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• W2077673859 abstract "In previous reports we demonstrated that glucose deprivation induces metabolic oxidative stress in drug-resistant human breast carcinoma MCF-7/ADR cells (Lee, Y. J., Galoforo, S. S., Berns, c. M., Chen, J. C., Davis, B. H., Swim, J. E., Corry, P. M., and Spitz, D. R. (1998)J. Biol. Chem. 273, 5294–5299). In the study described here, we investigated intracellular responses to metabolic oxidative stress. Northern blots show an increase in the level of HSP70 and HSP28 mRNA in cells exposed to glucose-free medium for 1 h. One- and two-dimensional polyacrylamide gel analyses confirmed that glucose deprivation induced a family of HSPs, particularly an inducible HSP70. Overexpression of bcl-2 suppressed glucose deprivation-induced HSP70 gene expression, heat shock transcription factor-heat shock element binding activity, as well as c-Jun NH2-terminal kinase (JNK1) activation. Expression of a dominant-negative mutant of JNK1 also suppressed glucose deprivation-induced JNK1 activation as well asHSP70 gene expression. Taken together, the stress-activated protein kinase signal transduction pathway is involved in glucose deprivation-induced heat shock gene expression. In previous reports we demonstrated that glucose deprivation induces metabolic oxidative stress in drug-resistant human breast carcinoma MCF-7/ADR cells (Lee, Y. J., Galoforo, S. S., Berns, c. M., Chen, J. C., Davis, B. H., Swim, J. E., Corry, P. M., and Spitz, D. R. (1998)J. Biol. Chem. 273, 5294–5299). In the study described here, we investigated intracellular responses to metabolic oxidative stress. Northern blots show an increase in the level of HSP70 and HSP28 mRNA in cells exposed to glucose-free medium for 1 h. One- and two-dimensional polyacrylamide gel analyses confirmed that glucose deprivation induced a family of HSPs, particularly an inducible HSP70. Overexpression of bcl-2 suppressed glucose deprivation-induced HSP70 gene expression, heat shock transcription factor-heat shock element binding activity, as well as c-Jun NH2-terminal kinase (JNK1) activation. Expression of a dominant-negative mutant of JNK1 also suppressed glucose deprivation-induced JNK1 activation as well asHSP70 gene expression. Taken together, the stress-activated protein kinase signal transduction pathway is involved in glucose deprivation-induced heat shock gene expression. heat shock transcription factor heat shock element c-Jun NH2-terminal kinase stress-activated protein kinase polyacrylamide gel electrophoresis oxidized glutathione reduced glutathione glyceraldehyde-3-phosphate dehydrogenase. It is well established that the transcriptional induction of heat shock genes in eukaryotes is mediated by the heat shock transcription factor (HSF)1 (1Kingston R.E. Schuetz T.J. Larin Z. Mol. Cell. Biol. 1987; 7: 1530-1534Crossref PubMed Scopus (155) Google Scholar, 2Parker C.S. Topol J.A. Cell. 1984; 37: 273-283Abstract Full Text PDF PubMed Scopus (246) Google Scholar, 3Sorger P.K. Pelham H.R.B. EMBO J. 1987; 6: 3035-3041Crossref PubMed Scopus (210) Google Scholar, 4Sorger P.K. Pelham H.R.B. Cell. 1988; 54: 855-864Abstract Full Text PDF PubMed Scopus (565) Google Scholar, 5Sorger P.K. Lewis M.J. Pelham H.R.B. Nature. 1987; 329: 81-84Crossref PubMed Scopus (305) Google Scholar, 6Wiederrecht G. Shuey D.J. Kibbe W.A. Parker C.S. Cell. 1987; 48: 507-515Abstract Full Text PDF PubMed Scopus (117) Google Scholar, 7Wu C. Nature. 1985; 317: 84-87Crossref PubMed Scopus (138) Google Scholar, 8Wu C. Wilson S. Walker B. Dawid I. Paisley T. Zimarino V. Ueda H. Science. 1987; 238: 247-1253Crossref Scopus (215) Google Scholar). This protein can be activated by a variety of stresses such as heat shock, heavy metals, or arsenite (4Sorger P.K. Pelham H.R.B. Cell. 1988; 54: 855-864Abstract Full Text PDF PubMed Scopus (565) Google Scholar, 9Gordon S. Bharadwaj S. Hnatov A. Ali A. Ovsenek N. Dev. Biol. 1997; 181: 47-63Crossref PubMed Scopus (30) Google Scholar, 10Hatayama T. Asai Y. Wakatsuki T. Kitamura T. Imahara H. J. Biochem. (Tokyo). 1993; 114: 592-597Crossref PubMed Scopus (36) Google Scholar, 11Larson J.S. Schuetz T.J. Kingston R.E. Nature. 1988; 335: 372-375Crossref PubMed Scopus (188) Google Scholar). The activated HSF binds to the promoters which contain the heat shock element (HSE) and then stimulates transcription (1Kingston R.E. Schuetz T.J. Larin Z. Mol. Cell. Biol. 1987; 7: 1530-1534Crossref PubMed Scopus (155) Google Scholar, 5Sorger P.K. Lewis M.J. Pelham H.R.B. Nature. 1987; 329: 81-84Crossref PubMed Scopus (305) Google Scholar, 12Zimarino V. Wu C. Nature. 1987; 327: 727-730Crossref PubMed Scopus (249) Google Scholar). A fundamental question which remains unanswered is how these stresses activate HSF. HSF is phosphorylated under normal growth conditions and is hyperphosphorylated subsequent to stress. It has been proposed that such phosphorylation may be mediated through the MAP kinase family such as c-Jun NH2-terminal kinase (JNK) (13Kim J. Nueda A. Meng Y.H. Dynan W.S. Mivechi N.F. J. Cell. Biochem. 1997; 67: 43-54Crossref PubMed Scopus (73) Google Scholar). Several reports have demonstrated that the JNK, also referred to as stress-activated protein kinase (SAPK), signal transduction pathway can be activated by oxidative stress (14Cui X.-L. Douglas J.G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3771-3776Crossref PubMed Scopus (163) Google Scholar, 15Jimenez L.A. Zanella C. Fung H. Janssen Y.M. Vacek P. Charland C. Goldberg J. Mossman B.T. Am. J. Physiol. 1997; 273: L1029-L1035PubMed Google Scholar, 16Qin S. Minami Y. Hibi M. Kurosaki T. Yamamura H. J. Biol. Chem. 1997; 272: 2098-2103Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Following activation, JNK phosphorylates several transcription factors including activating transcription factor-2 (17Gupta S. Campbell D. Derijard B. Davis R.J. Science. 1995; 267: 389-393Crossref PubMed Scopus (1336) Google Scholar), Elk-1 (18Cavigelli M. Dolfi F. Claret F.X. Karin M. EMBO J. 1995; 14: 5957-5964Crossref PubMed Scopus (487) Google Scholar), Sap-1a (19Janknecht R. Hunter T. J. Biol. Chem. 1997; 272: 4219-4224Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar), and c-Jun (20Derijard B. Hibi M. Wu I-H. Barrett T. Su B. Deng T. Karin M. Davis R.J. Cell. 1994; 76: 1025-1037Abstract Full Text PDF PubMed Scopus (2953) Google Scholar) which are involved in regulating numerous genes implicated in cell proliferation, transformation, differentiation, and DNA repair (21Angel P. Karin M. Biochim. Biophys. Acta. 1991; 1072: 129-157Crossref PubMed Scopus (3256) Google Scholar, 22Iwai S.A. Kosaka M. Nishina Y. Sumi T. Sakuda M. Nishimune Y. Exp. Cell Res. 1993; 205: 39-43Crossref PubMed Scopus (15) Google Scholar, 23Potapova O. Haghighi A. Bost F. Liu C. Birrer M.J. Gjerset R. Mercola D. J. Biol. Chem. 1997; 272: 14041-14044Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar, 24Thinakaran G. Ojala J. Bag J. FEBS Lett. 1993; 319: 271-276Crossref PubMed Scopus (24) Google Scholar). Recent studies also show that oxidative stress can activate HSF and increaseHSP70 gene expression (25Gomer C.J. Ryter S.W. Ferrario A. Rucker N. Wong S. Fisher A.M. Cancer Res. 1996; 56: 2355-2360PubMed Google Scholar). These observations suggest a possibility that oxidative stress-induced activation of HSF is mediated through the SAPK pathway. We have previously observed that glucose deprivation increases intracellular hydroperoxide production and oxidized glutathione in drug-resistant human breast carcinoma MCF-7/ADR cells (26Lee Y.J. Galoforo S.S. Berns C.M. Chen J.C. Davis B.H. Sim J.E. Corry P.M. Spitz D.R. J. Biol. Chem. 1998; 273: 5294-5299Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). These results led us to investigate the possibility that glucose deprivation-induced metabolic oxidative stress may activate HSF through the SAPK pathway and result in hsp gene expression. In this study, we demonstrate that glucose deprivation induces HSP genes, in particular inducible HSP70 gene expression as well as JNK activation. Studies with the bcl-2 gene or dominant-negative JNK1 mutant transfected cells indicate that metabolic oxidative stress-induced HSP70 gene expression is mediated through the SAPK pathway. Multidrug-resistant human breast carcinoma (MCF-7/ADR) cells were cultured in McCoy's 5a medium with 10% iron-supplemented bovine calf serum (HyClone, Logan, UT) and 26 mm sodium bicarbonate for monolayer cell culture. Two or three days prior to the experiment, cells were plated into 60-mm Petri dishes or T-25 flasks. The Petri dishes/flasks containing cells were kept in a 37 °C humidified incubator with a mixture of 95% air and 5% CO2. For survival determination, the T-25 flasks containing monolayers of asynchronous cells were trypsinized with ice-cold 0.05% trypsin in Hanks' balanced salt solution and 0.53 mm EDTA. After trypsinization, the cells were resuspended in 3 ml of McCoy's 5a medium containing 10% iron-supplemented bovine calf serum. Cell counts were determined with a Coulter counter, and appropriate dilutions were made. The appropriate number of cells were plated into two replicate T-25 flasks for clonogenic cell survival assay. After 1–2 weeks of incubation at 37 °C, colonies were stained and counted. Data were normalized to sham treated control plating efficiencies. Cells were rinsed three times with Hanks' balanced salt solution which took approximately 10 min. Cells were then treated with glucose-free McCoy's 5A medium (Life Technologies, Inc., Gaithersburg, MD). Monolayer cells were heated in a hot water bath as described previously (27Lee Y.J. Dewey W.C. Radiat. Res. 1986; 106: 98-110Crossref PubMed Scopus (62) Google Scholar). Exponentially growing cells were plated 2 days before experiments at a concentration of 4 × 105cells/60-mm culture dish. Cells were transfected with pcDNA3-FLAG-JNK1 (APF) or pCMV–bcl-2 vector (20 μg) by using LipofectinTM Reagent (Life Technologies, Inc.). The pcDNA3-FLAG-JNK1 (APF) vector contains a dominant-negative mutant of JNK1 with a NH2-terminal FLAG tag, which was kindly provided by Dr. R. Davis (Howard Hughes Medical Institute Research Laboratories, University of Massachusetts Medical Center, Worcester, MA). The pCMV-bcl-2 vector containing the bcl-2 gene was provided by Dr. S. J. Korsmeyer (Washington University, St. Louis, MO). Cells were labeled with 20 μCi/ml [3H]leucine (specific activity 160 Ci/mmol, Amersham) in leucine-free medium. After labeling, cells were washed twice with cold Hanks' balanced salt solution. For one-dimensional PAGE, samples were mixed with 2 × Laemmli lysis buffer (1 × = 2.4 m glycerol, 0.14 m Tris, pH 6.8, 0.21m SDS, 0.3 mm bromphenol blue), and boiled for 10 min. Protein content was measured with BCA* Protein Assay Reagent (Pierce, Rockford, IL). The samples were diluted with 1 × lysis buffer containing 1.28 m β-mercaptoethanol and an equal amount of protein (30 μg) was applied to a one-dimensional PAGE. Electrophoresis was carried out on a 10–18% linear gradient SDS-PAGE. For two-dimensional PAGE, samples were solubilized in sample buffer containing 8 m urea, 1.7% Nonidet P-40, and 4.3% β-mercaptoethanol. Proteins were first separated in isoelectric focusing gels (pH 3.5–10). These gels were then laid across the top of a 10–18% linear gradient SDS-polyacrylamide gel for two-dimensional analysis (28Walker J.M. Walker J.M. Methods in Molecular Biology. 1. Humana Press, Clifton, NJ1984: 57-61Google Scholar). After electrophoresis, gels were fixed in 30% trichloroacetic acid for 30 min. For fluorography, gels were dehydrated by washing for 15 min in each of 25% acetic acid, 50% acetic acid, and glacial acetic acid, consecutively. After fixation, gels were placed in 125 ml of PPO solution (20% (w/v) 2,5-diphenyloxazole in glacial acetic acid) for 2 h. The PPO solution was removed, and the gel was shaken gently overnight in distilled water and dried for 2.5 h at 60 °C. The gel was loaded into a cassette with Kodak SB-5 x-ray film and placed in a −70 °C freezer. After optimum exposure time, the fluorograph film was developed with Kodak GBX developer and fixed with Kodak GBX fixer. After electrophoresis, the proteins were transferred onto a nitrocellulose membrane by electroblotting using 0.12 A and 30 V overnight. The membrane was then incubated in blocking solution (3% bovine serum albumin for non-ECL system or 5% dry milk for ECL system) for 1 h, washed, and then incubated with anti-bcl-2 monoclonal antibody (diluted 1:1,000 for ECL system; Eastman Kodak Co., New Haven, CT), anti-ACTIVETM JNK polyclonal antibody (diluted 1:5,000 for ECL system; Promega, Madison, WI), or anti-actin monoclonal antibody (diluted 1:10,000; ICN, Costa Mesa, CA). After incubation with the primary antibody, the membrane was washed, and incubated with alkaline phosphatase-conjugated rabbit-anti-mouse IgG (diluted 2,000; Zymed Laboratories Inc., South San Francisco, CA) for 1–2 h or biotinylated sheep anti-mouse IgG/donkey anti-rabbit IgG (diluted 1:2, 500-7, 500; Amersham, Arlington Heights, IL) for 1–2 h followed by streptavidin-conjugated horseradish peroxidase (1:4,000; Amersham). The membrane was then stained using either nitro blue tetrazolium and 5′-bromo-4-chloro-3-indolylphosphate or the detection reagent of the ECL detection kit (Amersham). HSP70and HSP28 mRNA levels were determined using the Northern blot technique. Total cellular RNA was extracted by the LiCl-urea method of Tushinskiet al. (29Tushinski R. Sussman P., Yu, L. Bancroft F. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 2357-2361Crossref PubMed Scopus (104) Google Scholar). For RNA analysis, 30 μg of total RNA was electrophoresed in a 1% agarose-formaldehyde gel (30Lehrach H. Diamond L. Wozney J. Boedtker H. Biochemistry. 1977; 16: 4743-4751Crossref PubMed Scopus (2399) Google Scholar). The RNA was blotted from the gels onto nitrocellulose membranes and baked at 80 °C for 2 h in a vacuum oven. Membranes were prehybridized at 42 °C in 50% formamide, 1 × Denhardt's solution, 25 mm KPO4 (pH 7.4), 5 × SSC (1 × SSC = 150 mm NaCl, 15 mm sodium citrate), and 50 μg/ml denatured and fragmented salmon sperm DNA. Hybridizations were at 42 °C in prehybridization solution containing 10% dextran sulfate and radiolabeled HSP70, HSP28, GAPDH cDNA probes at a concentration of 1–2 × 106 cpm/ml. For post-hybridization, blots were washed twice in 2 × SSC for 15 min at room temperature, washed once in 0.5 × SSC and 0.1% SDS for 25 min at 50 °C, and washed twice in 0.2 × SSC and 0.1% SDS for 1 h at 50 °C. Blots were placed into a stainless steel cassette with intensifying screen and autoradiographed. Conditions for the gel mobility shift assay, a description of the 32P-labeled HSE oligonucleotide, and preparation of whole cell extracts were as published previously (31Demay M.B. Kiernan M.S. DeLuca H.F. Kronenberg H.M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8097-8101Crossref PubMed Scopus (368) Google Scholar, 32Lee Y.J. Galoforo S.S. Berns C.M. Erdos G. Gupta A.K. Ways D.K. Corry P.M. J. Biol. Chem. 1995; 270: 28790-28796Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 33Mosser D.D. Theodorakis N.G. Morimoto R.I. Mol. Cell. Biol. 1988; 8: 4736-4744Crossref PubMed Scopus (339) Google Scholar). A double-stranded HSE (upper strand 5′-CTTAACGAGAGAAGGTTCCAGATGAGGGCTGAA-3′, Ref. 34Hunt C. Morimoto R.I. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 6455-6459Crossref PubMed Scopus (697) Google Scholar) oligonucleotide of the human HSP70 gene promoter was used. Bold nucleotides represent essential sites for HSF. Binding reactions with 20 μg of whole cell extracts for HSE were performed for 15 min at 25 °C in a final volume of 25 μl of binding buffer (10 mm Tris-HCl, pH 7.5, 50 mm NaCl, 1 mm EDTA, 5% glycerol, 1 mm dithiothreitol for HSF; 10 mm HEPES, pH 8.0, 15% glycerol, 2 mmEDTA, 0.5 mm spermidine, 20 mm NaCl, 4 mm MgCl2, 2 mm dithiothreitol for HSE) containing about 0.5 ng of radiolabeled probe, 10 μg of yeast tRNA (Boehringer Mannheim, Indianapolis, IN), 1 μg of Escherichia coli DNA (Sigma), 2 μg of poly(d[I-C]) (Pharmacia LKB, Piscataway, NJ), 50 μg of bovine serum albumin (Sigma). Samples were electrophoresed on a nondenaturing 4.5% polyacrylamide gel for 2.5 h at 140 V. After electrophoresis, gels were fixed with 7.5% acetic acid for 15 min and rinsed with water for 3 min. For autoradiography, gels were dried in a slab gel dryer (Model 483, Bio-Rad) for 1.5 h at 80 °C and placed into a stainless steel cassette with an intensifying screen. Gels were autoradiographed on Fuji RX x-ray film. After an exposure of 2–4 days at −70 °C, autoradiographic film was developed with Kodak GBX developer, and fixed with Kodak GBX fixer. To investigate whether glucose deprivation can activate heat shock gene expression, MCF-7/ADR cells were exposed to glucose-free medium for 1 h and then incubated in complete medium before harvest. Relative levels of HSP70 and HSP28 mRNA were determined using the Northern blot technique. Equal loading of the RNA was confirmed by rehybridizing the nitrocellulose membrane with a probe for the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Northern blots in Fig. 1 show significant differences in the intrinsic level of HSP70 and HSP28 mRNA. A low level of HSP70 mRNA was detected in untreated control cells. In contrast, a much higher level of HSP28mRNA was detected in these cells. The levels of both mRNAs increased rapidly and reached a maximal value within 2 h after glucose deprivation for 1 h. The level of HSP70 orHSP28 mRNA increased approximately 22- or 1.5-fold, respectively, as determined by densitometry. Similar results were observed by one-dimensional SDS-PAGE analysis (Fig. 2). To investigate the glucose deprivation-induced synthesis of heat shock proteins, cells were labeled for 2–8 h with [3H]leucine after glucose deprivation for 1 h. Fluorography of an SDS-PAGE of [3H]leucine-labeled proteins shows that the level of HSP70 significantly increased and reached its maximum value within 4 h after glucose deprivation for 1 h. Unlike HSP70, the level of HSP28 protein was not detectably altered. These results were confirmed by two-dimensional SDS-PAGE analysis (Fig. 3). For this study, cells were either heated at 45 °C for 15 min or exposed to glucose-free medium for 1 h and then labeled with [3H]leucine for 6 h. The results from two-dimensional SDS-PAGE clearly demonstrated that glucose deprivation induced a significant increase in the level of inducible HSP70 but not constitutive HSP70 (Fig. 3 C). In contrast, the level of both HSP70s increased after heat shock (Fig. 3 B). These two forms of HSP70 which can be separated by their molecular size and charge are highly related to each other. The constitutive HSP70, 73 kDa, is an abundant protein in the normal unstressed cell. The inducible HSP70, 72 kDa, is synthesized at very high levels after stress. In most cells, synthesis of the inducible HSP70 occurs only after stress. However, in human cells, synthesis of both constitutive and inducible HSP70 is observed in cells grown under normal conditions (Fig. 3 A).Figure 2One-dimensional SDS-polyacrylamide gel electrophoretic analysis of proteins. MCF-7/ADR cells were exposed to glucose-free medium for 1 h and then labeled with 20 μCi/ml [3H]leucine in complete medium for 2–8 h, as indicated at the bottom of each lane. Lysates from cells were analyzed and 3H-labeled proteins were detected by fluorography. The location of inducible HSP70 is identified. C, untreated control cells.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Two-dimensional SDS-polyacrylamide gel electrophoretic analysis of proteins. MCF-7/ADR cells were heated at 45 °C for 15 min (panel B) or exposed to glucose-free medium for 1 h (panel C) and then labeled with 20 μCi/ml [3H]leucine for 6 h in leucine-free medium. Lysates from cells were analyzed and 3H-labeled proteins were detected by fluorography. Only a section of the fluorograph is shown. The locations of HSP28a (a), HSP28b (b), constitutive HSP70 (a), inducible HSP70 (b), HSP90, HSP110, and actin (A) are identified. Panel A, unheated control cells.View Large Image Figure ViewerDownload Hi-res image Download (PPT) It is well known that heat shock proteins are involved in the development of tolerance to stresses (35Li G.C. Mak J.Y. Cancer Res. 1985; 45: 3816-3824PubMed Google Scholar). To examine whether glucose deprivation-induced heat shock proteins can enhance resistance to stresses, MCF-7/ADR cells were exposed to glucose-free medium for 1 h and then incubated for various times in complete medium before being challenged to glucose deprivation for 4 h (Fig. 4) or heat shock at 45 °C for 1 h (Fig. 5). Survival fraction was plotted as a function of various times (2–16 h) between pretreatment and challenge treatment. Data from Figs. 4 and 5show that tolerance rapidly developed within 5 h and was maintained over 16 h. The time course for development of tolerance was similar for the two types of stress. For example, tolerance to glucose deprivation-induced cytotoxicity was observed as a 15-fold increase in survival from 4 × 10−2 to 6 × 10−1 after 8 h in complete medium (Fig. 4). In parallel, thermotolerance was observed as a 17-fold increase in survival from 4 × 10−2 to 7 × 10−1 after 8 h in complete medium (Fig. 5).Figure 5The time course for thermotolerance development by glucose deprivation. ▪, cells were incubated for various intervals (2–16 h) in complete medium after pretreatment with glucose-free medium for 1 h and then challenged to heat shock at 45 °C for 1 h. ■, cells were heated at 45 °C for 1 h without pretreatment. The survival fraction was plotted as a function of various incubation intervals in complete medium. The data are a compilation of two separate experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Previous studies have shown that glucose deprivation induces an increase in intracellular hydroperoxide production (26Lee Y.J. Galoforo S.S. Berns C.M. Chen J.C. Davis B.H. Sim J.E. Corry P.M. Spitz D.R. J. Biol. Chem. 1998; 273: 5294-5299Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar) which may lead to activation of heat shock gene expression. We have also observed that overexpression of bcl-2 prevents an increase in oxidized glutathione content which is an indicator of oxidative stress (data not shown). In this study, we investigated whether overexpression of bcl-2 prevents glucose deprivation-induced heat shock gene expression. For this study, cells were transfected with plasmid pCMV-bcl-2 containing the human BCL-2 cDNA gene and then stable transfectants were selected. Fig. 6 shows an overexpression of bcl-2 in pCMV-bcl-2 (pBcl-2) vector transfected cells. The level of bcl-2 did not significantly change during incubation in complete medium after 1 h of glucose deprivation in control vector pCMV (pNeo) or pBcl-2-transfected cells. Effects of overexpression of bcl-2 on glucose deprivation-induced synthesis of heat shock proteins was observed in these transfected cells (Fig. 7). Data from two-dimensional SDS-PAGE show that glucose deprivation-induced inducible HSP70 gene expression was markedly reduced in pBcl-2 transfected cells (Fig. 7). To determine whether the suppression of HSP70 gene expression was due to the alteration of upstream regulation of transcription, HSF-HSE binding activity was measured by gel mobility shift assay. It is known that the binding of HSF to HSE is necessary for transcriptional activation of eukaryotic heat shock genes (5Sorger P.K. Lewis M.J. Pelham H.R.B. Nature. 1987; 329: 81-84Crossref PubMed Scopus (305) Google Scholar, 36Pelham H.R.B. Cell. 1982; 30: 517-528Abstract Full Text PDF PubMed Scopus (647) Google Scholar). Gel mobility shift analysis of whole cell extracts from glucose-deprived cells showed the formation of the HSF-HSE complex (Fig. 8). The HSF binding activity was markedly reduced in pBcl-2 transfected cells. This may be due to inhibition of the signal transduction pathway by reducing oxidative damage in pBcl-2-transfected cells. It is well known that metabolic oxidative stress induces the SAPK pathway (37Mendelson K.G. Contois L.-R. Tevosian S.G. Davis R.J. Paulson K.E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12908-12913Crossref PubMed Scopus (211) Google Scholar) which has been suggested to be involved in the activation of HSF (13Kim J. Nueda A. Meng Y.H. Dynan W.S. Mivechi N.F. J. Cell. Biochem. 1997; 67: 43-54Crossref PubMed Scopus (73) Google Scholar). Western blots in Fig. 9 show that glucose deprivation indeed activated JNK1 and this activation was markedly reduced in pBcl-2-transfected cells.Figure 7Two-dimensional SDS-polyacrylamide gel electrophoretic analysis of proteins in pCMV (pNeo) or pCMV-bcl-2 transfected (pBcl-2) MCF-7/ADR cells. Panels B and D, cells were exposed to glucose-free medium for 1 h and then labeled with 20 μCi/ml [3H]leucine for 6 h in leucine-free medium. Lysates from cells were analyzed and 3H-labeled proteins were detected by fluorography. Only a section of the fluorograph is shown. The locations of constitutive HSP70 (a), inducible HSP70 (b), and actin (A) are identified. Panels A and C, untreated control cells.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 8Detection of a HSF-HSE in control plasmid pCMV (pNeo) or pCMV-bcl-2 (pBcl-2) transfected MCF-7/ADR cells. Cells were exposed to glucose-free medium for 1 h and then incubated in complete medium for various intervals (0–2 h). The gel mobility shift assay was performed with a32P-labeled HSE and whole cell extracts (20 μg of protein) prepared from untreated control cells (C) or treated cells (0–2). Closed arrow indicates the position of the HSF-HSE complex. Open arrow indicates a free32P-labeled oligonucleotide fragment.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 9Immunoblot detection of active form of JNK1 or actin in transfected MCF-7/ADR cells. Cells were stably transfected with pNeo or pBcl-2. Transfectants were exposed to glucose-free medium for various intervals (5–120 min) as indicated at the bottom of each lane. Western blot analysis was done as described in the legend to Fig. 6 with anti-ACTIVE JNK antibody or anti-actin antibody. C, untreated control cells.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To further evaluate the involvement of the signal transduction pathway in glucose deprivation-induced heat shock gene expression, cells were transfected with the plasmid pcDNA3-FLAG-JNK1 (APF) containing the dominant-negative mutant of JNK1 which blocks the SAPK pathway (17Gupta S. Campbell D. Derijard B. Davis R.J. Science. 1995; 267: 389-393Crossref PubMed Scopus (1336) Google Scholar). Stable transfectants were exposed to glucose-free medium for various times (5–120 min) and the active form of JNK1 was detected by immunoblot assay (Fig. 10) or HSP70 synthesis was determined by one-dimensional SDS-PAGE analysis (Fig. 11). Figs. 10 and 11 show that glucose deprivation-induced JNK1 activation as well as HSP70 synthesis was markedly reduced in pcDNA3-FLAG-JNK1 (APF)-transfected cells.Figure 11Fluorograph of a SDS-polyacrylamide slab gel of [3H]leucine-labeled proteins. Stably transfected MCF-7/ADR cells with control plasmid (pNeo) or dominant-negative mutant of JNK1 expression plasmid pcDNA3-FLAG-JNK1 (APF) were exposed to glucose-free medium for 1 h and then labeled with 20 μCi/ml [3H]leucine in complete medium for 2–8 h, as indicated at the bottom of each lane. Lysates from cells were analyzed and 3H-labeled proteins were detected by fluorography. The location of inducible HSP70 is identified. C, untreated control cells.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Several conclusions can be drawn upon consideration of the data presented. Glucose deprivation which is known to induce metabolic oxidative stress activates HSF and subsequently induces heat shock gene expression, in particular inducible HSP70 gene expression (Figs. Figure 1, Figure 2, Figure 3 and 8). These results are consistent with observations which demonstrate that oxidative stress can activate HSF and induceHSP70 gene expression (25Gomer C.J. Ryter S.W. Ferrario A. Rucker N. Wong S. Fisher A.M. Cancer Res. 1996; 56: 2355-2360PubMed Google Scholar). These observations were confirmed by overexpression of bcl-2 which may regulate an antioxidant pathway at sites of free radical generation (38Hockenbery D. Nunez G. Milliman C. Schreiber R.D. Korsmeyer S.J. Nature. 1990; 348: 334-336Crossref PubMed Scopus (3530) Google Scholar, 39Hockenbery D.M. Oltvai Z.N. Yin X.-M. Milliman C.L. Korsmeyer S.J. Ce" @default.
• W2077673859 created "2016-06-24" @default.
• W2077673859 creator A5040610316 @default.
• W2077673859 creator A5081161443 @default.
• W2077673859 date "1998-11-01" @default.
• W2077673859 modified "2023-10-18" @default.
• W2077673859 title "Metabolic Oxidative Stress-induced HSP70 Gene Expression Is Mediated through SAPK Pathway" @default.
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