- Email: [email protected]
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relationship was also observed for the sulfoxidation of S-benzyl-L-cysteine by rabbit FMO-1 and FMO-3, previouslyY Conclusion These methods can be used to easily and sensitively assess the fl elimination and oxidative deamination activities of SeCys conjugates by several enzymes. Using selective enzyme inhibitors, the relative contributions of the different enzymes in tissues can be delineated. This study demonstrates that SeCys conjugates are metabolized by at least three different enzyme systems, namely cysteine conjugate fl-lyase/GTK, L-AAO, and FMO. The present results implicate that four biologically active products can be formed from SeCys conjugates by these enzymes: selenols, selenenic acids, selenoxides, and hydrogen peroxide (Fig. 3). Theoretically, seleninic acids, of which methylseleninic acid displays chemopreventive activity,2 can also be formed via oxidation of selenenic acids by hydrogen peroxide. Which of these compounds accounts for the potent chemopreventive activity of SeCys conjugates remains to be established. 25 R. J. Duescher, M. E Lawton, R. M. Philpot, and A. A. Elfarra, J. Biol. Chem. 269, 17525 (1994).
[20] Gene Expression a n d Thiol Redox State By CAROLE KRETZ-REMY a n d ANDRE-PATRICK ARRIGO
Introduction Cells have developed sophisticated mechanisms to maintain redox state homeostasis and/or to try to cope with the reactive oxygen species (ROS) produced during oxidative stress. 1'2 These mechanisms either scavenge and/or detoxify ROS, block their production, or sequester transition metals that are a source of free electrons. They include detoxifing enzymes, vitamins C and E, or thiol-containing molecules such as glutathione or thioredoxin. Glutathione exists in either a reduced (GSH) or an oxidized (glutathione disulfide, GSSG) form and participates in redox reactions through the reversible oxidation of its active thiol. In addition, it acts as a coenzyme of numerous enzymes involved in cell defense (i.e., glutathione peroxidases, glutathione S-transferases, thiol transferases, formaldehyde dehydrogenase, I H, Sies, Am. J. Med. 91, 31S (1991). 2 H, Sies, Eur J. Biochem. 215, 213 (1993).
METHODSIN ENZYMOLOGY,VOL.348
Copyright© 2002by AcademicPress. All rightsof reproductionin any formreserved. 0076-6879/02 $35.00
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glyoxalase I, and maleylacetoacetate isomerase). In unstressed cells, the majority of glutathione is in its reduced form and its concentration is in the range of 0.5 to l0 mM depending on the cell type. Studies aimed at understanding the mechanisms by which oxidative stress and/or variation in the intracellular redox state modulate gene expression have focused on the crucial role played by transcription factors. Once activated, these proteins bind to the promoter regions of target genes and allow their transcription by RNA polymerase II. Two steps in the transcriptional activation of several eukaryotic transcription factors are regulated by the redox balance: (i) the mechanism of activation of the transcription factor, which induces its redistribution into the nucleus, and (ii) the binding of the transcription factor to the promoter region located in the 5' end of the target genes. For the first point, many different steps of the pathways leading to the activation of transcription factors are under the control of intracellular redox state and are discussed later. Conceming the second point, it is well known that some transcription factors contain cysteine residues (which are redox sensitive through their thiol group) localized in their DNA-binding domain. These cysteines are often essential for the recognition of the transcription factorbinding site through electrostatic interactions with specific DNA bases. Oxidation of these cysteines usually inhibits the ability of the trancription factor to transactivate gene expression. The presence of thioredoxin (TRX) 3 and/or redox factor 1 (Ref-1)4 is often required to locally promote a reducing environment that avoids the oxidation of these cysteines. It is also possible that oxidized cysteines regulate the tridimensional structure of the transcription factor or its binding to a cofactor (a transcription factor that is active as a dimer). Finally, cysteines and/or histidines, localized at specific sites can interact with a zinc atom to form a "zinc finger" structure that fits in the large DNA groove and are responsible for the binding of the protein to DNA. Consequently, because of the sensitivity of the thiol group to redox state variations, the activity of several transcription factors can be modified profoundly if their cysteine residues are oxidized or if they are unable to form appropriate disulfide bounds. Moreover, simple oxidation of the sulfur atom of cysteine by oxygen atoms (in -SOH or -SO2H) can be critical. Variations in the intracellular redox state mediated by oxidative stress can therefore transiently modify the activity of several transcription factors. Depending on the transcription factor and of its own mechanism of activation, this modulation can be positive or negative and therefore can upregulate or downregulate gene expression. The goal of this article is to review different practical approaches that are commonly used to analyze eukaryotic transcription factors whose activation and DNA binding are controlled by the thiol redox status of the cell. Emphasis will be
3 A. Holmgren, J. Biol. Chem. 264, 13963 (1989). 4 S. Xanthoudakis and T. Curran, EMBO J. 11, 653 (1992).
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given to two well-defined transcription factors: NF-tcB (nuclear factor KB)5 and HSF1,6 which confer redox state-dependent inducible gene expression. NF-KB The more frequent form of NF-KB is a heterodimeric complex that contains p50 and p65 polypeptides. Except for B lymphocytes, where it is constitutively active, in most cells NF-~cB is cytoplasmic and needs extracellular signals to be activated and to migrate in the nucleus where it binds KB sites (5'-GGGAN/NYYCC-3') on the DNA. 5 In the cytoplasm, NF-~cB is associated with a family of cytoplasmic inhibitors, the most frequent one being IKB-ot. On activation by oxidative stress, NF-KB/IKB-ot complexes are destroyed, allowing free NF-KB dimers to migrate into the nucleus and activate the transcription of tcB sites containing genes. This process is triggered by the phosphorylation of IKB-ot by IKB kinase. This event induces the ubiquitinylation of boB-or and its subsequent degradation by the proteasome. 5 Several studies have reached the conclusion that oxidative conditions in the cytoplasm stimulate the phosphorylation and subsequent degradation of boB-or, leading to the activation of NF-JcB. 6'7 Reducing nuclear conditions provided by TRX are required in some cellular types to favor the binding of NF-KB to DNA 8 (Fig. 1). Heat Shock Factor 1 Heat shock factor 1 (HSF1) is a transcription factor that regulates the expression of stress (heat shock) genes. HSF1 cytoplasmic activation is mediated by stresses, such as heat shock, or numerous different agents or conditions that generate abnormally folded proteins 9 (Fig. 1). Transcription of heat shock (or stress) genes requires the formation of a homotrimeric HSF1 complex that binds to the heat shock promoter element (HSE). HSE is characterized as multiple adjacent and inverse iterations of the 5'-nGAAn-3' motif. 6 The conversion of HSF1 from a monomer to a trimer state is induced by heat shock. It has been reported that agents or conditions that trigger the oxidation of thiol-containing molecules, leading to the formation of oxidized glutathione disulfide (GSSG), glutathione-protein mixed disulfides, and protein-protein disulfides, induce the trimerization of HSF1 and its binding to DNA) ° In contrast, incubation of cells (but not cell extracts) 5 p. A. Baeuerle and D. Baltimore, Cell 87, 13 (1996). 6 R. I. Morimoto, Genes Dev. 12, 3788 (1998). 7 C. Kretz-Remy, P. Mehlen, M. E. Mirault, and A.-P. Arrigo, J. CellBiol. 133, 1083 (1996). 8 M. T. Anderson, F. J. Staal, C. Gitler, and L. A. Herzenberg, Proc. Natl. Acad. Sci. U.S.A. 91, 11527 (1994). 9 j. T. Westwood and C. Wu, Mol. Cell. Biol. 13, 3481 (1993). 10 j. Zou, W. F. Salminen, S. M. Roberts, and R. Voellmy, Cell Stress Chaperones 3, 130 (1998).
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Nucleus
FIG. 1. Schematic illustration of redox-dependent steps in the activation of NF-~cB and HSF1. Thiol-containing agents can have a positive action by facilitating the binding of NF-xB to DNA (indicated in italics). In contrast, in heat-shocked cells, thiol-containing agents counteract the oxidation of intracellular thiols and block HSF1 activation. Thiols can also inhibit ROS accumulation and therefore inhibit NF-KB activation by oxidative stress. P, phosphorylated serine; Ub, ubiquitin, IKK, IxB kinase, TRX, thioredoxin. Adapted from A.-P. Arrigo, Free Radic. Biol. Med. 27, 936 (1999).
with dithiothreitol (DTT) inhibits HSF1 trimerization and the transcriptional activation of heat shock genes. 11 Two steps in HSFI activation have been identified: (i) disruption of intracellular thiol-disulfite redox homeostasis, which leads to the formation of disulfide-linked aggregates of cellular proteins, and (ii) recognition of denatured proteins by preexisting protein chaperones, a phenomenon that triggers HSF1 trimerization without its apparent oxidation. 1° Concerning HSF1 activation by hydrogen peroxide or other types of oxidants, it has been shown that these agents stimulate the nuclear translocation of HSF 1 but interfere with HSF1-DNA-binding activity by oxidizing critical HSFI cysteine residues. The intensity of the latter phenomenon is decreased by TRX, which is upregulated by oxidative stress prior to HSF1 activation. Hence, reducing conditions generated by TRX upregulation help HSF1 DNA-binding activity during oxidative stress, la
11 L. E. Huang, H. Zhang, S. W. Bae, and A. Y. Liu, J. Biol. Chem. 269, 30718 (1994). 12 M. R. Jacquier-Sarlin and B. S. PoUa, Biochem. J. 318, 187 (1996).
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D e t e r m i n a t i o n o f I n t r a c e l l u l a r G l u t a t h i o n e Level Glutathione is a naturally occurring tripeptide whose nucleophilic and reducing properties play a central role in the antioxidant system of most aerobic cells. The total cellular glutathione level can be measured by fluorescence-activated cell sorter (FACS) analysis using a monochlorobimane (MCB) fluorescent probe. 13,14About 5 × 105 cells/ml are incubated for 5 min with 40 mM MCB added to the growth medium. Fluorescence at 425 nm is analyzed in response to an excitation at 395 nm in a Becton Dickinson Vantage FACS (Le-Pont-de-Claix, France) equipped with a 37 ° sample chamber. Total glutathione content can also be estimated using the Bioxitech GSH-400 colorimetric assay of Oxis International (Portland, OR). The GSH-400 method is based on a chemical two-step reaction. The first step leads to the formation of thioethers adducts between a patented reagent and all mercaptans present in the sample. The second step takes advantage of the structure of the glutathione, which undergoes fl elimination at high pH, leaving the reagent in the form of a chromophoric thione, which has a maximal absorbance wavelength at 400 nm. Briefly, total cellular protein material from 6 × 106 cells is precipitated in 5% (w/v) metaphosphoric acid and the resulting supernatant is used for the test. The level of glutathione in each sample is calculated according to standard curves of increasing GSH concentrations. The enzymatic method described by Eyer and Podhrasky 15is used to determine the level of glutathione in its reduced or oxidized form. Harvested cells, washed in phosphate-buffered saline (PBS) are resuspended in 1 Mperchloric acid containing 1 mM EDTA. The mixture is kept at - 2 0 ° for at least 2 hr before adding an equal volume of 1.3 M K2HPO4 to the samples. It is then stirred for 30 min before insoluble crystals of KC104 salt are removed by centrifugation and analyzed according to Eyer and Podhrasky. 15 A procedure leading to the elimination of GSSG from total glutathione can be used to estimate the level of oxidized glutathione disulfide. In this case, cells are resuspended in a medium containing 1 M perchloric acid, 1 mM EDTA, and 0.02 M NEM (N-ethylmalemide). GSSG and NEM are then removed by five extractions with ice-cold, H20-saturated ethyl acetate.
Analysis of Formation of Glutathione-Protein Disulfites The method described by Zou e t al. lo can be used. Ceils are incubated for at least 5 hr in growth medium lacking cysteine, cystine, and methionine, but containing
13 D. C. Shrieve, E. A. Bump, and G. C. Rice, J. Biol. Chem. 263, 14107 (1988). 14 D. J. Kane, T. A. Sarafian, R. Anton, H. Hahn, E. B. Gralla, J. S. Valentine, T. Ord, D. E. Bredesen, Science 262, 1274 (1993). 15 p. Eyer and D. Podhradsky, Anal Biochem. 153, 57 (1986).
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10% fetal calf serum. Cycloheximide (50/zg/ml) is added for 30 min before the cells are incubated for 2 hr with 20 IzCi/ml of [35S]cystine (113 Ci/mmol). Cells are then exposed to different agents or conditions (i.e., heat shock) whose actions are tested. Cells are washed with phosphate-buffered saline (PBS) and lysed by scraping in cold 10 mM sodium phosphate buffer (pH 7.0) containing 15 mM N-ethylmaleimide, 5 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride (PMSF). Cell lysates are clarified by a 5-min spin at 8700g. The protein concentration is determined using the DC protein assay of Bio-Rad. Samples are mixed with twotime concentrated SDS-PAGE sample buffer lacking reducing agents before being boiled and applied to 7.5% SDS-PAGE gels. Gels are soaked in 1 M sodium salicylate (pH 6.0) before being dried and exposed to X-ray films for fluorography.
In Vivo F l u o r e s c e n t M e a s u r e m e n t of I n t r a c e U u l a r R e a c t i v e Oxygen Species 2 r,71-Dichlorofluorescin Diacetate Probe 2',7'-Dichlorofluorescin diacetate (DCFH-DA) diffuses rapidly into the cells, is hydrolyzed to 2',7'-dichlorofluorescin (DCFH), and is thereby trapped within the cells. Intracellular DCFH is converted to highly fluorescent 2',7'-dichlorofluorescein (DCF) by ROS-mediated oxidation. 16 Adherent cells 5 x 105 are trypsinized and rinsed three times with PBS. Thereafter, cells are incubated at 37 ° for various periods of time (from 10 to 60 min) with PBS containing 0.1 mg/ml of DCFH-DA. DCF fluorescence analysis is performed using a FACScalibur cytometer (Becton Dickinson) using a 488-nm excitation wavelength and a 530-nm emission filter bandpassed for DCE The slope of each curve obtained is representative of the level of ROS produced.
Hydroethidine Probe Hydroethidine, the sodium borohydride reduced form of ethidium, is freely permeable to cells and can be oxidized to fluorescent ethidium bromide by ROS. Cells (1.5 x 106) are trypsinized and resuspended into PBS containing 40/zg/ml of hydroethidine. Cells are then incubated at 37 ° for 10 min; the reaction is stopped by incubation on ice. Flow cytometric analysis is performed using a FACScalibur cytometer using a 488-nm excitation wavelength. The emission filter is 610 nm bandpassed for ethidium bromide fluorescence.
16D. Bass, J. W. Parce,L. R. Dechatelet,E Szejda,M. C. Seeds,and M. Thomas,J. lmmunol. 130, 1910 (1983).
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TABLE I COMPOUNDSCOMMONLYUSED TO STUDY EFFECTS OF INTRACELLULAR REDOX STATE ON GENE EXPRESSION Antioxidants Pyrrolidine dithiocarbamate (PDTC) N-Acetyl-L-cysteine (NAC) Glutathione ethyl ester
Oxidants Methyl viologen (paraquat) L-Buthionine (S,R) sulfoximine (BSO) Menadione(2-methyl- 1,4-naphthoquinone) Hydrogen peroxide Diamide [diazenedicarboxylic acid bis(N,N-dimethylamide)]
A n t i o x i d a n t a n d O x i d a n t D r u g s C o m m o n l y U s e d to T e s t R e d o x State Dependence of Gene Expression Antioxidants
PDTC and NAC are commonly used antioxidant drugs (see Table I). The former is a component carrying thiol groups and the latter is a precursor of GSH synthesis. Treatments of HeLa cells with 100/zM PDTC or 30 mM NAC for 1 hr is responsible for the inhibition of NF-~cB activation by oxidative stress. 7 GSH ethyl ester is freely permeable to cells and increases the intracellular GSH level. A 5 mM GSH ethyl ester treatment for 12 hr increases the level of GSH in L929 cells by 20%J 7 Oxidants
BSO is a glutathione synthesis inhibitor. A 24-hr treatment with I mM BSO depleted 99% of glutathione in murine L929 cells.18 Diamide is a specific oxidant for thiol groups: a 2-hr treatment with 2 mM diamide drastically decreased the GSH level in benefit of GSSG in human red blood cells. 19 A 2 mM treatment with hydrogen peroxide is also responsible for the rapid oxidation of GSH to GSSG. 19 Menadione is a quinone reagent that produces oxidative modifications in biological systems mainly through redox cyclin reactions with oxygen (it binds to thiol groups to form a conjugate able to redox cycle like menadione itself) and is responsible, in human red blood cells, for the rapid disappearance of GSH with a negligible production of GSSGJ 9 Paraquat (methyl viologen) is an oxidative stress inducer and decreases the level of GSH and the GSH/GSSG ratio. 2° 17 X. Preville, E Salvemini, S. Giraud, S. Chaufour, C. Paul, G. Stepien, M. V. Ursini, and A. E Arrigo, Exp. Cell Res. 247, 61 (1999). 18 E Mehlen, X. Pr6ville, C. Kretz-Remy, and A.-E Arrigo, EMBO J. 15, 2695 (1996). 19 R. Rossi, A. Milzani, I. Dalle-Donne, E Giannerini, D. Giustarini, L. Lusini, R. Colombo, and E I. Di Simplicio, J. Biol. Chem. 276, 7004 (2001). 20 S. G. Konstantinova and E. M. Russanov, Acta Physiol. Pharmacol. Bulg. 24, 107 (1999).
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A c t i v a t i o n of NF-KB a n d H S F I a n d T h e i r M i g r a t i o n into the Nucleus
NF-KB NF-xB activators induce the phosphorylation and subsequent degradation of IxB-ot, which result in the destruction of the cytoplasmic NF-KB/IxB complex. Free NF-tcB can then migrate into the nucleus. This complex phenomenon can be detected using several approaches. IxB-ot Phosphorylation and Degradation. To date, except in minor cases, NF-xB activation promoted by modulation of the redox state follows a universal pathway, which begins with IKB-ot phosphorylation, multiubiquitination, and degradation by the 26S proteasome. Those events can therefore be considered as markers of NF-KB activation. ANALYSISAFTERONE-DIMENSIONALSDS-PAGE. Cells (1.5 × 106) grown in 60-mm dishes are washed with cold PBS. Cells are then scraped from the dishes and pelleted at 1000g for 5 min. The cellular pellet is lysed and boiled in Laemmli sample buffer (50 mM Tris-HCl, pH 6.8, 2% SDS, 50 mM 2-mercaptoethanol, 7.5% glycerol, and 0.05% bromphenol blue) before being analyzed in SDS-PAGE made of 10% acrylamide running and 5% acrylamide stacking gels. The gel is electrophoresed at 30 mA (cooling system required). ANALYSISAFTERTWO-DIMENSIONALSDS-PAGE. Cellular extracts lysed and boiled in Laemmli sample buffer are incubated for 2 hr at room temperature with DNase (10,000 U) and RNase (10/xg) and thereafter heated at 70 ° for 5 min. After lyophilization, samples are resuspended in isoelectrofocusing sample buffer (9.95 M urea, 4% NP-40, 2% pH 6-8 ampholines, 0.1 M DTT). Samples are loaded at the anodic end of an isoelectrofocusing gel made up with 5.5 g urea, 1.26 m130% acrylamide, 1.07 ml 2% bisacrylamide, 2 ml NP-40, 0.97 ml H20, 0.2 mlpH 3-10 ampholines, 0.3 ml pH 4--6 ampholines, 10/zl N,N,N',N'-tetramethylethylenediamine (TEMED), and 10/~1 of 10% ammonium persulfate. Electrofocusing is for 600 V for 16 hr and 1000 V for 1 hr. SDS-PAGE in the second dimension is performed as mentioned previously. Immunoblot Analysis. Immunoblot blot analysis of one- or two-dimensional SDS-PAGE is performed by transferring the separated proteins from the acrylamide gel to a PVDF membrane (Immobilon P, pore size 0.45/zm, Millipore, Bedford, MA) using a liquid or semidry blotting system (Bio-Rad, Hercules, CA) in 25 mM "iris, 190 mM glycine, and 10% (v/v) methanol buffer. The membrane is then incubated in TBS (20 mM Tris-HC1, pH 7.6, 137 mM NaC1) 0.1% Tween, and 5% dry milk for 2 hr at room temperature to block unspecific binding sites. Thereafter, the membrane is incubated for 1 hr at room temperature with anti-IxB-~ (Santacruz Biotechnology) diluted at 1/zg/ml in TBS 0.1% Tween, 0.1% bovine serum albumin (BSA) as the primary antibody. After three washes in TBS 0.1% Tween, the immunoblot is probed with a goat anti-rabbit immunoglobulin diluted
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1/10,000 in T B S 0.1% Tween, 0.1% B S A and r e v e a l e d with the E C L (enhanced c h e m i l u m i n e s c e n c e ) kit f r o m A m e r s h a m (Orsay, France). The duration o f exposure is calculated to be in the linear response o f the film. In the E C L revealed i m m u n o b l o t presented in Fig. 2A, it is possible to observe a slow migrating IKB-ot phospho i s o f o r m after H202 treatment and its rapid disappearance due to proteolytic degradation. NF-tc B Migration in the Nucleus IMMUNOFLUORESCENCE APPROACH. Cells g r o w i n g on glass coverslips coated with 0.1% type B gelatin are submitted to various NF-~cB activators. Thereafter, the cells are rinsed twice with PBS at 37 ° and fixed for 90 sec with c o l d methanol. A f t e r two washes in cold PBS, coverslips are incubated for 1 hr at r o o m temperature with anti-p65 or anti-p50 antibodies (Santa Cruz B i o t e c h n o l o g y ) diluted 1/100 in PBS containing 0.1% B S A . Coverslips are then w a s h e d three times in c o l d PBS and incubated for 1 hr at r o o m temperature with goat anti-rabbit imm u n o g l o b u l i n c o u p l e d to isothiocyanate diluted 1/200 in P B S 0.1% B S A . Staining specificity is assessed by control coverslips solely incubated with the second
FIG. 2. Analysis of IKB-a phosphorylation and degradation in response to hydrogen peroxide treatment. (A) HeLa cells were treated for various time periods with 250 #M of hydrogen peroxide in the presence or absence of 60 mM of the proteasome inhibitor N-Cnz-lleu-Glu [o-tert-butyl]-Ala-leucinal (PSI). Cells were harvested, and equal amounts of total cellular proteins were separated in SDS-PAGE. The cellular contents of IKB-ct isoforms were analyzed in an immunoblot probed with the IrB-ot antibody. Note the presence of a slow migrating l~cB-otphospho isoform after 5 min of treatment with hydrogen peroxide. 1KB-otis no more detectable after 15 min of treatment due to proteolytic degradation by the proteasome, a phenomenon not observed in the presence of the proteasome inhibitor PSI. Reproduced from Kretz et al. (I 998), by copyright permission of the American Society of Microbiology. (B) Analysis of NF-xB binding to the KB DNA site. Murine L929 cells were either left untreated (C) or treated with tumor necrosis factor a (TNFot) (20, 200, and 2000 U/ml for 2 hr) in the presence or absence of 30 mM of N-acetylcysteine (NAC) added 30 min before TNFot treatment. Nuclear extracts were prepared, and equal amounts of nuclear proteins were incubated with a 32p-labeled DNA probe encompassing the xB sites. Samples were analyzed on a native 4% polyacrylamide gel. An autoradiograph of the gel is presented. Supershift (sps) was performed by adding an antiserum recognizing the p65 subunit of NF-KB to the binding mixture of 2000 U/ml TNFa-treated cell extracts. (C and D) Analysis of ~B-dependent transcription using a reporter gene assay. Control (C) and glutathione peroxidase-overexpressing (D) human breast cancer T47D cells were cotransfected with either pTKluc (black plot) or pKB6-TKIuc (hatched plot) plasmid, pKB6-TKIuc contains a luciferase gene reporter placed under control of a thymidine kinase promoter coupled to six ~cBelements. Luciferase expression from this construct was compared to that from a similar plasmid but without xB elements (pTKluc). Twenty hours after transfection and replating, cells were either kept untreated or treated for 2 hr with 250/zM H202 or 2000 U/ml TNFot. The medium was changed, and the cells were allowed to recover for 14 hr prior to harvesting and luciferase detection. Note the strong stimulation of luciferase activity in control cells treated with hydrogen peroxide or TNFot, which was only faintly detectable in glutathione peroxidase-expressing cells. Reproduced from C. Kretz-Remy, P. Mehlen, M. E. Mirault, and A. P. Arrigo, J, Cell Biol. 133, 1083 (1996) by copyright permission of the Rockefeller University Press.
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antibody. Stained cells are examined and photographed with a Zeiss axioscop photomicroscope. Cell Fractionation Approach. Cells growing in 60-mm dishes are scraped from the dishes and pelleted for 5 min at 1000g at 37 °. Thereafter, the cells are lysed for 10 min at 4 ° in a buffer containing 10 mM Tris, pH 7.4, 10 mM NaC1, 1.5 mM MgC12, and 0.5% Triton X-100. The lysates are then clarified for 10 min at 12,000g at 37 °. This procedure is repeated until nuclei present in the pellet fraction are free of cytoplasmic contaminations, as judged by microscopical analysis with a TMS-inverted photomicroscope (Nikon Inc.) equipped with phase contrast. The crude nuclear pellets are resuspended or diluted in similar amounts in Laemmli SDS buffer. Cytoplasmic and nuclear samples are then analyzed in 10% SDS-PAGE. After transferring the gel, blots are incubated for 1 hr at room temperature with anti-p65 or anti-p50 antibodies diluted at 2/zg/ml in TBS 0.1% Tween, 0.1% BSA. Revelation of the immunoblot is performed as described earlier.
HSF1 HSF1 Trimerization. Like many inducible factors, HSF1 is synthesized constitutively and stored in a latent cytoplasmic form under normal conditions. HSF1 is activated in response to heat shock by a conversion of HSF1 protein from a monomer to a heterotrimer.9 The HSF trimer migrates in the nucleus and binds to HSEs upstream of heat shock (stress) genes. The trimerization of HSF1 can be observed by gel filtration analysis of cell extracts. 9 To prepare cytoplasmic and nuclear extracts, cells are resuspended and lysed using a Dounce homogenizer in a buffer containing 10 mM N-2-hydroxyethylpiperazine N'-2-ethanesulfonic acid (HEPES; pH 7.9), 1.5 mM MgC12, 10 mM KC1, 0.5 mM DTT, and 0.5 mM PMSE The homogenate is centrifuged at 25,000g for 20 min at 4 °. The resulting nuclear pellet is extracted for 30 min with two original pellet volumes of a buffer containing 10 mM HEPES, pH 7.9, 0.4 M NaCI, 20% glycerol, 0.5 mM DTT, and 0.5 mM PMSF before being centrifuged for 60 min at 100,000g at 4 °. The supernatant of this second step is the heat-shocked nuclear extract, which contains trimerized HSF1. The supernatant of the 25,000g centrifugation step is centrifuged again at 100,000g for 60 min at 4 °. The supematant of this centrifugation is the cytoplasmic extract and contains the bulk of monomeric inactive HSF1. Analysis of native molecular masses of latent and activated HSF1 is performed by gel-filtration chromatography on a Superose 6 HR 10/30 column (Pharmacia, Piscataway, N J). Five hundred microliters of cytoplasmic or heat-shocked nuclear extracts is adjusted to 200 mM KC1 and is applied to the column. The mobile phase contains 200 mM KC1, 20 mM Tris, pH 7.9, 5% (v/v) glycerol, 0.1 mM EDTA, 0.5 mM DTT, and 0.5 mM PMSF. Detection of the HSF1 polypeptide in the different fractions of the
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column is by SDS-PAGE/immunoblot using an antibody that specifically recognizes this protein. HSF1 Migration into the Nucleus. The immnofluorescence approach and cell fractionation approach are performed as described earlier using antibodies that specifically recognize HSF1.
B i n d i n g of T r a n s c r i p t i o n F a c t o r s to DNA This property of transcription factors was analyzed by DNA binding and electrophoretic shift assays as describedl NF-KB Preparation of Nuclear Extracts. Cells (107) are harvested and centrifuged at 1000g for 5 min. The cell pellet is washed twice with ice-cold PBS and resuspended in 400 /zl of ice-cold buffer A (10 mM HEPES, pH 7.5, 1.5 mM MgCI2, 10 mM KC1, 0.5 mM DTT, 0.5 mM PMSF, and 2% aprotinin). DTT, PMSF, and aprotinin should be added extemporaneously. The cell suspension is kept on ice for 15 min. Thereafter, NP-40 is added to a 10% final concentration, and the suspension is vortexed vigorously and centrifuged at 13,000 rpm for 1 min (Eppendorf centrifuge). The supernatant is discarded, and the pellet of nuclei is resuspended in 100/xl of buffer C (20 mM HEPES, pH 7.5, 25% glycerol, 0.42 M NaC1, 1.5 mM MgC12, 0.2 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF, and 2% aprotinin). DTT, PMSF, and aprotinin should be added fresh. The nuclear suspension is kept on ice for 40 min, and the nuclear lysate is centrifuged at 10,000 rpm for 10 min at 4 °. The resulting supernatant is stored at - 8 0 °. An aliquot (5 #1) is taken from the supernatant to determine the protein concentration (Bradford protein assay reagent, Bio-Rad). Labeling of KB Probe. The 5'-overhangs of KB double-stranded oligonucleotides (5'-AGCTTCAGAGGGGACTTTCCGAGAGG-3' and 3'-AGTCTCCCCTG AAAGGCTCTCCAGCT-5') are used to detect the DNA-binding activity of NFKB as described previously.21 Four micrograms of KB oligonucleotides is incubated at room temperature for 15 min with 1 mM dATP, dTTP, and dGTP, 40/zCi of [ot-32p]dCTP in the presence of 2U of Klenow polymerase, and 1X Klenow buffer supplied with the enzyme (Roche Diagnostics). The reaction is stopped by a 5-min incubation at 70 °. Nonincorporated 32p is removed by Sephadex G-50 chromatography. The labeled oligonucleotide is precipitated overnight at - 2 0 ° . 21U. Zabel,R. Schreck,and P. A. Baeuerle,J. Biol. Chem.266, 252 (1991).
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DNA-Binding Reaction, Competition, and Supershifi Assays. Ten micrograms of protein from nuclear extracts is incubated with a 20,000 cpm (Cerenkov) 32p-labeled xB-DNA probe in the presence of 4/zg of poly(dl-dC) (Pharmacia) and 5 mM Tris-HC1, pH 7.5, 0.5 mM DTT, 0.5 mM EDTA, 25 mM NaC1, 1% Ficoll 400 in a final volume of 10 #1. The binding reaction is kept at room temperature for 15 min. To assess the specificity of NF-xB DNA binding, competition experiments are performed with 10 or 40 ng of the cold KB probe added to the binding mix including proteins, just before incubation with the 32p-labeled KB probe. The subunit composition of DNA-binding proteins can be ascertained by supershift experiments in which 2 #g of antibodies raised against the p50 or p65 NF-KB subunits is added to the binding reaction for an additional 30 min. Electrophoresis. Following incubation, samples are analyzed into native 4% acrylamide gel in TBE 0.25X. Autoradiographs of the gel are recorded onto BioMax MR films (Eastman Kodak Co., Rochester, NY). An example of the binding of NF-KB to tcB DNA after TNFot treatment is presented in Fig. 2B. HSF1 Preparation of Nuclear Extracts. Cells are resuspended in cold 20 mM HEPES-KOH buffer, pH 7.9, containing 25% (v/v) glycerol, 0.5 M NaC1, 1.5 mM MgC12, 0.2 mM EDTA, 0.5 mM DTT, 0.5 mg/ml pepstatin A, and 20/zg/ml Pefabloc SC (Merck, Fontenay sous Boix, France). The cell suspension is sonicated at 0 ° for 20 sec and then centrifuged at 4 ° at 125,000g for 10 min. Soluble extracts are frozen in liquid nitrogen and stored at - 8 0 ° . Labeling of HSE Probe. Fifty nanograms of an HSE oligonucleotide (5'-GCCT CGAATGTTCGCGAAGTTTCG-3') is incubated in 20/zl. of 1x kinase buffer (40 mM Tris-HCl, pH 8.5, 10 mM MgC12, and 10 mM 2-mercaptoethanol) with 50/zCi of [y-32p]ATP(9300 Ci/mmol) and 20 U of T4 polynucleotide kinase (Roche Diagnostic). The reaction is stopped by the addition of 2/zl of 0.2 M EDTA. Nonincorporated 32p is removed as mentioned earlier. DNA-Binding Reaction, Competition, and Supershift Assays. Twenty microliters of the reaction mixture contains 10 /zl of a 2× binding buffer (24 mM HEPES, pH 7.9, 4 mM MgC12, 0.24 mM EDTA, 0.6 mM PMSE 0.6 mM DTT, 24% glycerol), 0.2/xl of sonicated salmon sperm DNA (10 mg/ml), 4/zl water, and 5/zl of nuclear extract (about 15/zg of protein). The mixture is incubated for 15 min on ice before the 32p-labeled HSE double-stranded oligonucleotide probe (about 20,000 cpm Cerenkov) and 0.5/zg of poly(dI-dC) are added. Incubation with the probe is for 15 min at room temperature. To assess the specificity of HSE DNA binding, competition experiments can be performed as described earlier using 10 or 40 ng of cold HSE probe added to the binding mix
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just before incubation with the 32p-labeled probe. Supershift experiments are performed using 2/zg of antibodies raised against HSF1 added to the binding reaction. Eleetrophoresis. The experiment is performed as described earlier.
T r a n s a c t i v a t i o n of NF-KB- a n d H S F - 1 - D e p e n d e n t G e n e s
NF-xB Modulation of the activation of NF-xB transcription factor by the thiol redox state will have repercussion on the expression of xB-dependent genes. Hence, the transcriptional activity of NF-xB can be studied in cells transiently transfected with plasmids bearing ~cB consensus sites in front of a reporter gene, such as chloramphenicol acetyltransferase or luciferase. Transient Transfections. Cells are transiently transfected using the LipofectAMINE reagent (Life Technologies). Briefly, HeLa cells are seeded out the day before transfection at a density of 2 × 106 cells/100-mm dishes. Cells are then transfected with 11 #g of the desired plasmids. The lipid-DNA complex is applied to the cultured cells for 3 to 4 hr Two hours later, cells are trypsinized and replated into 60-ram dishes. After 12 hr cells are submitted to various treatments and are harvested after a 24-hr period of recovery. Assessement of transfection efficiency is performed using a pCMV/3 plasmid, which contains the gene encoding/%galactosidase under the control of the cytomegalovirus (CMV) promoter (Clontech). Reporter Genes. The transcriptional activity of NF-xB can be analyzed with pxB6-TKluc or p2xxB-37TKcat vectors that are plasmids bearing luciferase (for the former) and chloramphenicol acetyltransferase (CAT) (for the latter) reporter genes under the control of the herpes simplex thymidine kinase promotor and, respectively, six or two NF-xB-binding sites. 7'22 Reporter Activity Assays. Analysis of reporter gene expression is assessed by the luciferase assay or the CAT-ELISA test. Luciferase activity is analyzed with the Steady-Glo Luciferase assay system (Promega, Madison, WI) according to the manufacturer's instructions. For the CAT-ELISA test, transfected cells are lysed, and 50/zg of total cellular proteins is analyzed using the Roche CAT-ELISA test according to the manufacturer's instructions (Roche Diagnostics). An example of NF-xB-mediated transcriptional activation of pxB6-TKluc by oxidative stress and its downregulation by gluthatione peroxidase overexpression is presented in Fig. 2C.
22 C. Kretz-Remy, E. E. M. Bates, and A. E Arrigo, J. Biol. Chem. 273, 3180 (1998).
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[9-0]
150
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+
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Not treated
Azetidine
Heat shock
FIG. 3. Analysis of HSE-dependent transcription using a reporter gene assay. HeLa cells transfected transiently with a Hsp70 (HSE) promoter-dependent reporter Cat construct (pl 7-cat-neo) were either left untreated or pretreated for 1 hr with 100/zm PDTC. The cells were then heat shock treated for 90 min at 43 ° or treated for 6 hr with 10 mM azetidine. Azetidine is an amino acid analog known to activate the transcription of the HSE-dependent gene via the HSF1 factor. Cells were allowed to recover for 24 hr before being analyzed for the production of Cat polypeptide. Note the opposite effect mediated by the antioxidant agent PDTC on the transcriptional activation of the human Hsp70 gene promoter in response to heat shock or azetidine.
HSF1 Transcriptional activity mediated by the binding of HSF1 to HSE elements can be studied in cells transiently transfected with plasmids beating HSE consensus sites in front of reporter genes. Transient Transfections, Reporter Genes, and Reporter Activity Assay. As described earlier, but in this case cells are transfected with the pl 7-Cat neo plasmid, which contains the Cat gene under the control of the human hsp70 promoter. This DNA construct is a derivative from pLTR-Cat and pl7-hGH neo plasmids. 23-26 23 M. Dreano, J. Brochot, A. Myers, C. Cheng-Meyer, D. Rungger, R. Voellmy, and P. Bromley, Gene 49, 1 (1986). 24 M. Dreano, X. Fouillet, J. Brochot, J. M. Vallet, M. L. Michel, D. Rungger, and P. Bromley, ~rus Res. 8, 43 (1987). 25 p. Schiller, J. Amin, J. Ananthan, M. E. Brown, W. A. Scott, and R. Voellmy, Z Mol. Biol. 203, 97 (1988). 26 M. Dreano, E Fischbach, M. Montandon, C. Salina, P. Padieu, and P. Bromley, Bio/Technology 6, 953 (1988).
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Controls are performed using the pSV2-Cat plasmid, which contains the chloramphenicol acetyltransferase gene under control of the constitutive early promoter of the SV40 virus (Clontech). An example of HSFl-mediated transcriptional activation by heat shock and the amino acid analog azetidine, as well as its modulation by the antioxidant agent pyrrolidine dithiocarbamate (PDTC), are presented in Fig. 3. Conclusion Studies have described redox-sensitive transcription, which modulates the expression of specific genes depending on the balance between prooxidant and antioxidant conditions within the cell. Thiols can play a role in the signaling pathway that activates transcription factors that reside in the cytoplasm in a latent form. However, the unraveling of the precise role played by thiol-containing molecules during the different steps that control transcription factor activation (signal transduction, nuclear migration) is far from being achieved. DNA-protein interaction is another important step modulated by the oxidation status of crucial cysteine residues located in the DNA-binding domain of several transcription factors. The techniques described here in the case of HSF1 and NF-KB can be. adapted to study other transcription factors suspected of being regulated by the intracellular thiol redox state (i.e., AP-1, P53, NF-1, USE E2E IIIC, kU, Egr-1, vEts, HoxB5, the bovine papillomavirus type 1 E2 protein, Spl, PB2/CBF, and glucocorticoid and estrogen receptors). Future studies will certainly bring us more information concerning putative effects mediated by the thiol redox state at the level of downstream events, such as transcription, mRNA processing, transport, and translation.
PROTEIN SENSORS AND REACTIVE OXYGEN SPECIES
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relationship was also observed for the sulfoxidation of S-benzyl-L-cysteine by rabbit FMO-1 and FMO-3, previouslyY Conclusion These methods can be used to easily and sensitively assess the fl elimination and oxidative deamination activities of SeCys conjugates by several enzymes. Using selective enzyme inhibitors, the relative contributions of the different enzymes in tissues can be delineated. This study demonstrates that SeCys conjugates are metabolized by at least three different enzyme systems, namely cysteine conjugate fl-lyase/GTK, L-AAO, and FMO. The present results implicate that four biologically active products can be formed from SeCys conjugates by these enzymes: selenols, selenenic acids, selenoxides, and hydrogen peroxide (Fig. 3). Theoretically, seleninic acids, of which methylseleninic acid displays chemopreventive activity,2 can also be formed via oxidation of selenenic acids by hydrogen peroxide. Which of these compounds accounts for the potent chemopreventive activity of SeCys conjugates remains to be established. 25 R. J. Duescher, M. E Lawton, R. M. Philpot, and A. A. Elfarra, J. Biol. Chem. 269, 17525 (1994).
[20] Gene Expression a n d Thiol Redox State By CAROLE KRETZ-REMY a n d ANDRE-PATRICK ARRIGO
Introduction Cells have developed sophisticated mechanisms to maintain redox state homeostasis and/or to try to cope with the reactive oxygen species (ROS) produced during oxidative stress. 1'2 These mechanisms either scavenge and/or detoxify ROS, block their production, or sequester transition metals that are a source of free electrons. They include detoxifing enzymes, vitamins C and E, or thiol-containing molecules such as glutathione or thioredoxin. Glutathione exists in either a reduced (GSH) or an oxidized (glutathione disulfide, GSSG) form and participates in redox reactions through the reversible oxidation of its active thiol. In addition, it acts as a coenzyme of numerous enzymes involved in cell defense (i.e., glutathione peroxidases, glutathione S-transferases, thiol transferases, formaldehyde dehydrogenase, I H, Sies, Am. J. Med. 91, 31S (1991). 2 H, Sies, Eur J. Biochem. 215, 213 (1993).
METHODSIN ENZYMOLOGY,VOL.348
Copyright© 2002by AcademicPress. All rightsof reproductionin any formreserved. 0076-6879/02 $35.00
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glyoxalase I, and maleylacetoacetate isomerase). In unstressed cells, the majority of glutathione is in its reduced form and its concentration is in the range of 0.5 to l0 mM depending on the cell type. Studies aimed at understanding the mechanisms by which oxidative stress and/or variation in the intracellular redox state modulate gene expression have focused on the crucial role played by transcription factors. Once activated, these proteins bind to the promoter regions of target genes and allow their transcription by RNA polymerase II. Two steps in the transcriptional activation of several eukaryotic transcription factors are regulated by the redox balance: (i) the mechanism of activation of the transcription factor, which induces its redistribution into the nucleus, and (ii) the binding of the transcription factor to the promoter region located in the 5' end of the target genes. For the first point, many different steps of the pathways leading to the activation of transcription factors are under the control of intracellular redox state and are discussed later. Conceming the second point, it is well known that some transcription factors contain cysteine residues (which are redox sensitive through their thiol group) localized in their DNA-binding domain. These cysteines are often essential for the recognition of the transcription factorbinding site through electrostatic interactions with specific DNA bases. Oxidation of these cysteines usually inhibits the ability of the trancription factor to transactivate gene expression. The presence of thioredoxin (TRX) 3 and/or redox factor 1 (Ref-1)4 is often required to locally promote a reducing environment that avoids the oxidation of these cysteines. It is also possible that oxidized cysteines regulate the tridimensional structure of the transcription factor or its binding to a cofactor (a transcription factor that is active as a dimer). Finally, cysteines and/or histidines, localized at specific sites can interact with a zinc atom to form a "zinc finger" structure that fits in the large DNA groove and are responsible for the binding of the protein to DNA. Consequently, because of the sensitivity of the thiol group to redox state variations, the activity of several transcription factors can be modified profoundly if their cysteine residues are oxidized or if they are unable to form appropriate disulfide bounds. Moreover, simple oxidation of the sulfur atom of cysteine by oxygen atoms (in -SOH or -SO2H) can be critical. Variations in the intracellular redox state mediated by oxidative stress can therefore transiently modify the activity of several transcription factors. Depending on the transcription factor and of its own mechanism of activation, this modulation can be positive or negative and therefore can upregulate or downregulate gene expression. The goal of this article is to review different practical approaches that are commonly used to analyze eukaryotic transcription factors whose activation and DNA binding are controlled by the thiol redox status of the cell. Emphasis will be
3 A. Holmgren, J. Biol. Chem. 264, 13963 (1989). 4 S. Xanthoudakis and T. Curran, EMBO J. 11, 653 (1992).
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given to two well-defined transcription factors: NF-tcB (nuclear factor KB)5 and HSF1,6 which confer redox state-dependent inducible gene expression. NF-KB The more frequent form of NF-KB is a heterodimeric complex that contains p50 and p65 polypeptides. Except for B lymphocytes, where it is constitutively active, in most cells NF-~cB is cytoplasmic and needs extracellular signals to be activated and to migrate in the nucleus where it binds KB sites (5'-GGGAN/NYYCC-3') on the DNA. 5 In the cytoplasm, NF-~cB is associated with a family of cytoplasmic inhibitors, the most frequent one being IKB-ot. On activation by oxidative stress, NF-KB/IKB-ot complexes are destroyed, allowing free NF-KB dimers to migrate into the nucleus and activate the transcription of tcB sites containing genes. This process is triggered by the phosphorylation of IKB-ot by IKB kinase. This event induces the ubiquitinylation of boB-or and its subsequent degradation by the proteasome. 5 Several studies have reached the conclusion that oxidative conditions in the cytoplasm stimulate the phosphorylation and subsequent degradation of boB-or, leading to the activation of NF-JcB. 6'7 Reducing nuclear conditions provided by TRX are required in some cellular types to favor the binding of NF-KB to DNA 8 (Fig. 1). Heat Shock Factor 1 Heat shock factor 1 (HSF1) is a transcription factor that regulates the expression of stress (heat shock) genes. HSF1 cytoplasmic activation is mediated by stresses, such as heat shock, or numerous different agents or conditions that generate abnormally folded proteins 9 (Fig. 1). Transcription of heat shock (or stress) genes requires the formation of a homotrimeric HSF1 complex that binds to the heat shock promoter element (HSE). HSE is characterized as multiple adjacent and inverse iterations of the 5'-nGAAn-3' motif. 6 The conversion of HSF1 from a monomer to a trimer state is induced by heat shock. It has been reported that agents or conditions that trigger the oxidation of thiol-containing molecules, leading to the formation of oxidized glutathione disulfide (GSSG), glutathione-protein mixed disulfides, and protein-protein disulfides, induce the trimerization of HSF1 and its binding to DNA) ° In contrast, incubation of cells (but not cell extracts) 5 p. A. Baeuerle and D. Baltimore, Cell 87, 13 (1996). 6 R. I. Morimoto, Genes Dev. 12, 3788 (1998). 7 C. Kretz-Remy, P. Mehlen, M. E. Mirault, and A.-P. Arrigo, J. CellBiol. 133, 1083 (1996). 8 M. T. Anderson, F. J. Staal, C. Gitler, and L. A. Herzenberg, Proc. Natl. Acad. Sci. U.S.A. 91, 11527 (1994). 9 j. T. Westwood and C. Wu, Mol. Cell. Biol. 13, 3481 (1993). 10 j. Zou, W. F. Salminen, S. M. Roberts, and R. Voellmy, Cell Stress Chaperones 3, 130 (1998).
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Nucleus
FIG. 1. Schematic illustration of redox-dependent steps in the activation of NF-~cB and HSF1. Thiol-containing agents can have a positive action by facilitating the binding of NF-xB to DNA (indicated in italics). In contrast, in heat-shocked cells, thiol-containing agents counteract the oxidation of intracellular thiols and block HSF1 activation. Thiols can also inhibit ROS accumulation and therefore inhibit NF-KB activation by oxidative stress. P, phosphorylated serine; Ub, ubiquitin, IKK, IxB kinase, TRX, thioredoxin. Adapted from A.-P. Arrigo, Free Radic. Biol. Med. 27, 936 (1999).
with dithiothreitol (DTT) inhibits HSF1 trimerization and the transcriptional activation of heat shock genes. 11 Two steps in HSFI activation have been identified: (i) disruption of intracellular thiol-disulfite redox homeostasis, which leads to the formation of disulfide-linked aggregates of cellular proteins, and (ii) recognition of denatured proteins by preexisting protein chaperones, a phenomenon that triggers HSF1 trimerization without its apparent oxidation. 1° Concerning HSF1 activation by hydrogen peroxide or other types of oxidants, it has been shown that these agents stimulate the nuclear translocation of HSF 1 but interfere with HSF1-DNA-binding activity by oxidizing critical HSFI cysteine residues. The intensity of the latter phenomenon is decreased by TRX, which is upregulated by oxidative stress prior to HSF1 activation. Hence, reducing conditions generated by TRX upregulation help HSF1 DNA-binding activity during oxidative stress, la
11 L. E. Huang, H. Zhang, S. W. Bae, and A. Y. Liu, J. Biol. Chem. 269, 30718 (1994). 12 M. R. Jacquier-Sarlin and B. S. PoUa, Biochem. J. 318, 187 (1996).
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D e t e r m i n a t i o n o f I n t r a c e l l u l a r G l u t a t h i o n e Level Glutathione is a naturally occurring tripeptide whose nucleophilic and reducing properties play a central role in the antioxidant system of most aerobic cells. The total cellular glutathione level can be measured by fluorescence-activated cell sorter (FACS) analysis using a monochlorobimane (MCB) fluorescent probe. 13,14About 5 × 105 cells/ml are incubated for 5 min with 40 mM MCB added to the growth medium. Fluorescence at 425 nm is analyzed in response to an excitation at 395 nm in a Becton Dickinson Vantage FACS (Le-Pont-de-Claix, France) equipped with a 37 ° sample chamber. Total glutathione content can also be estimated using the Bioxitech GSH-400 colorimetric assay of Oxis International (Portland, OR). The GSH-400 method is based on a chemical two-step reaction. The first step leads to the formation of thioethers adducts between a patented reagent and all mercaptans present in the sample. The second step takes advantage of the structure of the glutathione, which undergoes fl elimination at high pH, leaving the reagent in the form of a chromophoric thione, which has a maximal absorbance wavelength at 400 nm. Briefly, total cellular protein material from 6 × 106 cells is precipitated in 5% (w/v) metaphosphoric acid and the resulting supernatant is used for the test. The level of glutathione in each sample is calculated according to standard curves of increasing GSH concentrations. The enzymatic method described by Eyer and Podhrasky 15is used to determine the level of glutathione in its reduced or oxidized form. Harvested cells, washed in phosphate-buffered saline (PBS) are resuspended in 1 Mperchloric acid containing 1 mM EDTA. The mixture is kept at - 2 0 ° for at least 2 hr before adding an equal volume of 1.3 M K2HPO4 to the samples. It is then stirred for 30 min before insoluble crystals of KC104 salt are removed by centrifugation and analyzed according to Eyer and Podhrasky. 15 A procedure leading to the elimination of GSSG from total glutathione can be used to estimate the level of oxidized glutathione disulfide. In this case, cells are resuspended in a medium containing 1 M perchloric acid, 1 mM EDTA, and 0.02 M NEM (N-ethylmalemide). GSSG and NEM are then removed by five extractions with ice-cold, H20-saturated ethyl acetate.
Analysis of Formation of Glutathione-Protein Disulfites The method described by Zou e t al. lo can be used. Ceils are incubated for at least 5 hr in growth medium lacking cysteine, cystine, and methionine, but containing
13 D. C. Shrieve, E. A. Bump, and G. C. Rice, J. Biol. Chem. 263, 14107 (1988). 14 D. J. Kane, T. A. Sarafian, R. Anton, H. Hahn, E. B. Gralla, J. S. Valentine, T. Ord, D. E. Bredesen, Science 262, 1274 (1993). 15 p. Eyer and D. Podhradsky, Anal Biochem. 153, 57 (1986).
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10% fetal calf serum. Cycloheximide (50/zg/ml) is added for 30 min before the cells are incubated for 2 hr with 20 IzCi/ml of [35S]cystine (113 Ci/mmol). Cells are then exposed to different agents or conditions (i.e., heat shock) whose actions are tested. Cells are washed with phosphate-buffered saline (PBS) and lysed by scraping in cold 10 mM sodium phosphate buffer (pH 7.0) containing 15 mM N-ethylmaleimide, 5 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride (PMSF). Cell lysates are clarified by a 5-min spin at 8700g. The protein concentration is determined using the DC protein assay of Bio-Rad. Samples are mixed with twotime concentrated SDS-PAGE sample buffer lacking reducing agents before being boiled and applied to 7.5% SDS-PAGE gels. Gels are soaked in 1 M sodium salicylate (pH 6.0) before being dried and exposed to X-ray films for fluorography.
In Vivo F l u o r e s c e n t M e a s u r e m e n t of I n t r a c e U u l a r R e a c t i v e Oxygen Species 2 r,71-Dichlorofluorescin Diacetate Probe 2',7'-Dichlorofluorescin diacetate (DCFH-DA) diffuses rapidly into the cells, is hydrolyzed to 2',7'-dichlorofluorescin (DCFH), and is thereby trapped within the cells. Intracellular DCFH is converted to highly fluorescent 2',7'-dichlorofluorescein (DCF) by ROS-mediated oxidation. 16 Adherent cells 5 x 105 are trypsinized and rinsed three times with PBS. Thereafter, cells are incubated at 37 ° for various periods of time (from 10 to 60 min) with PBS containing 0.1 mg/ml of DCFH-DA. DCF fluorescence analysis is performed using a FACScalibur cytometer (Becton Dickinson) using a 488-nm excitation wavelength and a 530-nm emission filter bandpassed for DCE The slope of each curve obtained is representative of the level of ROS produced.
Hydroethidine Probe Hydroethidine, the sodium borohydride reduced form of ethidium, is freely permeable to cells and can be oxidized to fluorescent ethidium bromide by ROS. Cells (1.5 x 106) are trypsinized and resuspended into PBS containing 40/zg/ml of hydroethidine. Cells are then incubated at 37 ° for 10 min; the reaction is stopped by incubation on ice. Flow cytometric analysis is performed using a FACScalibur cytometer using a 488-nm excitation wavelength. The emission filter is 610 nm bandpassed for ethidium bromide fluorescence.
16D. Bass, J. W. Parce,L. R. Dechatelet,E Szejda,M. C. Seeds,and M. Thomas,J. lmmunol. 130, 1910 (1983).
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TABLE I COMPOUNDSCOMMONLYUSED TO STUDY EFFECTS OF INTRACELLULAR REDOX STATE ON GENE EXPRESSION Antioxidants Pyrrolidine dithiocarbamate (PDTC) N-Acetyl-L-cysteine (NAC) Glutathione ethyl ester
Oxidants Methyl viologen (paraquat) L-Buthionine (S,R) sulfoximine (BSO) Menadione(2-methyl- 1,4-naphthoquinone) Hydrogen peroxide Diamide [diazenedicarboxylic acid bis(N,N-dimethylamide)]
A n t i o x i d a n t a n d O x i d a n t D r u g s C o m m o n l y U s e d to T e s t R e d o x State Dependence of Gene Expression Antioxidants
PDTC and NAC are commonly used antioxidant drugs (see Table I). The former is a component carrying thiol groups and the latter is a precursor of GSH synthesis. Treatments of HeLa cells with 100/zM PDTC or 30 mM NAC for 1 hr is responsible for the inhibition of NF-~cB activation by oxidative stress. 7 GSH ethyl ester is freely permeable to cells and increases the intracellular GSH level. A 5 mM GSH ethyl ester treatment for 12 hr increases the level of GSH in L929 cells by 20%J 7 Oxidants
BSO is a glutathione synthesis inhibitor. A 24-hr treatment with I mM BSO depleted 99% of glutathione in murine L929 cells.18 Diamide is a specific oxidant for thiol groups: a 2-hr treatment with 2 mM diamide drastically decreased the GSH level in benefit of GSSG in human red blood cells. 19 A 2 mM treatment with hydrogen peroxide is also responsible for the rapid oxidation of GSH to GSSG. 19 Menadione is a quinone reagent that produces oxidative modifications in biological systems mainly through redox cyclin reactions with oxygen (it binds to thiol groups to form a conjugate able to redox cycle like menadione itself) and is responsible, in human red blood cells, for the rapid disappearance of GSH with a negligible production of GSSGJ 9 Paraquat (methyl viologen) is an oxidative stress inducer and decreases the level of GSH and the GSH/GSSG ratio. 2° 17 X. Preville, E Salvemini, S. Giraud, S. Chaufour, C. Paul, G. Stepien, M. V. Ursini, and A. E Arrigo, Exp. Cell Res. 247, 61 (1999). 18 E Mehlen, X. Pr6ville, C. Kretz-Remy, and A.-E Arrigo, EMBO J. 15, 2695 (1996). 19 R. Rossi, A. Milzani, I. Dalle-Donne, E Giannerini, D. Giustarini, L. Lusini, R. Colombo, and E I. Di Simplicio, J. Biol. Chem. 276, 7004 (2001). 20 S. G. Konstantinova and E. M. Russanov, Acta Physiol. Pharmacol. Bulg. 24, 107 (1999).
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A c t i v a t i o n of NF-KB a n d H S F I a n d T h e i r M i g r a t i o n into the Nucleus
NF-KB NF-xB activators induce the phosphorylation and subsequent degradation of IxB-ot, which result in the destruction of the cytoplasmic NF-KB/IxB complex. Free NF-tcB can then migrate into the nucleus. This complex phenomenon can be detected using several approaches. IxB-ot Phosphorylation and Degradation. To date, except in minor cases, NF-xB activation promoted by modulation of the redox state follows a universal pathway, which begins with IKB-ot phosphorylation, multiubiquitination, and degradation by the 26S proteasome. Those events can therefore be considered as markers of NF-KB activation. ANALYSISAFTERONE-DIMENSIONALSDS-PAGE. Cells (1.5 × 106) grown in 60-mm dishes are washed with cold PBS. Cells are then scraped from the dishes and pelleted at 1000g for 5 min. The cellular pellet is lysed and boiled in Laemmli sample buffer (50 mM Tris-HCl, pH 6.8, 2% SDS, 50 mM 2-mercaptoethanol, 7.5% glycerol, and 0.05% bromphenol blue) before being analyzed in SDS-PAGE made of 10% acrylamide running and 5% acrylamide stacking gels. The gel is electrophoresed at 30 mA (cooling system required). ANALYSISAFTERTWO-DIMENSIONALSDS-PAGE. Cellular extracts lysed and boiled in Laemmli sample buffer are incubated for 2 hr at room temperature with DNase (10,000 U) and RNase (10/xg) and thereafter heated at 70 ° for 5 min. After lyophilization, samples are resuspended in isoelectrofocusing sample buffer (9.95 M urea, 4% NP-40, 2% pH 6-8 ampholines, 0.1 M DTT). Samples are loaded at the anodic end of an isoelectrofocusing gel made up with 5.5 g urea, 1.26 m130% acrylamide, 1.07 ml 2% bisacrylamide, 2 ml NP-40, 0.97 ml H20, 0.2 mlpH 3-10 ampholines, 0.3 ml pH 4--6 ampholines, 10/zl N,N,N',N'-tetramethylethylenediamine (TEMED), and 10/~1 of 10% ammonium persulfate. Electrofocusing is for 600 V for 16 hr and 1000 V for 1 hr. SDS-PAGE in the second dimension is performed as mentioned previously. Immunoblot Analysis. Immunoblot blot analysis of one- or two-dimensional SDS-PAGE is performed by transferring the separated proteins from the acrylamide gel to a PVDF membrane (Immobilon P, pore size 0.45/zm, Millipore, Bedford, MA) using a liquid or semidry blotting system (Bio-Rad, Hercules, CA) in 25 mM "iris, 190 mM glycine, and 10% (v/v) methanol buffer. The membrane is then incubated in TBS (20 mM Tris-HC1, pH 7.6, 137 mM NaC1) 0.1% Tween, and 5% dry milk for 2 hr at room temperature to block unspecific binding sites. Thereafter, the membrane is incubated for 1 hr at room temperature with anti-IxB-~ (Santacruz Biotechnology) diluted at 1/zg/ml in TBS 0.1% Tween, 0.1% bovine serum albumin (BSA) as the primary antibody. After three washes in TBS 0.1% Tween, the immunoblot is probed with a goat anti-rabbit immunoglobulin diluted
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1/10,000 in T B S 0.1% Tween, 0.1% B S A and r e v e a l e d with the E C L (enhanced c h e m i l u m i n e s c e n c e ) kit f r o m A m e r s h a m (Orsay, France). The duration o f exposure is calculated to be in the linear response o f the film. In the E C L revealed i m m u n o b l o t presented in Fig. 2A, it is possible to observe a slow migrating IKB-ot phospho i s o f o r m after H202 treatment and its rapid disappearance due to proteolytic degradation. NF-tc B Migration in the Nucleus IMMUNOFLUORESCENCE APPROACH. Cells g r o w i n g on glass coverslips coated with 0.1% type B gelatin are submitted to various NF-~cB activators. Thereafter, the cells are rinsed twice with PBS at 37 ° and fixed for 90 sec with c o l d methanol. A f t e r two washes in cold PBS, coverslips are incubated for 1 hr at r o o m temperature with anti-p65 or anti-p50 antibodies (Santa Cruz B i o t e c h n o l o g y ) diluted 1/100 in PBS containing 0.1% B S A . Coverslips are then w a s h e d three times in c o l d PBS and incubated for 1 hr at r o o m temperature with goat anti-rabbit imm u n o g l o b u l i n c o u p l e d to isothiocyanate diluted 1/200 in P B S 0.1% B S A . Staining specificity is assessed by control coverslips solely incubated with the second
FIG. 2. Analysis of IKB-a phosphorylation and degradation in response to hydrogen peroxide treatment. (A) HeLa cells were treated for various time periods with 250 #M of hydrogen peroxide in the presence or absence of 60 mM of the proteasome inhibitor N-Cnz-lleu-Glu [o-tert-butyl]-Ala-leucinal (PSI). Cells were harvested, and equal amounts of total cellular proteins were separated in SDS-PAGE. The cellular contents of IKB-ct isoforms were analyzed in an immunoblot probed with the IrB-ot antibody. Note the presence of a slow migrating l~cB-otphospho isoform after 5 min of treatment with hydrogen peroxide. 1KB-otis no more detectable after 15 min of treatment due to proteolytic degradation by the proteasome, a phenomenon not observed in the presence of the proteasome inhibitor PSI. Reproduced from Kretz et al. (I 998), by copyright permission of the American Society of Microbiology. (B) Analysis of NF-xB binding to the KB DNA site. Murine L929 cells were either left untreated (C) or treated with tumor necrosis factor a (TNFot) (20, 200, and 2000 U/ml for 2 hr) in the presence or absence of 30 mM of N-acetylcysteine (NAC) added 30 min before TNFot treatment. Nuclear extracts were prepared, and equal amounts of nuclear proteins were incubated with a 32p-labeled DNA probe encompassing the xB sites. Samples were analyzed on a native 4% polyacrylamide gel. An autoradiograph of the gel is presented. Supershift (sps) was performed by adding an antiserum recognizing the p65 subunit of NF-KB to the binding mixture of 2000 U/ml TNFa-treated cell extracts. (C and D) Analysis of ~B-dependent transcription using a reporter gene assay. Control (C) and glutathione peroxidase-overexpressing (D) human breast cancer T47D cells were cotransfected with either pTKluc (black plot) or pKB6-TKIuc (hatched plot) plasmid, pKB6-TKIuc contains a luciferase gene reporter placed under control of a thymidine kinase promoter coupled to six ~cBelements. Luciferase expression from this construct was compared to that from a similar plasmid but without xB elements (pTKluc). Twenty hours after transfection and replating, cells were either kept untreated or treated for 2 hr with 250/zM H202 or 2000 U/ml TNFot. The medium was changed, and the cells were allowed to recover for 14 hr prior to harvesting and luciferase detection. Note the strong stimulation of luciferase activity in control cells treated with hydrogen peroxide or TNFot, which was only faintly detectable in glutathione peroxidase-expressing cells. Reproduced from C. Kretz-Remy, P. Mehlen, M. E. Mirault, and A. P. Arrigo, J, Cell Biol. 133, 1083 (1996) by copyright permission of the Rockefeller University Press.
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antibody. Stained cells are examined and photographed with a Zeiss axioscop photomicroscope. Cell Fractionation Approach. Cells growing in 60-mm dishes are scraped from the dishes and pelleted for 5 min at 1000g at 37 °. Thereafter, the cells are lysed for 10 min at 4 ° in a buffer containing 10 mM Tris, pH 7.4, 10 mM NaC1, 1.5 mM MgC12, and 0.5% Triton X-100. The lysates are then clarified for 10 min at 12,000g at 37 °. This procedure is repeated until nuclei present in the pellet fraction are free of cytoplasmic contaminations, as judged by microscopical analysis with a TMS-inverted photomicroscope (Nikon Inc.) equipped with phase contrast. The crude nuclear pellets are resuspended or diluted in similar amounts in Laemmli SDS buffer. Cytoplasmic and nuclear samples are then analyzed in 10% SDS-PAGE. After transferring the gel, blots are incubated for 1 hr at room temperature with anti-p65 or anti-p50 antibodies diluted at 2/zg/ml in TBS 0.1% Tween, 0.1% BSA. Revelation of the immunoblot is performed as described earlier.
HSF1 HSF1 Trimerization. Like many inducible factors, HSF1 is synthesized constitutively and stored in a latent cytoplasmic form under normal conditions. HSF1 is activated in response to heat shock by a conversion of HSF1 protein from a monomer to a heterotrimer.9 The HSF trimer migrates in the nucleus and binds to HSEs upstream of heat shock (stress) genes. The trimerization of HSF1 can be observed by gel filtration analysis of cell extracts. 9 To prepare cytoplasmic and nuclear extracts, cells are resuspended and lysed using a Dounce homogenizer in a buffer containing 10 mM N-2-hydroxyethylpiperazine N'-2-ethanesulfonic acid (HEPES; pH 7.9), 1.5 mM MgC12, 10 mM KC1, 0.5 mM DTT, and 0.5 mM PMSE The homogenate is centrifuged at 25,000g for 20 min at 4 °. The resulting nuclear pellet is extracted for 30 min with two original pellet volumes of a buffer containing 10 mM HEPES, pH 7.9, 0.4 M NaCI, 20% glycerol, 0.5 mM DTT, and 0.5 mM PMSF before being centrifuged for 60 min at 100,000g at 4 °. The supernatant of this second step is the heat-shocked nuclear extract, which contains trimerized HSF1. The supernatant of the 25,000g centrifugation step is centrifuged again at 100,000g for 60 min at 4 °. The supematant of this centrifugation is the cytoplasmic extract and contains the bulk of monomeric inactive HSF1. Analysis of native molecular masses of latent and activated HSF1 is performed by gel-filtration chromatography on a Superose 6 HR 10/30 column (Pharmacia, Piscataway, N J). Five hundred microliters of cytoplasmic or heat-shocked nuclear extracts is adjusted to 200 mM KC1 and is applied to the column. The mobile phase contains 200 mM KC1, 20 mM Tris, pH 7.9, 5% (v/v) glycerol, 0.1 mM EDTA, 0.5 mM DTT, and 0.5 mM PMSF. Detection of the HSF1 polypeptide in the different fractions of the
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column is by SDS-PAGE/immunoblot using an antibody that specifically recognizes this protein. HSF1 Migration into the Nucleus. The immnofluorescence approach and cell fractionation approach are performed as described earlier using antibodies that specifically recognize HSF1.
B i n d i n g of T r a n s c r i p t i o n F a c t o r s to DNA This property of transcription factors was analyzed by DNA binding and electrophoretic shift assays as describedl NF-KB Preparation of Nuclear Extracts. Cells (107) are harvested and centrifuged at 1000g for 5 min. The cell pellet is washed twice with ice-cold PBS and resuspended in 400 /zl of ice-cold buffer A (10 mM HEPES, pH 7.5, 1.5 mM MgCI2, 10 mM KC1, 0.5 mM DTT, 0.5 mM PMSF, and 2% aprotinin). DTT, PMSF, and aprotinin should be added extemporaneously. The cell suspension is kept on ice for 15 min. Thereafter, NP-40 is added to a 10% final concentration, and the suspension is vortexed vigorously and centrifuged at 13,000 rpm for 1 min (Eppendorf centrifuge). The supernatant is discarded, and the pellet of nuclei is resuspended in 100/xl of buffer C (20 mM HEPES, pH 7.5, 25% glycerol, 0.42 M NaC1, 1.5 mM MgC12, 0.2 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF, and 2% aprotinin). DTT, PMSF, and aprotinin should be added fresh. The nuclear suspension is kept on ice for 40 min, and the nuclear lysate is centrifuged at 10,000 rpm for 10 min at 4 °. The resulting supernatant is stored at - 8 0 °. An aliquot (5 #1) is taken from the supernatant to determine the protein concentration (Bradford protein assay reagent, Bio-Rad). Labeling of KB Probe. The 5'-overhangs of KB double-stranded oligonucleotides (5'-AGCTTCAGAGGGGACTTTCCGAGAGG-3' and 3'-AGTCTCCCCTG AAAGGCTCTCCAGCT-5') are used to detect the DNA-binding activity of NFKB as described previously.21 Four micrograms of KB oligonucleotides is incubated at room temperature for 15 min with 1 mM dATP, dTTP, and dGTP, 40/zCi of [ot-32p]dCTP in the presence of 2U of Klenow polymerase, and 1X Klenow buffer supplied with the enzyme (Roche Diagnostics). The reaction is stopped by a 5-min incubation at 70 °. Nonincorporated 32p is removed by Sephadex G-50 chromatography. The labeled oligonucleotide is precipitated overnight at - 2 0 ° . 21U. Zabel,R. Schreck,and P. A. Baeuerle,J. Biol. Chem.266, 252 (1991).
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DNA-Binding Reaction, Competition, and Supershifi Assays. Ten micrograms of protein from nuclear extracts is incubated with a 20,000 cpm (Cerenkov) 32p-labeled xB-DNA probe in the presence of 4/zg of poly(dl-dC) (Pharmacia) and 5 mM Tris-HC1, pH 7.5, 0.5 mM DTT, 0.5 mM EDTA, 25 mM NaC1, 1% Ficoll 400 in a final volume of 10 #1. The binding reaction is kept at room temperature for 15 min. To assess the specificity of NF-xB DNA binding, competition experiments are performed with 10 or 40 ng of the cold KB probe added to the binding mix including proteins, just before incubation with the 32p-labeled KB probe. The subunit composition of DNA-binding proteins can be ascertained by supershift experiments in which 2 #g of antibodies raised against the p50 or p65 NF-KB subunits is added to the binding reaction for an additional 30 min. Electrophoresis. Following incubation, samples are analyzed into native 4% acrylamide gel in TBE 0.25X. Autoradiographs of the gel are recorded onto BioMax MR films (Eastman Kodak Co., Rochester, NY). An example of the binding of NF-KB to tcB DNA after TNFot treatment is presented in Fig. 2B. HSF1 Preparation of Nuclear Extracts. Cells are resuspended in cold 20 mM HEPES-KOH buffer, pH 7.9, containing 25% (v/v) glycerol, 0.5 M NaC1, 1.5 mM MgC12, 0.2 mM EDTA, 0.5 mM DTT, 0.5 mg/ml pepstatin A, and 20/zg/ml Pefabloc SC (Merck, Fontenay sous Boix, France). The cell suspension is sonicated at 0 ° for 20 sec and then centrifuged at 4 ° at 125,000g for 10 min. Soluble extracts are frozen in liquid nitrogen and stored at - 8 0 ° . Labeling of HSE Probe. Fifty nanograms of an HSE oligonucleotide (5'-GCCT CGAATGTTCGCGAAGTTTCG-3') is incubated in 20/zl. of 1x kinase buffer (40 mM Tris-HCl, pH 8.5, 10 mM MgC12, and 10 mM 2-mercaptoethanol) with 50/zCi of [y-32p]ATP(9300 Ci/mmol) and 20 U of T4 polynucleotide kinase (Roche Diagnostic). The reaction is stopped by the addition of 2/zl of 0.2 M EDTA. Nonincorporated 32p is removed as mentioned earlier. DNA-Binding Reaction, Competition, and Supershift Assays. Twenty microliters of the reaction mixture contains 10 /zl of a 2× binding buffer (24 mM HEPES, pH 7.9, 4 mM MgC12, 0.24 mM EDTA, 0.6 mM PMSE 0.6 mM DTT, 24% glycerol), 0.2/xl of sonicated salmon sperm DNA (10 mg/ml), 4/zl water, and 5/zl of nuclear extract (about 15/zg of protein). The mixture is incubated for 15 min on ice before the 32p-labeled HSE double-stranded oligonucleotide probe (about 20,000 cpm Cerenkov) and 0.5/zg of poly(dI-dC) are added. Incubation with the probe is for 15 min at room temperature. To assess the specificity of HSE DNA binding, competition experiments can be performed as described earlier using 10 or 40 ng of cold HSE probe added to the binding mix
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just before incubation with the 32p-labeled probe. Supershift experiments are performed using 2/zg of antibodies raised against HSF1 added to the binding reaction. Eleetrophoresis. The experiment is performed as described earlier.
T r a n s a c t i v a t i o n of NF-KB- a n d H S F - 1 - D e p e n d e n t G e n e s
NF-xB Modulation of the activation of NF-xB transcription factor by the thiol redox state will have repercussion on the expression of xB-dependent genes. Hence, the transcriptional activity of NF-xB can be studied in cells transiently transfected with plasmids bearing ~cB consensus sites in front of a reporter gene, such as chloramphenicol acetyltransferase or luciferase. Transient Transfections. Cells are transiently transfected using the LipofectAMINE reagent (Life Technologies). Briefly, HeLa cells are seeded out the day before transfection at a density of 2 × 106 cells/100-mm dishes. Cells are then transfected with 11 #g of the desired plasmids. The lipid-DNA complex is applied to the cultured cells for 3 to 4 hr Two hours later, cells are trypsinized and replated into 60-ram dishes. After 12 hr cells are submitted to various treatments and are harvested after a 24-hr period of recovery. Assessement of transfection efficiency is performed using a pCMV/3 plasmid, which contains the gene encoding/%galactosidase under the control of the cytomegalovirus (CMV) promoter (Clontech). Reporter Genes. The transcriptional activity of NF-xB can be analyzed with pxB6-TKluc or p2xxB-37TKcat vectors that are plasmids bearing luciferase (for the former) and chloramphenicol acetyltransferase (CAT) (for the latter) reporter genes under the control of the herpes simplex thymidine kinase promotor and, respectively, six or two NF-xB-binding sites. 7'22 Reporter Activity Assays. Analysis of reporter gene expression is assessed by the luciferase assay or the CAT-ELISA test. Luciferase activity is analyzed with the Steady-Glo Luciferase assay system (Promega, Madison, WI) according to the manufacturer's instructions. For the CAT-ELISA test, transfected cells are lysed, and 50/zg of total cellular proteins is analyzed using the Roche CAT-ELISA test according to the manufacturer's instructions (Roche Diagnostics). An example of NF-xB-mediated transcriptional activation of pxB6-TKluc by oxidative stress and its downregulation by gluthatione peroxidase overexpression is presented in Fig. 2C.
22 C. Kretz-Remy, E. E. M. Bates, and A. E Arrigo, J. Biol. Chem. 273, 3180 (1998).
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FIG. 3. Analysis of HSE-dependent transcription using a reporter gene assay. HeLa cells transfected transiently with a Hsp70 (HSE) promoter-dependent reporter Cat construct (pl 7-cat-neo) were either left untreated or pretreated for 1 hr with 100/zm PDTC. The cells were then heat shock treated for 90 min at 43 ° or treated for 6 hr with 10 mM azetidine. Azetidine is an amino acid analog known to activate the transcription of the HSE-dependent gene via the HSF1 factor. Cells were allowed to recover for 24 hr before being analyzed for the production of Cat polypeptide. Note the opposite effect mediated by the antioxidant agent PDTC on the transcriptional activation of the human Hsp70 gene promoter in response to heat shock or azetidine.
HSF1 Transcriptional activity mediated by the binding of HSF1 to HSE elements can be studied in cells transiently transfected with plasmids beating HSE consensus sites in front of reporter genes. Transient Transfections, Reporter Genes, and Reporter Activity Assay. As described earlier, but in this case cells are transfected with the pl 7-Cat neo plasmid, which contains the Cat gene under the control of the human hsp70 promoter. This DNA construct is a derivative from pLTR-Cat and pl7-hGH neo plasmids. 23-26 23 M. Dreano, J. Brochot, A. Myers, C. Cheng-Meyer, D. Rungger, R. Voellmy, and P. Bromley, Gene 49, 1 (1986). 24 M. Dreano, X. Fouillet, J. Brochot, J. M. Vallet, M. L. Michel, D. Rungger, and P. Bromley, ~rus Res. 8, 43 (1987). 25 p. Schiller, J. Amin, J. Ananthan, M. E. Brown, W. A. Scott, and R. Voellmy, Z Mol. Biol. 203, 97 (1988). 26 M. Dreano, E Fischbach, M. Montandon, C. Salina, P. Padieu, and P. Bromley, Bio/Technology 6, 953 (1988).
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Controls are performed using the pSV2-Cat plasmid, which contains the chloramphenicol acetyltransferase gene under control of the constitutive early promoter of the SV40 virus (Clontech). An example of HSFl-mediated transcriptional activation by heat shock and the amino acid analog azetidine, as well as its modulation by the antioxidant agent pyrrolidine dithiocarbamate (PDTC), are presented in Fig. 3. Conclusion Studies have described redox-sensitive transcription, which modulates the expression of specific genes depending on the balance between prooxidant and antioxidant conditions within the cell. Thiols can play a role in the signaling pathway that activates transcription factors that reside in the cytoplasm in a latent form. However, the unraveling of the precise role played by thiol-containing molecules during the different steps that control transcription factor activation (signal transduction, nuclear migration) is far from being achieved. DNA-protein interaction is another important step modulated by the oxidation status of crucial cysteine residues located in the DNA-binding domain of several transcription factors. The techniques described here in the case of HSF1 and NF-KB can be. adapted to study other transcription factors suspected of being regulated by the intracellular thiol redox state (i.e., AP-1, P53, NF-1, USE E2E IIIC, kU, Egr-1, vEts, HoxB5, the bovine papillomavirus type 1 E2 protein, Spl, PB2/CBF, and glucocorticoid and estrogen receptors). Future studies will certainly bring us more information concerning putative effects mediated by the thiol redox state at the level of downstream events, such as transcription, mRNA processing, transport, and translation.