accumulation of hsp27 and a B crystallin. Stress-induced accumulation of hsp70, estimated by Western blotting analysis, was also enhanced by these agents. Northern blotting analysis revealed increase in levels of mRNAs for hsp27, a B crystallin and hsp70. The period of heat shock element ŽHSE.-binding activity of heat shock factor ŽHSF. stimulated by arsenite was extended by addition of diethyl maleate and buthionine sulfoximine. The induced phosphorylated state of HSF1 was also prolonged by diethyl maleate. Although exposure of cells to diethyl maleate alone for 1 h caused neither accumulation of hsp27, a B crystallin and hsp70 nor expression of mRNAs for these proteins, HSE-binding activity of HSF was stimulated. However, the activated HSF was not phosphorylated. These results suggest that diethyl maleate induces an intermediate state of HSF that binds to HSE but is transcriptionally inert. The mechanism is unclear but the levels of glutathione in cells that were exposed to diethyl maleate or buthionine sulfoximine were markedly decreased. q 1998 Elsevier Science B.V. Keywords: Stress response; Small heat shock protein; Glutathione; Redox; hsp27; Crystallin
1. Introduction The cellular response to stress is well conserved. Physical and chemical stresses induce the expression of stress responsive genes whose products are named heat shock or stress proteins Ž hsps. . The hsps are
) Corresponding author. Department of Biochemistry, Institute for Developmental Research, Aichi Human Service Center, 713-8, Kamiya, Kasugai, Aichi 480-03, Japan. Fax: q81-568-88-0811; E-mail: [email protected]
highly conserved and are elicited by a variety of stresses including heat, amino acid analogues, heavy metals, oxidative agents or thiol-reactive species w1,2x. The underlying mechanisms are thought to be as follows: heat shock transcription factor 1 ŽHSF1., which exists as a monomer in normal cells polymerizes to form a trimer in response to stress, then binds to a consensus sequence, the heat shock element ŽHSE. , located in the promoter regions of hsp genes w3x. Recently, several studies have revealed a relationship between the stress response and redox processes.
0167-4781r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 7 - 4 7 8 1 Ž 9 8 . 0 0 0 1 0 - 4
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For example, Caltabiano et al. w4x reported that thiol reactive agents Ž p-chloromercuribenzoate and iodoacetamide. can induce stress proteins in human or murine melanoma cells and Chen et al. w5x described an increase in synthesis of hsp70 in an epithelial cell line, LLC-PK1, due to reactive electrophiles. Huang et al. w6x further reported inhibition of the heat shock response in human HeLa and mouse C2C12 myogenic cells by a high concentration Ž 2 mM. of dithiothreitol. Iodoacetamide w7x or diamide w8x can activate the heat shock transcription factor, resulting in induction of hsp70 protein. We previously reported that a high concentration of dithiothreitol Ž2 mM. inhibits the arsenite-induced stress responses of hsps, whereas a low concentration Ž 0.03–0.1 mM. causes enhancement w9x. Moreover, Mehlen et al. w10x found that overexpression of hsp27 in mouse L929 fibrosarcoma cells resulted in a decrease of reactive oxygen species and an increase in the levels of glutathione w10x. The present study was conducted to determine the effect of glutathione-depleting drugs such as diethyl maleate, a compound that binds the free thiol groups w11x, and buthionine sulfoximine, a specific and essentially irreversible inhibitor of g-glutamyl-cysteine synthetase w12x, on stress-induced expression of hsp27, a B crystallin and hsp70.
that were preincubated for 10–15 min in 5% CO 2 in air with or without diethyl maleate or buthionine sulfoximine were floated in a water bath at 428C for 30 min. Then the medium was replaced with the standard culture medium and cells were cultured at 378C until harvesting. Cells in each dish were washed twice with phosphate-buffered saline Ž PBS, containing 8 g of NaCl, 0.2 g of KCl, 1.15 g of Na 2 HPO4 , and 0.2 g of KH 2 PO4 in 1000 ml of H 2 O. and stored frozen at y208C for a few days prior to analysis. The frozen cells on each dish were collected and suspended in 0.3 ml of PBS, and each suspension was sonicated and centrifuged at 125,000 = g for 20 min
2. Materials and methods 2.1. Cell culture and treatment C6 cells Žobtained from the Japanese Cancer Research Resources Bank, Tokyo. were grown in Dulbecco’s modified Eagle’s medium Ž DMEM; Nissui Pharmaceutical, Tokyo, Japan., supplemented with 10% fetal calf serum ŽLife Technologies, Tokyo, Japan. at 378C in a humidified atmosphere of 95% air and 5% CO 2 . The cells were seeded on 35-mm dishes for analysis of proteins, on 60-mm dishes for assays of the binding of HSF to HSEs and on 90-mm dishes for extraction of RNA for Northern blotting analysis of mRNAs. In each case the medium was changed every 2 or 3 days. When cells reached confluence, they were exposed to NaAsO 2 or CdCl 2 at 378C for 1 h in the presence or absence of diethyl maleate or buthionine sulfoximine Ž Nacalai Tesque, Kyoto, Japan. . For heat treatment, dishes with cells
Fig. 1. Enhancement by diethyl maleate ŽDM. and buthionine sulfoximine ŽBS. of arsenite- ŽA., heat- or CdCl 2 ŽB.-induced accumulation of hsp27 and a B crystallin in C6 cells. ŽA. C6 cells were exposed to 100 m M arsenite in the presence or absence of 1 mM diethyl maleate ŽDM. or 2.5 mM buthionine sulfoximine ŽBS. for 1 h and then cultured for 16 h in the standard medium. ŽB. Cells were exposed to heat Ž428C for 30 min, lanes 2–4. or 100 m M CdCl 2 Žlanes 5–7. in the presence or absence Žlanes 2 and 5. of 0.3 mM Žlanes 3 and 6. or 1.0 mM Žlanes 4 and 7. diethyl maleate for 1 h and then cultured in the standard medium for 16 h. Lane 1: untreated control cells. Concentrations of hsp27 and a B crystallin were determined by specific immunoassays and each column and bar show the mean "SD of the results for five dishes.
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in 12.5% Ž for analysis of hsp27 and a B crystallin. or 10% Žfor analysis of hsp70. polyacrylamide slab gels. Western blotting analysis of hsp27 and a B crystallin was carried out as described previously w16x using affinity-purified antibodies Ž0.05 m grml. raised in rabbits against rat hsp27 or against the carboxyterminal decapeptide of a B crystallin and peroxidase-labeled antibodies raised in goats against rabbit IgG as second antibodies. For detection of hsp70, we used a mouse monoclonal antibody specific for the inducible form of hsp70 Ž C92F3A-5; StressGen Biotechnologies, Victoria, BC, Canada. . The second antibody was against mouse IgG ŽMedical and Biological Laboratories, Nagoya, Japan. . Peroxidase activity on nitrocellulose sheets was visualized on X-ray
Fig. 2. Effects of diethyl maleate on arsenite-induced accumulation of hsp27 and a B crystallin in C6 cells. ŽA. Cells were exposed to 100 m M arsenite in the presence Žsolid circles. or absence Žopen circles. of 1 mM diethyl maleate for 1 h and then cultured in the standard medium for the indicated periods of time. Open squares: untreated control cells. Each point and vertical bar show the mean"SD of the results for three to four dishes. ŽB. Cells were exposed to 100 m M arsenite in the presence or absence of the indicated concentrations of diethyl maleate ŽDM. for 1 h and then cultured in the standard medium for 16 h. Each column and bar show the mean"SD of the results for three to four dishes.
at 48C. The supernatants were used for assays of hsp27, a B crystallin and hsp70. 2.2. Immunoassays of hsp27 and a B crystallin Concentrations of hsp27 w13x and a B crystallin w14x in extracts of cells were determined by specific immunoassays, as described previously. 2.3. Electrophoresis and Western blotting analysis of hsp27, a B crystallin, and hsp70 SDS-polyacrylamide gel electrophoresis Ž SDSPAGE. was performed as described by Laemmli w15x
Fig. 3. Effects of 1 mM diethyl maleate ŽA. or 2.5 mM buthionine sulfoximine ŽB. on the accumulation of hsp27 and a B crystallin in response to various concentrations of arsenite. Cells were exposed to the indicated concentrations of arsenite in the presence or absence of 1 mM diethyl maleate ŽDM; A. or 2.5 mM buthionine sulfoximine ŽBSO; B. for 1 h and then cultured in the standard medium for 16 h. Each column and bar show the mean"SD of the results for three to four dishes.
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films using a Western blotting chemiluminescence reagent Ž Renaissance; Dupont NEN, Boston, MA, USA..
2.4. Isolation of RNA and Northern blotting analysis Total RNA was isolated from cells with a QuickPrep Total RNA Extraction kit ŽPharmacia Biotech, Tokyo, Japan. . The 20-m g samples of total RNA were subjected to electrophoresis on 0.9% agarose– 2.2 M formaldehyde gels and blotted onto nitrocellulose membranes w17x. For Northern blotting analysis, membranes were allowed to hybridize with cDNA probes for hsp27 Ž BamHI–HindIII fragment of cDNA for mouse hsp27, kindly provided by Dr. L.F. Cooper, University of North Carolina. w18x, a B crystallin Ž Pst I fragment of cDNA for bovine a B crystallin, kindly provided by Dr. H. Bloemendal, University of Nijmegen. w19x and hsp70 Ž EcoRI fragment of cDNA for human hsp70, obtained from StressGen Biotechnologies., which were labeled with a Multiprime DNA labeling system ŽAmersham, Buckinghamshire, UK., as described previously w20x.
2.5. Gel mobility shift assays and Western blotting analysis of HSF Cells were suspended at 08C in 20 mM HEPES– KOH buffer, pH 7.9, containing 25% Ž vrv. glycerol, 0.5 M NaCl, 1.5 mM MgCl 2 , 0.2 mM EDTA, 0.5 mM dithiothreitol, 0.5 mgrml pepstatin A, and 20 m grml PefaBlock SC ŽMerck, Darmstad, Germany.. The suspensions were sonicated at 08C for 20 s and then centrifuged at 48C at 125,000 = g for 10 min. Supernatants were collected and aliquots were frozen in liquid N2 and stored at y808C. For Western blotting analysis of HSF, aliquots of extracts containing 40 m g of protein were subjected to SDS-PAGE in 6% polyacrylamide slab gels. HSF1 was detected with rabbit antiserum raised against HSF1, which was kindly provided by Dr. A. Nakai Ž Kyoto University.. For gel mobility shift assays, aliquots of cell extracts containing 15 m g of protein were incubated for 20 min at 08C with 0.1 ng of a 32 P-labeled oligonucleotide that included the sequence of the HSE from the promoter of the gene for hsp70 in Drosophila Ž5XGCCTCGAATGTTCGCGAAGTTTCG-3X . w32x and 0.5 m g of polyŽ dI–dC. Ž Pharmacia Biotech. in 10
Fig. 4. Western blotting analysis of levels of hsp70, hsp27 and a B crystallin ŽA. and Northern blotting analysis of the respective mRNAs ŽB. in C6 cells. ŽA. Cells were exposed to 1 mM diethyl maleate Žlanes 3 and 4., or 100 m M arsenite Žlanes 5 and 6. in the presence of 0.3 mM Žlanes 7 and 8. or 1 mM Žlanes 9 and 10. of diethyl maleate for 1 h and then cultured for 16 h in the standard medium. Extracts of cells containing 20 m g of protein were subjected to SDS-PAGE followed by Western blotting analysis with antibodies against hsp70, hsp27, and a B crystalline, as indicated. Lanes 1 and 2: untreated control cells. ŽB. Cells were exposed to 1 mM diethyl maleate alone Žlane 2., or 100 m M arsenite in the presence Žlane 4. or absence Žlane 3. of 1 mM diethyl maleate for 1 h and then cultured for 6 h in the standard medium. A total of 20-m g of the total RNA from each sample were analyzed by Northern blotting with cDNA probes for hsp70, hsp27 and a B crystallin. Lane 1: control cells. Bands of 28S RNA are shown for reference.
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mM Tris–HCl buffer, pH 8.0, containing 50 mM NaCl, 1 mM EDTA, 5% glycerol, and 0.5 mM dithiothreitol in a final volume of 25 m l. The reaction mixtures were subjected to electrophoresis on 4% polyacrylamide gels and the dried gels were autoradiographed with X-ray films.
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cules, CA, USA. using bovine serum albumin as the standard. Rat hsp27 and a B crystallin, which were used as the standards for immunoassays and electrophoresis, were purified from rat skeletal muscle w13,23x.
2.6. Quantitation of glutathione and protein thiols Reduced glutathione and oxidized glutathione were determined using enzymatic recycling and the Ellman’s reagent as described by Eyer and Podhradsky w21x. Protein thiols were determined using Ellman’s reagent as described by Matsumura and Matthews w22x. 2.7. Other methods Concentrations of protein in soluble extracts were estimated with a protein assay kit Ž Bio-Rad, Her-
3. Results 3.1. Diethyl maleate and buthionine sulfoximine enhance stress-induced accumulation of hsp27 and a B crystallin Levels of hsp27 and a B crystallin in C6 cells in confluent culture were low. Exposure of cells to 100 m M arsenite for 1 h and subsequent culture in the
Fig. 5. Effects of diethyl maleate on activation of HSF. ŽA. Cells were exposed to 1 mM diethyl maleate ŽDM., 100 m M arsenite or 100 m M arsenite plus 1 mM diethyl maleate for 1 h and cultured in the standard medium for the indicated periods of time. Extracts of cells containing 15 m g of protein were subjected to gel mobility shift assays of HSF. Lane C: untreated control cells. Arrowheads: specific binding. Ns: nonspecific binding. ŽB. Cells were exposed to 1 mM diethyl maleate ŽDM., 100 m M arsenite or 100 m M arsenite plus 1 mM diethyl maleate for 1 h and then cultured in the standard medium for the indicated periods of time. Extracts of cells containing 40 m g of protein were subjected to SDS-PAGE followed by Western blotting with antibody against HSF1. Lane C: untreated control cells.
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normal medium at 378C for 16 h increased both ŽFig. 1A.. When cells were exposed to arsenite in the presence of 1 mM diethyl maleate or 2.5 mM buthionine sulfoximine, accumulation of the two proteins was enhanced ŽFig. 1A. . This was particularly the case with diethyl maleate. However, exposure of cells to these reagents alone for 1 h did not significantly increase the levels of either of the proteins ŽFig. 1A.. Diethyl maleate also enhanced heat- or CdCl 2-induced accumulation of hsp27 and a B crystallin although it suppressed CdCl 2-induction of a B crystallin ŽFig. 1B. . The levels of hsp27 and a B crystallin reached a maximum at 16 h after arsenite treatment regardless of the presence or absence of diethyl maleate Ž Fig. 2A.. Stimulatory effects of diethyl maleate on the arsenite-induced accumulation of hsp27 and a B crystallin were observed even at 0.1 mM, with the maximum effect seen at 0.3–1 mM Ž Fig. 2B. . In contrast, the effects of buthionine sulfoximine were similar at concentrations between 0.5 mM and 5 mM under the conditions Ždata not shown.. The extent of stimulation by diethyl maleate or buthionine sulfoximine was greatest when the concentration of arsenite was 50–100 m M ŽFig. 3A and B.. 3.2. Western and Northern blotting analyses of hsp27, a B crystallin and hsp70 To determine whether the arsenite-induced accumulation of hsp70, another representative hsp, was also affected by diethyl maleate, Western blotting analysis was performed. It was confirmed that enhancement of arsenite-induced hsp27 and a B crystallin accumulation was accompanied by a similar stimulation with regards to hsp70 ŽFig. 4A.. Heat-induced accumulation of hsp70 was also enhanced by diethyl maleate and buthionine sulfoximine demonstrated equivalent influence on accumulation of hsp70 in both arsenite and heat stresses Ž data not shown.. Treatment with diethyl maleate alone did not cause accumulation of any of the three proteins. Ž Fig. 4A.. Northern blotting analysis indicated that the arsenite-induced expression of mRNAs for hsp70, hsp27 and a B crystallin in C6 cells was enhanced by diethyl maleate but treatment with diethyl maleate
alone did not induce the expression of mRNAs for three proteins ŽFig. 4B. . Arsenite-induced increase in levels of mRNAs for the three proteins was also augmented by buthionine sulfoximine Ždata not shown.. 3.3. Effects of diethyl maleate and buthionine sulfoximine on HSF actiÕation When cells were exposed to 100 m M arsenite for 1 h and subsequent culture at 378C in the standard medium, HSE-binding activity of HSF in cell extracts was observed, reaching a maximum within 2 h and then decreases Ž Fig. 5A. . In contrast, when cells were exposed to 100 m M arsenite for 1 h with 1 mM diethyl maleate, HSE-binding activity of HSF in whole cell extract reached a maximum within 4 h and was sustained for a longer period Ž Fig. 5A. . A similar prolongation of HSE-binding activity of HSF by buthionine sulfoximine was also observed Ž Fig. 6. . Significant HSE-binding activity was detected in extracts of cells that were exposed to 1 mM diethyl maleate alone for 1 h ŽFig. 5A.. It has been shown that HSF1 is phosphorylated by heat or other stress w3x. We, therefore, estimated the effects of diethyl maleate on the phosphorylation state of HSF. The phosphorylated form of HSF1 with a high molecular mass Žabout 90 kDa. was detected
Fig. 6. Effects of buthionine sulfoximine on activation of HSF. Cells were exposed to 2.5 mM buthionine sulfoximine ŽBSO., 100 m M arsenite or 100 m M arsenite plus 2.5 mM buthionine sulfoximine for 1 h and then cultured in the standard medium for the indicated periods of time. Extracts of cells containing 15 m g of protein were subjected to gel mobility shift assays of HSF. Lane C: untreated control cells. Arrowheads: specific binding. Ns: nonspecific binding.
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Table 1 Effects of exposure to diethyl maleate and buthionine sulfoximine on the levels of glutathione ŽGSH. and protein thiols ŽPSH. in cells Treatment
GSH Žnmolrmg protein.
PSH Žnmolrmg protein.
control diethyl maleate Ž1 mM. buthionine sulfoximine Ž2.5 mM. arsenite Ž100 m M. arsenite plus diethyl maleate arsenite plus buthionine sulfoximine
15.8 " 0.47) 3.49 " 0.18 3.42 " 0.26 19.7 " 1.13 5.09 " 0.12 3.74 " 0.09
93 " 4) 127 " 6 105 " 1 88 " 15 126 " 32 124 " 26
Cells were exposed to 100 m M arsenite for 30 min in the presence or absence of 1 mM diethyl maleate or exposed to 2.5 mM buthionine sulfoximine for 2 h with or without 100 m M arsenite during the last 30 min. Cells were then washed three times with PBS and frozen at y808C until analysis. Concentrations of glutathione ŽGSH. and protein thiols ŽPSH. were determined as described in Section 2 and each value is expressed as nmolrmg soluble protein. )Mean" SD of values for three dishes.
in cells that were exposed to arsenite or arsenite plus diethyl maleate; and diethyl maleate prolonged the arsenite-induced phosphorylated state of HSF1 for a longer period ŽFig. 5B. . In contrast, it was not observed in cells that were exposed for 1 h to diethyl maleate alone ŽFig. 5B.. 3.4. Effects of exposure to diethyl maleate or buthionine sulfoximine on leÕels of glutathione and protein thiols in cells Diethyl maleate is a compound that binds to free thiol groups and buthionine sulfoximine is a specific and essentially irreversible inhibitor of g-glutamylcysteine synthetase w12x. Exposure of cells to 100 m M arsenite for 30 min increased the level of glutathione slightly compared to that in control cells ŽTable 1. . When cells were exposed to 1 mM diethyl maleate for 30 min or 2.5 mM buthionine sulfoximine for 2 h, with or without 100 m M arsenite, the levels of glutathione in cells were decreased regardless of the presence or absence of arsenite. However, levels of protein thiols in cells were barely affected by diethyl maleate and buthionine sulfoximine under the conditions ŽTable 1..
4. Discussion The present study demonstrated that the glutathione-depleting agents such as diethyl maleate and buthionine sulfoximine, enhanced the stress-induced
responses of C6 cells, while themselves neither increasing the expression of mRNAs for hsps nor their accumulation. Glutathione has numerous functions which include reactive oxygen species detoxification w24x and regulation of cell death w25x. We showed that the stress responses of cells to insult by arsenite, CdCl 2 or heat is markedly enhanced under reduced cellular glutathione conditions. In addition, diethyl maleate and buthionine sulfoximine prolonged the arsenite-induced HSE-binding activity of HSF and its phosphorylated state Ž Fig. 5B.. Although the exposure to diethyl maleate alone caused HSF to bind to HSE ŽFig. 5A. , it was not phosphorylated Ž Fig. 5B. and transcriptionally inert. It has been shown that the binding of HSF to HSE does not always result in the activation of the transcription of genes for hsps. For example, an alkaline shift in pH of the culture w26x, oxidative injury w27x, and anti-inflammatory drugs, namely, salicylate and indomethacin w28,29x, activate HSF binding to HSE, but do not induce hsp transcription. The present results suggest that diethyl maleate may induce an intermediate state of HSF that is bound to HSE but is transcriptionally inert, similar to that reported for salicylate and indomethacin by Cotto et al. w29x. Such an intermediate state of HSF might be expected to becomes transcriptionally active in the presence of arsenite or other stress, and together with the directly activated HSF synergistically contribute to transcription of hsp genes. It has been suggested that loss of redox control with a shift to an oxidative state due to reaction of
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arsenite with protein thiols might trigger expression of hsps w30,31x. However, in the present experiment, exposure of cells to arsenite did not decrease the cellular levels of glutathione and protein thiols but rather caused a slight increase when expressed on the basis of mg soluble protein under the assay conditions. In contrast, exposure of cells to diethyl maleate or buthionine sulfoximine decreased the levels of glutathione in cells regardless of the presence or absence of arsenite ŽTable 1. . These results suggest that stress responses are not elicited solely by reduction in the levels of glutathione in cells, but that such decrease acts to strongly enhance the reaction to arsenite, CdCl 2 and heat. Acknowledgements This work was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Science and Culture, Japan. References w1x L. Nover, in: L. Nover ŽEd.., Heat Shock Response, CRC Press, Boca Raton, FL, 1991, pp. 5–40. w2x L. Nover, K.-D. Scharf, in: L. Nover ŽEd.., Heat Shock Response, CRC Press, Boca Raton, FL, 1991, pp. 41–128. w3x P.K. Sorger, Cell 65 Ž1991. 363–366. w4x M.M. Caltabiano, T.P. Koestler, G. Poste, R.G. Greig, J. Biol. Chem. 261 Ž1986. 13381–13386. w5x Q. Chen, K. Yu, J.L. Stevens, J. Biol. Chem. 267 Ž1992. 24322–24327. w6x L.E. Huang, H. Zhang, S.W. Bae, A.Y.-C. Liu, J. Biol. Chem. 48 Ž1994. 30718–30725. w7x H. Liu, R. Lightfoot, J.L. Stevens, J. Biol. Chem. 271 Ž1996. 4805–4812. w8x M.L. Freeman, M.J. Borrelli, K. Syed, G. Senisterra, D.M. Stafford, J.R. Lepock, J. Cell Physiol. 164 Ž1995. 356–366. w9x K. Kato, H. Ito, K. Okamoto, Cell Stress and Chaperones 2 Ž1997. 199–209.
w10x P. Mehlen, C. Kretz-Remy, X. Preville, A.-P. Arrigo, EMBO ´ J. 15 Ž1996. 2695–2706. w11x R.H. Ku, R.E. Billings, Arch. Biochem. Biophys. 183 Ž1986. 183–189. w12x O.W. Griffith, J. Biol. Chem. 257 Ž1982. 13704–13717. w13x Y. Inaguma, S. Goto, H. Shinohara, K. Hasegawa, K. Ohshima, K. Kato, J. Biochem. 114 Ž1993. 378–384. w14x K. Kato, H. Shinohara, N. Kurobe, Y. Inaguma, K. Shimizu, K. Ohshima, Biochim. Biophys. Acta 1074 Ž1991. 201–208. w15x U.K. Laemmli, Nature 227 Ž1970. 680–685. w16x K. Kato, S. Goto, K. Hasegawa, H. Shinohara, Y. Inaguma, Biochim. Biophys. Acta 1175 Ž1993. 257–262. w17x G.M. Wahl, M. Stern, G.R. Stark, Proc. Natl. Acad. Sci. U.S.A. 76 Ž1979. 3683–3687. w18x L.F. Cooper, K. Uoshima, J. Biol. Chem. 269 Ž1994. 7869– 7873. w19x Y. Quax-Jeuken, W. Quax, G. van Rens, P.M. Khan, H. Bloemendal, Proc. Natl. Acad. Sci. U.S.A. 82 Ž1985. 5819– 5823. w20x H. Ito, K. Hasegawa, Y. Inaguma, O. Kozawa, K. Kato, J. Cell. Physiol. 166 Ž1996. 332–339. w21x P. Eyer, D. Podhradsky, Anal. Biochem. 153 Ž1986. 57–66. w22x M. Matsumura, B.W. Matthews, Methods Enzymol. 202 Ž1991. 336–356. w23x K. Kato, H. Shinohara, S. Goto, Y. Inaguma, R. Morishita, T. Asano, J. Biol. Chem. 267 Ž1992. 7718–7725. w24x A. Meister, M.E. Anderson, Annu. Rev. Biochem. 52 Ž1983. 711–760. w25x D.J. Kane, T.A. Sarafian, R. Anton, H. Hahn, E.B. Gralla, J.S. Valentine, T. Ord, D.E. Bredesen, Science 262 Ž1993. 1274–1277. w26x P.G. Petronini, R. Alfieri, C. Campanini, A.F. Borghetti, J. Cell. Physiol. 162 Ž1995. 322–329. w27x J.L. Bruce, B.D. Price, C.N. Coleman, S.K. Calderwood, Cancer Res. 53 Ž1993. 12–15. w28x D.A. Jurivich, L. Sistonen, R.A. Kroes, R.I. Morimoto, Science 255 Ž1992. 1243–1245. w29x J.J. Cotto, M. Kline, R.I. Morimoto, J. Biol. Chem. 271 Ž1996. 3355–3358. w30x N.S. Klemperer, C.M. Pickart, J. Biol. Chem. 264 Ž1989. 19245–19252. w31x R.P. Beckmann, M. Lovett, W.J. Welch, J. Cell Biol. 117 Ž1992. 1137–1150. w32x M.J. Blake, R. Udelsman, G.J. Feulner, D.D. Norton, N.J. Holbrook, Proc. Natl. Acad. Sci. U.S.A. 88 Ž1991. 9873– 9877.
Enhancement of expression of stress proteins by agents that lower the levels of glutathione in cells Hidenori Ito, Keiko Okamoto, Kanefusa Kato
)
Department of Biochemistry, Institute for DeÕelopmental Research, Aichi Human SerÕice Center, Kamiya, Kasugai, Aichi 480-03, Japan Received 21 November 1997; accepted 8 January 1998
Abstract The effects of diethyl maleate and buthionine sulfoximine, agents that lower cellular levels of glutathione, on expression of hsp27 and a B crystallin in response to stress were studied. When C6 rat glioma cells were treated with 100 m M arsenite for 1 h, accumulation of the two proteins, estimated by specific immunoassays, was markedly enhanced by additional exposure to 1 mM diethyl maleate or 2.5 mM buthionine sulfoximine. The latter also increased heat- and CdCl 2-induced accumulation of hsp27 and a B crystallin. Stress-induced accumulation of hsp70, estimated by Western blotting analysis, was also enhanced by these agents. Northern blotting analysis revealed increase in levels of mRNAs for hsp27, a B crystallin and hsp70. The period of heat shock element ŽHSE.-binding activity of heat shock factor ŽHSF. stimulated by arsenite was extended by addition of diethyl maleate and buthionine sulfoximine. The induced phosphorylated state of HSF1 was also prolonged by diethyl maleate. Although exposure of cells to diethyl maleate alone for 1 h caused neither accumulation of hsp27, a B crystallin and hsp70 nor expression of mRNAs for these proteins, HSE-binding activity of HSF was stimulated. However, the activated HSF was not phosphorylated. These results suggest that diethyl maleate induces an intermediate state of HSF that binds to HSE but is transcriptionally inert. The mechanism is unclear but the levels of glutathione in cells that were exposed to diethyl maleate or buthionine sulfoximine were markedly decreased. q 1998 Elsevier Science B.V. Keywords: Stress response; Small heat shock protein; Glutathione; Redox; hsp27; Crystallin
1. Introduction The cellular response to stress is well conserved. Physical and chemical stresses induce the expression of stress responsive genes whose products are named heat shock or stress proteins Ž hsps. . The hsps are
) Corresponding author. Department of Biochemistry, Institute for Developmental Research, Aichi Human Service Center, 713-8, Kamiya, Kasugai, Aichi 480-03, Japan. Fax: q81-568-88-0811; E-mail: [email protected]
highly conserved and are elicited by a variety of stresses including heat, amino acid analogues, heavy metals, oxidative agents or thiol-reactive species w1,2x. The underlying mechanisms are thought to be as follows: heat shock transcription factor 1 ŽHSF1., which exists as a monomer in normal cells polymerizes to form a trimer in response to stress, then binds to a consensus sequence, the heat shock element ŽHSE. , located in the promoter regions of hsp genes w3x. Recently, several studies have revealed a relationship between the stress response and redox processes.
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For example, Caltabiano et al. w4x reported that thiol reactive agents Ž p-chloromercuribenzoate and iodoacetamide. can induce stress proteins in human or murine melanoma cells and Chen et al. w5x described an increase in synthesis of hsp70 in an epithelial cell line, LLC-PK1, due to reactive electrophiles. Huang et al. w6x further reported inhibition of the heat shock response in human HeLa and mouse C2C12 myogenic cells by a high concentration Ž 2 mM. of dithiothreitol. Iodoacetamide w7x or diamide w8x can activate the heat shock transcription factor, resulting in induction of hsp70 protein. We previously reported that a high concentration of dithiothreitol Ž2 mM. inhibits the arsenite-induced stress responses of hsps, whereas a low concentration Ž 0.03–0.1 mM. causes enhancement w9x. Moreover, Mehlen et al. w10x found that overexpression of hsp27 in mouse L929 fibrosarcoma cells resulted in a decrease of reactive oxygen species and an increase in the levels of glutathione w10x. The present study was conducted to determine the effect of glutathione-depleting drugs such as diethyl maleate, a compound that binds the free thiol groups w11x, and buthionine sulfoximine, a specific and essentially irreversible inhibitor of g-glutamyl-cysteine synthetase w12x, on stress-induced expression of hsp27, a B crystallin and hsp70.
that were preincubated for 10–15 min in 5% CO 2 in air with or without diethyl maleate or buthionine sulfoximine were floated in a water bath at 428C for 30 min. Then the medium was replaced with the standard culture medium and cells were cultured at 378C until harvesting. Cells in each dish were washed twice with phosphate-buffered saline Ž PBS, containing 8 g of NaCl, 0.2 g of KCl, 1.15 g of Na 2 HPO4 , and 0.2 g of KH 2 PO4 in 1000 ml of H 2 O. and stored frozen at y208C for a few days prior to analysis. The frozen cells on each dish were collected and suspended in 0.3 ml of PBS, and each suspension was sonicated and centrifuged at 125,000 = g for 20 min
2. Materials and methods 2.1. Cell culture and treatment C6 cells Žobtained from the Japanese Cancer Research Resources Bank, Tokyo. were grown in Dulbecco’s modified Eagle’s medium Ž DMEM; Nissui Pharmaceutical, Tokyo, Japan., supplemented with 10% fetal calf serum ŽLife Technologies, Tokyo, Japan. at 378C in a humidified atmosphere of 95% air and 5% CO 2 . The cells were seeded on 35-mm dishes for analysis of proteins, on 60-mm dishes for assays of the binding of HSF to HSEs and on 90-mm dishes for extraction of RNA for Northern blotting analysis of mRNAs. In each case the medium was changed every 2 or 3 days. When cells reached confluence, they were exposed to NaAsO 2 or CdCl 2 at 378C for 1 h in the presence or absence of diethyl maleate or buthionine sulfoximine Ž Nacalai Tesque, Kyoto, Japan. . For heat treatment, dishes with cells
Fig. 1. Enhancement by diethyl maleate ŽDM. and buthionine sulfoximine ŽBS. of arsenite- ŽA., heat- or CdCl 2 ŽB.-induced accumulation of hsp27 and a B crystallin in C6 cells. ŽA. C6 cells were exposed to 100 m M arsenite in the presence or absence of 1 mM diethyl maleate ŽDM. or 2.5 mM buthionine sulfoximine ŽBS. for 1 h and then cultured for 16 h in the standard medium. ŽB. Cells were exposed to heat Ž428C for 30 min, lanes 2–4. or 100 m M CdCl 2 Žlanes 5–7. in the presence or absence Žlanes 2 and 5. of 0.3 mM Žlanes 3 and 6. or 1.0 mM Žlanes 4 and 7. diethyl maleate for 1 h and then cultured in the standard medium for 16 h. Lane 1: untreated control cells. Concentrations of hsp27 and a B crystallin were determined by specific immunoassays and each column and bar show the mean "SD of the results for five dishes.
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in 12.5% Ž for analysis of hsp27 and a B crystallin. or 10% Žfor analysis of hsp70. polyacrylamide slab gels. Western blotting analysis of hsp27 and a B crystallin was carried out as described previously w16x using affinity-purified antibodies Ž0.05 m grml. raised in rabbits against rat hsp27 or against the carboxyterminal decapeptide of a B crystallin and peroxidase-labeled antibodies raised in goats against rabbit IgG as second antibodies. For detection of hsp70, we used a mouse monoclonal antibody specific for the inducible form of hsp70 Ž C92F3A-5; StressGen Biotechnologies, Victoria, BC, Canada. . The second antibody was against mouse IgG ŽMedical and Biological Laboratories, Nagoya, Japan. . Peroxidase activity on nitrocellulose sheets was visualized on X-ray
Fig. 2. Effects of diethyl maleate on arsenite-induced accumulation of hsp27 and a B crystallin in C6 cells. ŽA. Cells were exposed to 100 m M arsenite in the presence Žsolid circles. or absence Žopen circles. of 1 mM diethyl maleate for 1 h and then cultured in the standard medium for the indicated periods of time. Open squares: untreated control cells. Each point and vertical bar show the mean"SD of the results for three to four dishes. ŽB. Cells were exposed to 100 m M arsenite in the presence or absence of the indicated concentrations of diethyl maleate ŽDM. for 1 h and then cultured in the standard medium for 16 h. Each column and bar show the mean"SD of the results for three to four dishes.
at 48C. The supernatants were used for assays of hsp27, a B crystallin and hsp70. 2.2. Immunoassays of hsp27 and a B crystallin Concentrations of hsp27 w13x and a B crystallin w14x in extracts of cells were determined by specific immunoassays, as described previously. 2.3. Electrophoresis and Western blotting analysis of hsp27, a B crystallin, and hsp70 SDS-polyacrylamide gel electrophoresis Ž SDSPAGE. was performed as described by Laemmli w15x
Fig. 3. Effects of 1 mM diethyl maleate ŽA. or 2.5 mM buthionine sulfoximine ŽB. on the accumulation of hsp27 and a B crystallin in response to various concentrations of arsenite. Cells were exposed to the indicated concentrations of arsenite in the presence or absence of 1 mM diethyl maleate ŽDM; A. or 2.5 mM buthionine sulfoximine ŽBSO; B. for 1 h and then cultured in the standard medium for 16 h. Each column and bar show the mean"SD of the results for three to four dishes.
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films using a Western blotting chemiluminescence reagent Ž Renaissance; Dupont NEN, Boston, MA, USA..
2.4. Isolation of RNA and Northern blotting analysis Total RNA was isolated from cells with a QuickPrep Total RNA Extraction kit ŽPharmacia Biotech, Tokyo, Japan. . The 20-m g samples of total RNA were subjected to electrophoresis on 0.9% agarose– 2.2 M formaldehyde gels and blotted onto nitrocellulose membranes w17x. For Northern blotting analysis, membranes were allowed to hybridize with cDNA probes for hsp27 Ž BamHI–HindIII fragment of cDNA for mouse hsp27, kindly provided by Dr. L.F. Cooper, University of North Carolina. w18x, a B crystallin Ž Pst I fragment of cDNA for bovine a B crystallin, kindly provided by Dr. H. Bloemendal, University of Nijmegen. w19x and hsp70 Ž EcoRI fragment of cDNA for human hsp70, obtained from StressGen Biotechnologies., which were labeled with a Multiprime DNA labeling system ŽAmersham, Buckinghamshire, UK., as described previously w20x.
2.5. Gel mobility shift assays and Western blotting analysis of HSF Cells were suspended at 08C in 20 mM HEPES– KOH buffer, pH 7.9, containing 25% Ž vrv. glycerol, 0.5 M NaCl, 1.5 mM MgCl 2 , 0.2 mM EDTA, 0.5 mM dithiothreitol, 0.5 mgrml pepstatin A, and 20 m grml PefaBlock SC ŽMerck, Darmstad, Germany.. The suspensions were sonicated at 08C for 20 s and then centrifuged at 48C at 125,000 = g for 10 min. Supernatants were collected and aliquots were frozen in liquid N2 and stored at y808C. For Western blotting analysis of HSF, aliquots of extracts containing 40 m g of protein were subjected to SDS-PAGE in 6% polyacrylamide slab gels. HSF1 was detected with rabbit antiserum raised against HSF1, which was kindly provided by Dr. A. Nakai Ž Kyoto University.. For gel mobility shift assays, aliquots of cell extracts containing 15 m g of protein were incubated for 20 min at 08C with 0.1 ng of a 32 P-labeled oligonucleotide that included the sequence of the HSE from the promoter of the gene for hsp70 in Drosophila Ž5XGCCTCGAATGTTCGCGAAGTTTCG-3X . w32x and 0.5 m g of polyŽ dI–dC. Ž Pharmacia Biotech. in 10
Fig. 4. Western blotting analysis of levels of hsp70, hsp27 and a B crystallin ŽA. and Northern blotting analysis of the respective mRNAs ŽB. in C6 cells. ŽA. Cells were exposed to 1 mM diethyl maleate Žlanes 3 and 4., or 100 m M arsenite Žlanes 5 and 6. in the presence of 0.3 mM Žlanes 7 and 8. or 1 mM Žlanes 9 and 10. of diethyl maleate for 1 h and then cultured for 16 h in the standard medium. Extracts of cells containing 20 m g of protein were subjected to SDS-PAGE followed by Western blotting analysis with antibodies against hsp70, hsp27, and a B crystalline, as indicated. Lanes 1 and 2: untreated control cells. ŽB. Cells were exposed to 1 mM diethyl maleate alone Žlane 2., or 100 m M arsenite in the presence Žlane 4. or absence Žlane 3. of 1 mM diethyl maleate for 1 h and then cultured for 6 h in the standard medium. A total of 20-m g of the total RNA from each sample were analyzed by Northern blotting with cDNA probes for hsp70, hsp27 and a B crystallin. Lane 1: control cells. Bands of 28S RNA are shown for reference.
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mM Tris–HCl buffer, pH 8.0, containing 50 mM NaCl, 1 mM EDTA, 5% glycerol, and 0.5 mM dithiothreitol in a final volume of 25 m l. The reaction mixtures were subjected to electrophoresis on 4% polyacrylamide gels and the dried gels were autoradiographed with X-ray films.
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cules, CA, USA. using bovine serum albumin as the standard. Rat hsp27 and a B crystallin, which were used as the standards for immunoassays and electrophoresis, were purified from rat skeletal muscle w13,23x.
2.6. Quantitation of glutathione and protein thiols Reduced glutathione and oxidized glutathione were determined using enzymatic recycling and the Ellman’s reagent as described by Eyer and Podhradsky w21x. Protein thiols were determined using Ellman’s reagent as described by Matsumura and Matthews w22x. 2.7. Other methods Concentrations of protein in soluble extracts were estimated with a protein assay kit Ž Bio-Rad, Her-
3. Results 3.1. Diethyl maleate and buthionine sulfoximine enhance stress-induced accumulation of hsp27 and a B crystallin Levels of hsp27 and a B crystallin in C6 cells in confluent culture were low. Exposure of cells to 100 m M arsenite for 1 h and subsequent culture in the
Fig. 5. Effects of diethyl maleate on activation of HSF. ŽA. Cells were exposed to 1 mM diethyl maleate ŽDM., 100 m M arsenite or 100 m M arsenite plus 1 mM diethyl maleate for 1 h and cultured in the standard medium for the indicated periods of time. Extracts of cells containing 15 m g of protein were subjected to gel mobility shift assays of HSF. Lane C: untreated control cells. Arrowheads: specific binding. Ns: nonspecific binding. ŽB. Cells were exposed to 1 mM diethyl maleate ŽDM., 100 m M arsenite or 100 m M arsenite plus 1 mM diethyl maleate for 1 h and then cultured in the standard medium for the indicated periods of time. Extracts of cells containing 40 m g of protein were subjected to SDS-PAGE followed by Western blotting with antibody against HSF1. Lane C: untreated control cells.
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normal medium at 378C for 16 h increased both ŽFig. 1A.. When cells were exposed to arsenite in the presence of 1 mM diethyl maleate or 2.5 mM buthionine sulfoximine, accumulation of the two proteins was enhanced ŽFig. 1A. . This was particularly the case with diethyl maleate. However, exposure of cells to these reagents alone for 1 h did not significantly increase the levels of either of the proteins ŽFig. 1A.. Diethyl maleate also enhanced heat- or CdCl 2-induced accumulation of hsp27 and a B crystallin although it suppressed CdCl 2-induction of a B crystallin ŽFig. 1B. . The levels of hsp27 and a B crystallin reached a maximum at 16 h after arsenite treatment regardless of the presence or absence of diethyl maleate Ž Fig. 2A.. Stimulatory effects of diethyl maleate on the arsenite-induced accumulation of hsp27 and a B crystallin were observed even at 0.1 mM, with the maximum effect seen at 0.3–1 mM Ž Fig. 2B. . In contrast, the effects of buthionine sulfoximine were similar at concentrations between 0.5 mM and 5 mM under the conditions Ždata not shown.. The extent of stimulation by diethyl maleate or buthionine sulfoximine was greatest when the concentration of arsenite was 50–100 m M ŽFig. 3A and B.. 3.2. Western and Northern blotting analyses of hsp27, a B crystallin and hsp70 To determine whether the arsenite-induced accumulation of hsp70, another representative hsp, was also affected by diethyl maleate, Western blotting analysis was performed. It was confirmed that enhancement of arsenite-induced hsp27 and a B crystallin accumulation was accompanied by a similar stimulation with regards to hsp70 ŽFig. 4A.. Heat-induced accumulation of hsp70 was also enhanced by diethyl maleate and buthionine sulfoximine demonstrated equivalent influence on accumulation of hsp70 in both arsenite and heat stresses Ž data not shown.. Treatment with diethyl maleate alone did not cause accumulation of any of the three proteins. Ž Fig. 4A.. Northern blotting analysis indicated that the arsenite-induced expression of mRNAs for hsp70, hsp27 and a B crystallin in C6 cells was enhanced by diethyl maleate but treatment with diethyl maleate
alone did not induce the expression of mRNAs for three proteins ŽFig. 4B. . Arsenite-induced increase in levels of mRNAs for the three proteins was also augmented by buthionine sulfoximine Ždata not shown.. 3.3. Effects of diethyl maleate and buthionine sulfoximine on HSF actiÕation When cells were exposed to 100 m M arsenite for 1 h and subsequent culture at 378C in the standard medium, HSE-binding activity of HSF in cell extracts was observed, reaching a maximum within 2 h and then decreases Ž Fig. 5A. . In contrast, when cells were exposed to 100 m M arsenite for 1 h with 1 mM diethyl maleate, HSE-binding activity of HSF in whole cell extract reached a maximum within 4 h and was sustained for a longer period Ž Fig. 5A. . A similar prolongation of HSE-binding activity of HSF by buthionine sulfoximine was also observed Ž Fig. 6. . Significant HSE-binding activity was detected in extracts of cells that were exposed to 1 mM diethyl maleate alone for 1 h ŽFig. 5A.. It has been shown that HSF1 is phosphorylated by heat or other stress w3x. We, therefore, estimated the effects of diethyl maleate on the phosphorylation state of HSF. The phosphorylated form of HSF1 with a high molecular mass Žabout 90 kDa. was detected
Fig. 6. Effects of buthionine sulfoximine on activation of HSF. Cells were exposed to 2.5 mM buthionine sulfoximine ŽBSO., 100 m M arsenite or 100 m M arsenite plus 2.5 mM buthionine sulfoximine for 1 h and then cultured in the standard medium for the indicated periods of time. Extracts of cells containing 15 m g of protein were subjected to gel mobility shift assays of HSF. Lane C: untreated control cells. Arrowheads: specific binding. Ns: nonspecific binding.
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Table 1 Effects of exposure to diethyl maleate and buthionine sulfoximine on the levels of glutathione ŽGSH. and protein thiols ŽPSH. in cells Treatment
GSH Žnmolrmg protein.
PSH Žnmolrmg protein.
control diethyl maleate Ž1 mM. buthionine sulfoximine Ž2.5 mM. arsenite Ž100 m M. arsenite plus diethyl maleate arsenite plus buthionine sulfoximine
15.8 " 0.47) 3.49 " 0.18 3.42 " 0.26 19.7 " 1.13 5.09 " 0.12 3.74 " 0.09
93 " 4) 127 " 6 105 " 1 88 " 15 126 " 32 124 " 26
Cells were exposed to 100 m M arsenite for 30 min in the presence or absence of 1 mM diethyl maleate or exposed to 2.5 mM buthionine sulfoximine for 2 h with or without 100 m M arsenite during the last 30 min. Cells were then washed three times with PBS and frozen at y808C until analysis. Concentrations of glutathione ŽGSH. and protein thiols ŽPSH. were determined as described in Section 2 and each value is expressed as nmolrmg soluble protein. )Mean" SD of values for three dishes.
in cells that were exposed to arsenite or arsenite plus diethyl maleate; and diethyl maleate prolonged the arsenite-induced phosphorylated state of HSF1 for a longer period ŽFig. 5B. . In contrast, it was not observed in cells that were exposed for 1 h to diethyl maleate alone ŽFig. 5B.. 3.4. Effects of exposure to diethyl maleate or buthionine sulfoximine on leÕels of glutathione and protein thiols in cells Diethyl maleate is a compound that binds to free thiol groups and buthionine sulfoximine is a specific and essentially irreversible inhibitor of g-glutamylcysteine synthetase w12x. Exposure of cells to 100 m M arsenite for 30 min increased the level of glutathione slightly compared to that in control cells ŽTable 1. . When cells were exposed to 1 mM diethyl maleate for 30 min or 2.5 mM buthionine sulfoximine for 2 h, with or without 100 m M arsenite, the levels of glutathione in cells were decreased regardless of the presence or absence of arsenite. However, levels of protein thiols in cells were barely affected by diethyl maleate and buthionine sulfoximine under the conditions ŽTable 1..
4. Discussion The present study demonstrated that the glutathione-depleting agents such as diethyl maleate and buthionine sulfoximine, enhanced the stress-induced
responses of C6 cells, while themselves neither increasing the expression of mRNAs for hsps nor their accumulation. Glutathione has numerous functions which include reactive oxygen species detoxification w24x and regulation of cell death w25x. We showed that the stress responses of cells to insult by arsenite, CdCl 2 or heat is markedly enhanced under reduced cellular glutathione conditions. In addition, diethyl maleate and buthionine sulfoximine prolonged the arsenite-induced HSE-binding activity of HSF and its phosphorylated state Ž Fig. 5B.. Although the exposure to diethyl maleate alone caused HSF to bind to HSE ŽFig. 5A. , it was not phosphorylated Ž Fig. 5B. and transcriptionally inert. It has been shown that the binding of HSF to HSE does not always result in the activation of the transcription of genes for hsps. For example, an alkaline shift in pH of the culture w26x, oxidative injury w27x, and anti-inflammatory drugs, namely, salicylate and indomethacin w28,29x, activate HSF binding to HSE, but do not induce hsp transcription. The present results suggest that diethyl maleate may induce an intermediate state of HSF that is bound to HSE but is transcriptionally inert, similar to that reported for salicylate and indomethacin by Cotto et al. w29x. Such an intermediate state of HSF might be expected to becomes transcriptionally active in the presence of arsenite or other stress, and together with the directly activated HSF synergistically contribute to transcription of hsp genes. It has been suggested that loss of redox control with a shift to an oxidative state due to reaction of
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arsenite with protein thiols might trigger expression of hsps w30,31x. However, in the present experiment, exposure of cells to arsenite did not decrease the cellular levels of glutathione and protein thiols but rather caused a slight increase when expressed on the basis of mg soluble protein under the assay conditions. In contrast, exposure of cells to diethyl maleate or buthionine sulfoximine decreased the levels of glutathione in cells regardless of the presence or absence of arsenite ŽTable 1. . These results suggest that stress responses are not elicited solely by reduction in the levels of glutathione in cells, but that such decrease acts to strongly enhance the reaction to arsenite, CdCl 2 and heat. Acknowledgements This work was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Science and Culture, Japan. References w1x L. Nover, in: L. Nover ŽEd.., Heat Shock Response, CRC Press, Boca Raton, FL, 1991, pp. 5–40. w2x L. Nover, K.-D. Scharf, in: L. Nover ŽEd.., Heat Shock Response, CRC Press, Boca Raton, FL, 1991, pp. 41–128. w3x P.K. Sorger, Cell 65 Ž1991. 363–366. w4x M.M. Caltabiano, T.P. Koestler, G. Poste, R.G. Greig, J. Biol. Chem. 261 Ž1986. 13381–13386. w5x Q. Chen, K. Yu, J.L. Stevens, J. Biol. Chem. 267 Ž1992. 24322–24327. w6x L.E. Huang, H. Zhang, S.W. Bae, A.Y.-C. Liu, J. Biol. Chem. 48 Ž1994. 30718–30725. w7x H. Liu, R. Lightfoot, J.L. Stevens, J. Biol. Chem. 271 Ž1996. 4805–4812. w8x M.L. Freeman, M.J. Borrelli, K. Syed, G. Senisterra, D.M. Stafford, J.R. Lepock, J. Cell Physiol. 164 Ž1995. 356–366. w9x K. Kato, H. Ito, K. Okamoto, Cell Stress and Chaperones 2 Ž1997. 199–209.
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