Identification of sodium salicylate as an hsp inducer using a simple screening system for stress response modulators in mammalian cells Keiichi Ishihara, Kenji Horiguchi, Nobuyuki Yamagishi and Takumi Hatayama Department of Biochemistry, Kyoto Pharmaceutical University, Japan As heat shock proteins (Hsps) are involved in protecting cells and also in the pathophysiology of diseases such as inflam- mation, cancer and neurodegenerative disorders, modula- tors of Hsp expression in mammalian cells would seem to be useful for the treatment of various diseases. In this study, we isolated mammalian cell lines for screening of Hsp modu- lators; mouse C3H10T1/2 cells stably transfected with a plasmid containing the mouse Hsp105 or human Hsp70B promoter upstream of a luciferase or b-galactosidase reporter gene, respectively. Using these cells, we examined the effect of sodium salicylate (SA), which may induce the transcription of hsp genes, on stress response in mammalian cells. When these cells were treated with SA for 1 h at 37 °C, both promoter activities were up-regulated by SA at con- centrations of more than 45 m M . The activation of heat shock factor and the subsequent accumulation of Hsp105a and Hsp70 were detected in cells treated with SA at con- centrations of more than 20 and 45 m M , respectively. Fur- thermore, SA induced resistance against a subsequent lethal stress. These findings suggested that SA is a potent hsp inducer, and may be used to protect cells against deleterious stressors. Keywords: cytoprotection; heat shock factor; heat shock promoter; heat shock proteins; sodium salicylate. Heat shock proteins (Hsp) are a set of highly conserved proteins that are produced in response to physiological and environmental stress [1]. Hsps are also expressed under physiological conditions and play important roles in normal cellular events such as the synthesis, translocation and degradation of proteins [2]. Hsps protect cells from the cytotoxic effects of aggregated proteins produced by various types of stress, and play a vital role in cell survival under both physiological and stressed conditions. Because cellular resistance against stress appears to be regulated by the expression levels of Hsps, selective modulators of Hsp expression could have medicinal applications. For instance, the induction of Hsps seems to improve the prognosis of patients after a massive operation. Geranylgeranylacetone, a nontoxic Hsp70 inducer, is suggested to prevent acute liver failure after massive hepatectomy, at least in part, by enhancing cellular levels of Hsp70 [3]. Moreover, as Hsp70 and Hsp40 protect against the aggregation of mutated proteins and cell death in neurodegenerative disorders such as Parkinson’s and Huntington’s disease [4,5], Hsp inducers are expected to be useful for the treatment of these diseases. On the other hand, a major problem with hyperthermia, which is one of the therapies applied for advanced cancers, is the development of a transient thermoresistance in cancer cells with recurrent heat treatments [6,7]. The acquisition of thermotolerance is expected to be suppressed by reducing the expression levels of Hsps in the cells. In mammalian cells, the transcription of hsp genes is mediated by the conversion of a pre-existing heat shock factor (HSF) from an inactive to an active form [8]. HSF presents as an inactive monomeric form in the cytoplasm under physiological conditions, and is converted to an active trimeric form that has sequence-specific DNA- binding activity under stressed conditions. Activated HSF relocalizes to the nucleus and binds to heat shock element (HSE) in the 5¢-flanking region of hsp genes, resulting in the trans-activation of hsp genes [8]. In the present study, we isolated mouse C3H10T1/2 cells stably transfected with a plasmid containing the mouse Hsp105 or human Hsp70B promoter upstream of a luciferase or b-galactosidase repor- ter gene, respectively, as a simple system for screening Hsp modulators. Furthermore, although sodium salicylate (SA) is widely used as a nonsteroidal anti-inflammatory drug, the mech- anism of action of SA is still a subject of debate. Several suggestions such as inhibition of cyclooxygenase, which is the rate-limiting enzyme in the conversion of arachidonic acid to prostaglandins [9] and inhibition of the activation of transcription factor nuclear factor-kappa B [10], have been made to describe how SA exerts its anti-inflammatory effects and also its side effects. In addition, SA has been found to activate HSF in mammalian cells, although the induction of transcription of hsp genes may not be induced by SA [11]. Correspondence to T. Hatayama, Department of Biochemistry, Kyoto Pharmaceutical University, 5 Nakauchi-cho, Misasagi, Yamashina-ku, Kyoto 607-8414, Japan. Fax: + 81 75 595 4758, Tel.: + 81 75 595 4653, E-mail: hatayama@mb.kyoto-phu.ac.jp Abbreviations: SA, sodium salicylate; hsp(s), heat shock protein(s); Hsp70, 70 kDa heat shock protein; Hsp40, 40 kDa heat shock pro- tein; Hsp105, 105 kDa heat shock protein; Hsp105a, a isoform of Hsp105; HSF, heat shock factor; HSE, heat shock element; Luc, luciferase; b-gal, b-galactosidase; DMEM, Dulbecco’s modified Eagle’s medium. (Received 14 April 2003, revised 30 June 2003, accepted3July2003) Eur. J. Biochem. 270, 3461–3468 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03740.x Here we examined the effects of SA on stress response in mammalian cells using a simple screening system, and revealed that SA is a potent Hsp inducer in mammalian cells, thereby protecting cells against deleterious stress. Experimental procedures Cell culture Mouse fibroblast C3H10T1/2 and mouse embryonic F9 cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Nissui Pharmaceutical, Tokyo, Japan) supple- mented with 10% foetal bovine serum in a humidified atmosphere of 5% (v/v) CO 2 in air at 37 °C. Human HeLa cells were grown in Eagle’s Minimum Essential medium (Nissui Pharmaceutical) containing 10% bovine serum in a CO 2 incubator at 37 °C. Screening for Hsp modulators A reporter plasmid containing the Hsp105 promoter upstream of a luciferase (luc) reporter gene was constructed by subcloning a 1.2-kb fragment of the 5¢-flanking region of the hsp105 gene [12] to pGL2-basic vector (Promega). The p173OR plasmid, which contains the hsp70B promoter upstream of the b-galactosidase (b-gal) reporter gene, was obtained from StressGen Biotechnologies (San Diego, CA, USA). pGL105 or p173OR plasmid (7 lg each) and pBK/ neo plasmid (Stratagene) containing a geneticin resistant gene (3 lg) were cotransfected into C3H10T1/2 cells (1 · 10 7 cells per 100 mm dish) with 30 lL lipofectAMINE reagent (Invitrogen) according to the manufacturer’s instructions, and incubated for 48 h. Cells were then maintained in DMEM containing 0.4 mgÆmL )1 G418 antibiotic reagent (Wako Pure Chemical, Osaka, Japan) for 3 weeks, and C3H10T1/2 cell lines stably transfected with pGL105 or p173OR plasmid, designated as pGL105/ C3H and p173OR/C3H, respectively, were obtained and maintained in DMEM containing 0.2 mgÆmL )1 G418. Measurement of Luc activity pGL105C3H cells (2 · 10 5 cells per 35 mm dish) were washed with NaCl/P i three times, lysed in 50 lLCellLysis Regent (Promega), and centrifuged at 20 000 g for 10 min. Aliquots (5 lL) of cell extracts were added to 50 lL Luciferase Assay Reagent (Promega), and the Luc activity was measured using a Turner Designs model TD-20/20 Luminometer. Measurement of b-gal activity p173OR/C3H cells (2 · 10 5 cells per 35 mm dish) washed with NaCl/P i were suspended in 50 lL0.25 M Tris/HCl pH 8.0, and lysed by freeze-thawing (frozen at )80 °Cfor 30 min and thawed at 37 °Cfor3min,fivetimes).After centrifugation at 20 000 g for 10 min, aliquots of cell extracts (5 lg protein) were added to a final volume of 125 lL Z buffer (0.2 M sodium phosphate buffer pH 7.5, 10 m M KCl, 1 m M magnesium sulfate, 0.05 m M 2-merca- ptoethanol). Then, 25 lL15m M chlorophenolred-b- D -galactosidase were added, and the mixture was incubated at 37 °C for 30 min. The reaction was stopped by adding 60 lL1 M Na 2 CO 3 , and absorbance at 574 nm was measured. Gel mobility shift assay C3H10T1/2 cells (5 · 10 5 cells per 60 mm dish) were washed with NaCl/P i , and quickly frozen at )80 °C. Frozen cells were suspended in 100 lL extraction buffer (20 m M Hepes/KOH pH 7.9, 1.5 m M MgCl 2 ,0.2m M EDTA, 0.5 m M phenylmethanesulfonyl fluoride, 0.5 m M dithiothre- itol, 0.42 M NaCl and 25% glycerol, v/v), kept at 4 °Cfor 15 min, and vortexed for 15 min at 4 °C. After centrifuga- tion at 50 000 g for 5 min, aliquots of the supernatant (15 lg protein) were incubated in 25 lL buffer containing 10 m M Tris/HCl pH 7.8, 1 m M EDTA, 50 m M NaCl, 0.5 m M dithiothreitol, 5% (v/v) glycerol, 0.2 mgÆmL )1 BSA, 40 lgÆmL )1 poly[dI-dC] and 0.4 ngÆmL )132 P-labelled HSE corresponding to nucleotides )115 to )81 of the human hsp70 gene [13] at 25 °C for 20 min. The mixtures were then electrophoresed on a native 4% polyacrylamide gel, and the gel was dried and subjected to autoradiography. To define the specific HSF–HSE complex, unlabelled HSE was added to the reaction mixture in a 100-fold molar excess of the labelled HSE. Western blot analysis C3H10T1/2, F9 or HeLa cells were lysed in 100 lL0.1% SDS. Cellular proteins (15 lg) were separated by SDS/ PAGE, and blotted onto nitrocellulose membrane. The membrane was washed with Tris-buffered saline (0.1 M Tris/HCl pH 7.5, 0.9% NaCl) containing 0.1% Tween 20 (TTBS), and reacted with rabbit anti-Hsp105 [14,15] or mouse anti-Hsp70 Ig (Sigma) at room temperature for 1 h. After a wash with TTBS, the membrane was further incubated with horseradish peroxidase-conjugated anti- rabbit or anti-mouse IgG (Santa Cruz Biotechnology) at room temperature for 1 h. Hsp105a and Hsp70 were detected using enhanced chemiluminescence reagent (Santa Cruz Biotechnology). For quantification, films were digit- ized by scanning into Adobe PHOTOSHOP 5(AdobeSystems), and the intensities of the bands (Hsp105 and Hsp70) were quantified using the software program NIH IMAGE (http:// rsb.info.nih.gov/nih-image/). Examination of cell morphology C3H10T1/2 cells (7 · 10 4 cells per well) grown in 24-well plates containing collagen-coated coverslips were washed with NaCl/P i three times, fixed with 4% paraformaldehyde at room temperature for 20 min, and then observed using a phase-contrast microscope. Neutral red uptake assay C3H10T1/2 cells (7 · 10 4 cells/well) in 24-well plates were incubated for 3 h in the presence of 50 lgÆmL )1 neutral red, and fixed with 1% formaldehyde containing 1% CaCl 2 for 1 min. The dye incorporated into viable cells was extracted with 50% ethanol containing 1% acetic acid, and absorb- ance at 540 nm was measured. 3462 K. Ishihara et al. (Eur. J. Biochem. 270) Ó FEBS 2003 Results Isolation of mammalian cell lines for screening of Hsp modulators To facilitate the measurement of heat shock promoter activity, we isolated mouse C3H10T1/2 cells that were stably transfected with pGL105 reporter plasmid containing the luc gene linked to a 1.2 kb fragment of the 5¢-flanking region of the hsp105 gene [12] or p173OR reporter plasmid containing the b-gal gene linked to the Hsp70B promoter, and designated them pGL105/C3H and p173OR/C3H cells, respectively (Fig. 1A,C). Under nonstressed conditions, the Luc activity in pGL105/C3H cells was detected at low levels (Fig. 1B). When pGL105/C3H cells were incubated at 37 °Cfor6h after heat shock at 39, 41 or 43 °C for 1 h, Luc activity was enhanced approximately 4 and 10 times in cells heat- shocked at 41 and 43 °C, respectively, compared to control levels. During continuous heat shock at 39, 41 or 43 °Cfor 6 h, Luc activity was only enhanced in cells treated at 41 °C. Because firefly luciferase is thermosensitive and may be rapidly inactivated at high temperature, we analyzed the amount of Luc protein in the soluble and insoluble fractions of these cells treated at 39, 41 and 43 °Cfor6h(Fig.1B, part b, upper panel). Luc protein was detected in the soluble fractions but not in the insoluble fractions under these conditions, and the amounts of the protein were directly proportional to the Luc activity in cells, suggesting that levels of Luc activity at high temperatures also reflect the levels of transcription and translation of Hsp105. Furthermore, when these cells were treated with chem- ical stressors such as sodium arsenite, cupric chloride and zinc chloride, Luc activity was also enhanced in a dose- dependent manner. As Hsp105a, a major product of hsp105 gene, is constitutively expressed and also induced by various forms of stress in mammalian cells, the expression of Luc activity in pGL105/C3H cells seemed to reflect the Fig. 1. Stress-inducible Hsp105 and Hsp70B promoters in pGL105/C3H and p173OR/C3H cells. (A) The structure of pGL105 plasmid containing the Hsp105 promoter upstream of the luciferase reporter gene is shown schematically. (B) pGL105/C3H cells were incubated at 37 °Cfor6hafter heat shock at various temperatures for 1 h (a), incubated at various temperatures for 6 h (b), or treated with sodium arsenite (c), cupric chloride (d) or zinc chloride (e) at 37 °C for 6 h. Then, Luc activity was assayed, and relative activities are shown as ratios to that of untreated control cells. For detection of Luc protein, cells incubated at 37–43 °C for 6 h were lysed and the lysates were centrifuged at 20 000 g for 15 min, Luc protein in the supernatant (s) and pellet (p) fractions were detected by Western blotting using anti-Luc Ig [upper panels in (b)]. (C) The structure of p173OR plasmid containing the Hsp70B promoter up-stream of the b-galactosidase reporter gene is shown schematically. (D) p173OR/C3H cells were incubated at 37 °C for 6 h after heat shock at various temperatures for 1 h (a), incubated at various temperatures for 6 h (b), or treated with sodium arsenite (c), cupric chloride (d) or zinc chloride (e) at 37 °C for 6 h. Then, b-gal activity was assayed, and relative activities are shown (arbitrary unit, AU). Ó FEBS 2003 Sodium salicylate is a potent hsp inducer (Eur. J. Biochem. 270) 3463 expression of endogenous Hsp105a in mammalian cells [15,16]. In p173OR/C3H cells, b-gal activity was not detected under nonstressed conditions (Fig. 1D). The b-gal activity in the cells was induced by heat shock but not by chemical stressors. The induction of the enzyme activity was consis- tent with that of the hsp70B gene in mammalian cells [17]. Thus, the promoter activities of Hsp105 and Hsp70B can be measured easily using the pGL105/C3H and p173OR/C3H cells, and pGL105/C3H cells seemed to be more useful for screening modulators of stress response in mammalian cells. Induction of stress response by SA Enhancement of heat shock promoter activity by SA. SA induces the activation of HSF but does not enhance the transcription of hsp genes in human HeLa cells and Drosophila [11,18], whereas the drug is also shown to induce Hsp70 synthesis in mouse L929 cells [19]. Because transcription of hsp genes may be induced by SA in mammalian cells, we first examined the effect of SA on the heat shock promoter using pGL105/C3H or p173OR/C3H cells (Fig. 2). When pGL105/C3H cells were treated with 15–60 m M SA at 37 °C for 1 h and incubated further at 37 °C for 6 h without SA, the Luc activity increased approximately 10- and 30-fold in cells treated with 45 and 60 m M SA, respectively, compared with that of untreated cells (Fig. 2A). When the amounts of Luc protein in cells treatedwithSAat37°C were examined by Western blotting, the amounts of the protein were directly propor- tional to the Luc activity in cells, suggesting that the increase of Luc activity by SA reflect the levels of transcription and translation of Hsp105, not due to an indirect effect of SA on the basal activity of Luc. Enhance- ment of Luc activity was also detected at 45 and 60 m M SA, when pGL105/C3H cells were incubated at 39 °Cfor6h after the SA treatment. However, the enzyme activity was not enhanced in cells incubated at 41 °Cfor6hafter treatment with 60 m M SA, due to the markedly reduced viability of the cells. Furthermore, when p173OR/C3H cells were treated with 15–60 m M SA at 37 °C for 1 h and incubated further at 37 °C for 6 h without SA, the activity significantly increased in cells treated with 45 and 60 m M SA (Fig. 2B). Enhance- ment of b-gal activity was also observed in cells incubated at 39 or 41 °C for 6 h after SA treatment similarly to the Luc activity in pGL105/C3H cells. The enzyme activity was also not enhanced in cells incubated at 41 °Cfor6hafter treatment with 60 mm SA, due to the markedly reduced viability of the cells. These results suggested that Hsp105 and Hsp70B promoters were activated even at 37 °Cincells treatedwith45and60m M SA, followed by transcription and translation of the gene products. When the effects of other known Hsp-inducing com- pounds such as geldanamycin, curcumin and geranylgera- nylacetone on the heat shock promoter were examined using pGL105/C3H cells [20–22], the Luc activity increased approximately two- to fivefold in cells treated with these compounds at 37 °C for 6 h compared with that of untreated cells. SA seemed to activate the heat shock promoter markedly than these Hsp inducers in mammalian cells (Table 1). Fig. 2. Effect of SA on Hsp promoters in pGL105/C3H and p173OR cells. pGL105/C3H (A) or p173OR (B) cells were incubated with or without 15, 30, 45 and 60 m M SA at 37 °C for 1 h, and further incu- bated at 37, 39 or 41 °C for 6 h without SA. Then, luciferase or b-galactosidase activity was assayed. Each value represents the mean ± SD of three independent experiments. Statistical significance was determined with Student’s t-test; *, P < 0.01 vs. control cells incubated at 37, 39 or 41 °C for 6 h. Upper panel in (A) is Western blot of Luc protein in the supernatant of cells treated with or without 30, 45 and 60 m M SA at 37 °C. Table 1. Effect of geldanamycin, curcumin, or geranylgeranylacetone on Hsp105 promoter in pGL105/C3H cells. Cells were treated with com- pounds at 37 °C for 1 h, and further incubated at 37 °Cfor6h.Each value is average of results from two independent experiments. Treatment Concentration Relative luciferase activity Control 1.0 Sodium salicylate 45 m M 9.2 60 m M 27.6 Geldanamycin 10 l M 3.4 15 l M 4.5 Curcumin 50 l M 1.7 75 l M 4.2 Geranylgeranylacetone 0.5 l M 2.0 1 l M 2.3 3464 K. Ishihara et al. (Eur. J. Biochem. 270) Ó FEBS 2003 Activation of HSF by SA and accumulation of Hsp105a and Hsp70 To examine whether SA enhances heat shock promoter activity by activating HSF, a gel mobility shift assay using 32 P-labelled HSE was performed. When C3H10T1/2 cells were treated with 10–60 m M SA at 37 °C for 1 h, HSF was activated in cells treated with SA at concentrations of more than 20 m M (Fig. 3A). The kinetics of activation of HSF by SA revealed that the HSF–HSE complex was detected immediately after 30 m M SA treatment, and quickly diminished in 1–2 h at 37 °C (Fig. 3B). Furthermore, SA affected the activation of HSF by heat shock (Fig. 3C). When activated by heat shock at 41 °C for 1 h, the HSF decreased to the basal level within 2 h. However, when cells treated with 30 m M SA at 37 °C were continuously heat- shocked at 41 °C, the SA-activated HSF remained at a high level for 2 h, then diminished to the basal level within 3 h of heat shock. Thus, SA seems not only to activate HSF at 37 °C, but also to enhance the activation of HSF by heat shock. Next, we examined whether Hsp105a and Hsp70 proteins accumulatedincellstreatedwithSA(Fig.4).Mouse C3H10T1/2, mouse F9 and human HeLa cells were treated with 15–60 m M SA at 37 °C for 1 h and further incubated for 6 h at 37 °C. The levels of Hsp105a and Hsp70 significantly increased in cells treated with 45 and 60 m M SA. However, the levels remained unchanged or decreased, when these cells were incubated at 41 °C for 6 h after the SA treatment. These results suggested that SA treatment at 37 °C induced the expression of endogenous heat shock proteins such as Hsp105a and Hsp70 in various mammalian cells. Enhancement of thermoresistance of cells by SA Upon exposure to a sublethal heat treatment, mammalian cells acquire transient resistance to a subsequent heat shock that would normally be lethal [6,7]. The phenomenon is known as thermotolerance, and much evidence supports the idea that Hsps, especially Hsp70, play important roles in its development. Since SA seemed to induce the expression of Hsp105a and Hsp70 in mammalian cells, we examined whether SA induced resistance against subsequent lethal heat shock. As shown in Fig. 5A, the treatment of cells with 30 or 45 m M SA for 1 h did not induce marked changes of cell morphology and numbers of cells attached to culture dishes, although the attached cells were slightly reduced in number by 60 m M SA treatment. When these cells were incubated at 37 °C for 6 h and exposed to a lethal heat shock at 45 °C for 45 min, cells attached to dishes markedly decreased in number regardless of SA treatment. However, when these cells were further incubated at 37 °C for 24 h, numbers attached to culture dishes were increased in cells treatedwith45or60m M SA, but not 30 m M SA. Furthermore, the viability of cells was assessed based on the ability of living cells to incorporate neutral red into lysozomes (Fig. 5B). The uptake of neutral red into cells decreased gradually after heat shock at 45 °C for 45 min, although uptake of the dye was only slightly affected by 30–60 m M SA at 37 °C. However, the uptake again increased in cells pretreated with 45 and 60 m M SA for 24 and 120 h, respectively, after the heat shock. Thus, the resistance of cells against a subsequent heat shock seemed to be enhanced by 45 or 60 m M SA treatment. Discussion As Hsps play important roles in the folding, regulation and degradation of cellular proteins and also cellular resistance against stress as molecular chaperones, drugs that can regulate the expression levels of Hsps in cells seem to have various medicinal applications. In the present study, we isolated cell lines for screening of stress response modula- tors: mouse pGL105/C3H and p173OR/C3H cells. pGL105/C3H cells have a plasmid containing the Hsp105 promoter upstream of a luc reporter gene, while p173OR/ C3H cells have a plasmid containing the Hsp70B promoter upstream of a b-gal reporter gene. The Luc or b-gal activity in these cells was expressed and induced similarly to endogenous Hsp105a or Hsp70B, respectively, in mamma- lian cells. Using these cells, the activities of Hsp105 and Hsp70B promoters could be easily measured. Fig. 3. Effect of SA on activation of HSF in C3H10T1/2 cells. (A) C3H10T1/2 cells were treated with or without 10, 20, 30 and 60 m M SA at 37 °C for 1 h, or heat-shocked at 42 °C for 3 h as a positive control. (B and C) C3H10T1/2 cells were incubated with or without SA at 37 °C for 1 h, then further incubated at 37 °C (B) or at 41 °C(C) without SA for up to 6 h. Cell extracts from these cells were subjected to gel mobility shift assay using a radioactive HSE probe. HSF–HSE complexes were determined by adding a 100-fold excess of unlabelled HSE. Arrows indicate specific HSF–HSE complexes. Ó FEBS 2003 Sodium salicylate is a potent hsp inducer (Eur. J. Biochem. 270) 3465 Using these cells, we examined the effects of SA, a nonsteroidal anti-inflammatory drug, on the stress response of mammalian cells. Jurivich et al. have shown that 20 m M SA induces activation of HSF but not transcription of hsp genes in human cells [11]. In contrast, Liu et al. have shown that 60 m M SA induces not only activation of HSF but also accumulation of Hsp70 in mouse cells [19]. Here we showed that relatively high doses of SA (45–60 m M ) induced the activation of promoter activities of Hsp105 and Hsp70 as well as the accumulation of Hsp105a and Hsp70 in mouse and human cells, although relatively low doses of SA (20– 30 m M ) induced the activation of HSF but not transcription of hsp genes in cells. Thus, relatively high doses of SA seemed to be required for the induction of the transcription of hsp genes and the accumulation of Hsp in mammalian cells. HSF is a transcription factor which is converted to a trimeric and hyper-phosphorylated active form from inac- tive monomers in response to various forms of stress and induces the transcription of hsp genes [8]. SA is shown to trigger HSF differently than heat shock [20]. The SA- induced form of HSF is not hyperphosphorylated like the heat-induced form, and SA-induced threonine phosphory- lation of HSF, whereas heat shock led to a predominance of HSF serine phosphorylation [23]. However, the HSF activated by relatively high doses of SA did induce transcription of hsp genes in cells. The HSF activated by SA may be somehow different at levels of modification such as phosphorylation depending on the doses of SA, although further study is needed to elucidate the different effects of the SA-activated HSF on the induction of transcription of hsp genes. Hsps are suggested to play important roles in the acquisition of resistance of cells against various forms of stress [1]. Here we showed that SA induced the activation of HSF, the transcription of hsp genes and the accumu- lation of Hsps in various mammalian cells and a concomi- tant increase of thermoresistance of cells. Thus, SA may be used for the protection of cells against deleterious stressors. Furthermore, several neurodegenerative disorders including Alzheimer’s, polyglutamine and Parkinson’s disease are though to be caused by an accumulation of protein aggregates in the brain [24], and Hsps such as Hsp70 and Hsp40 are shown to suppress the toxicity of these diseases [25,26]. Recently, long-term use of non- steroidal anti-inflammatory drugs was shown to prevent the occurrence of Alzheimer’s disease [27,28]. Our finding that SA induces the expression of Hsps in mammalian cells may explain the protective effect of SA on Alzheimer’s disease. SA activated heat shock promoter and induced the expression of Hsps in mammalian cells at concentrations higher than those used for its anti-inflammatory effects. However, long-term use of therapeutic doses of the drug may induce the expression of Hsps in mammalian cells. SA has a potent anti-inflammatory effect mediated by suppres- sion of the production of inflammatory mediators by inhibition of cyclooxygenase and nuclear factor-kappa B activation [9,10]. In addition to the anti-inflammatory effects, it is noteworthy that SA which can activate HSF Fig. 4. Effect of SA on accumulation of Hsp105 and Hsp70 in mammalian cells. C3H10T1/2 (A), F9 (B), and HeLa cells (C) were treated with or without SA at 37 °Cfor 1 h, then further incubated at 37 or 41 °Cfor 6 h without SA. Cellular proteins (15 lg) were separated by SDS/PAGE (10% polyacryl- amide), blotted onto nitrocellulose membranes, and immunostained using anti- Hsp105 or anti-Hsp70 Ig (upper panels). Bands were quantified by densitometry, and relative levels of Hsp105a or Hsp70 are shown as ratios to that of untreated cells at 37 °Cor cells heat-shocked at 41 °C for 6 h, respect- ively (lower graphs). Each value in (A) and (B) represents the mean ± SD of three independent experiments. Statistical significance was determined with Student’s t-test; *, P < 0.01 vs. control cells incubated at 37 or 41 °Cfor6h. 3466 K. Ishihara et al. (Eur. J. Biochem. 270) Ó FEBS 2003 Fig. 5. Induction of thermoresistance of cells by SA. (A) C3H10T1/2 cells were treated with or without 30, 45 and 60 m M SA at 37 °C for 1 h (a). These cells were further incubated without SA at 37 °C for 24 h (b) or heat shocked at 45 °C for 45 min after incubation at 37 °C for 6 h (c). The heat-shocked cells were further incubated at 37 °C for 24 h (d). Then, these cells were fixed with 4% paraformaldhyde and observed using a phase contrast microscope (· 100). (B) C3H10T1/2 cells treated with or without 30, 45 and 60 m M SA at 37 °C for 1 h were incubated at 37 °Cfor6h without SA. Then, these cells were heat-shocked at 45 °C for 45 min, and further incubated at 37 °C for the indicated periods. Viability of cells was assessed by neutral red uptake assay. 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Identification of sodium salicylate as an hsp inducer using a simple screening system for stress response modulators in mammalian cells Keiichi Ishihara,. examined the effects of SA on stress response in mammalian cells using a simple screening system, and revealed that SA is a potent Hsp inducer in mammalian cells,