GlutathioneDepletionIsNecessaryforApoptosisin ... · hasprimarilybeenthoughttoreflectthegenerationofreactive species of oxygen (ROS) and nitrogen (RNS) (6, 7). Although, theroleofROSinapoptosishasbeenextensivelystudied

Glutathione Depletion Is Necessary for Apoptosis inLymphoid Cells Independent of Reactive OxygenSpecies Formation*

Received for publication, April 12, 2007, and in revised form, July 26, 2007 Published, JBC Papers in Press, August 27, 2007, DOI 10.1074/jbc.M703091200

Rodrigo Franco, Mihalis I. Panayiotidis1, and John A. Cidlowski2

From the Laboratory of Signal Transduction, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709

Changes in the intracellular redox environment of cells havebeen reported to be critical for the activation of apoptoticenzymes and the progression of programmed cell death. Gluta-thione (GSH) depletion is an early hallmark observed in apopto-sis, and we have demonstrated that GSH efflux during deathreceptor-mediated apoptosis occurs via a GSH transporter. Wenow evaluate the relationship between GSH depletion, the gen-eration of reactive oxygen species (ROS), and the progression ofapoptosis. Simultaneous single cell analysis of changes in GSHcontent and ROS formation by multiparametric FACS revealedthat loss of intracellularGSHwas paralleled by the generation ofdifferent ROS including hydrogen peroxide, superoxide anion,hydroxyl radical, and lipid peroxides. However, inhibition ofROS formation by a variety of antioxidants showed that GSHlosswas independent from the generation of ROS. Furthermore,GSH depletion was observed to be necessary for ROS genera-tion. Interestingly, high extracellular thiol concentration (GSHand N-acetyl-cysteine) inhibited apoptosis, whereas, inhibitionof ROS generation by other non-thiol antioxidantswas ineffectivein preventing cell death. Finally, GSH depletion was shown to be anecessary for theprogressionof apoptosis activatedbybothextrin-sic and intrinsic signalingpathways.These resultsdocumentanec-essary andcritical role forGSHloss inapoptosis andclearlyuncou-ple for the first timeGSH depletion fromROS formation.

Apoptosis or programmed cell death is a ubiquitous, evolu-tionary conserved process. Under physiological conditions it isimportant not only in the turnover of cells in all tissues, but alsoduring the normal development and senescence of the orga-nism.Moreover, its deregulation has been observed to occur aseither a cause or consequence of distinct pathologies (1, 2). Forexample, apoptosis mediated by Fas ligand (FasL)3 receptor

(Fas/CD95/Apo-1) activation is necessary for the immune sys-tem homeostasis because of its role in the rapid clearance ofimmunoreactive T cells maintaining the immune balance andreducing the risk of autoimmune diseases (3). Apoptosis is ahighly organized process characterized by the progressive acti-vation of precise pathways leading to specific biochemical andmorphological alterations. Initial stages of apoptosis are char-acterized by reactive oxygen species (ROS) formation, changesin intracellular ionic homeostasis, cell shrinkage, loss ofmembrane lipid asymmetry, and chromatin condensation.Later stages associated with the execution phase of apoptosisare characterized by activation of execution caspases andendonucleases, apoptotic body formation, and cell fragmen-tation (1, 4, 5).Apoptosis has been widely reported to be modulated by

changes in the redox status of the cell; however, the precisemechanisms involved are still unclear (6, 7). Reduced glutathi-one (GSH) is the most abundant low molecular weight thiol inanimal cells and is involved in many cellular processes includ-ing antioxidant defense, drug detoxification, cell signaling, andcell proliferation (8, 9). In addition, alterations in its concentra-tion have been demonstrated as a common feature of manypathological situations including AIDS, neurodegenerative dis-eases, and cancer (9, 10). Intracellular GSH loss is an early hall-mark in the progression of cell death in response to differentapoptotic stimuli (11–14), and has been associated with theactivation of a plasma membrane transport mechanismrather than to its oxidation (11–13, 15–17). Furthermore,several studies have shown a correlation between cellularGSH depletion and the progression of apoptosis (12, 13).Additionally, high GSH levels have also been associated withan apoptotic resistant phenotype in different cells (18–21).We have recently shown that Fas ligand (FasL)-induced GSHefflux is mediated by its extrusion across the plasma mem-brane in lymphoid cells with characteristics of GSH/organicanion transporter (13); however, themechanisms involved inthe regulation of apoptosis by changes in intracellular GSHare not completely understood.Because of its action as a primary intracellular antioxidant in

the cells, a reduction in the intracellular glutathione content

* This research was supported by the Intramural Research Program of theNIEHS, National Institutes of Health. The costs of publication of this articlewere defrayed in part by the payment of page charges. This article musttherefore be hereby marked “advertisement” in accordance with 18 U.S.C.Section 1734 solely to indicate this fact.

1 Current address: School of Public Health, University of Nevada at Reno,Reno, NV 89557.

2 To whom correspondence should be addressed: Laboratory of Signal Trans-duction, NIEHS, National Institutes of Health. P. O. Box 12233. 111. T. W.Alexander Drive. Research Triangle Park, NC 27709. Tel.: 919-541-1564; Fax:919-541-1367; E-mail: [email protected].

3 The abbreviations used are: FasL, Fas ligand; GSH, reduced glutathione;6556, glutathione disulfide; OA�, organic anion; ROS, reactive oxygen spe-cies; RNS, reactive nitrogen species; �O2

�, superoxide anion; �OH, hydroxyl rad-ical; 1O2, singlet oxygen; NO, nitric oxide; OONO�, peroxynitrite; H2O2, hydro-

gen peroxide; LPO, lipid peroxides; NAC, N-acetyl-cysteine; ANT-CK,antioxidant cocktail; PBS, phosphate-buffered saline; FACS, fluorescence-acti-vated cell sorting; FITC, fluorescein isothiocyanate; DIC, differential interfer-ence contrast; PARP, poly(ADP-ribose) polymerase; BSO, DL-buthionine-(S,R)-sulfoximine; DHR, dihydrorhodamine; DHE, dihydroethidium; HPF, 3�-(p-hydroxyphenyl) fluorescein; mBCl, monochlorobimane.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 282, NO. 42, pp. 30452–30465, October 19, 2007Printed in the U.S.A.

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has primarily been thought to reflect the generation of reactivespecies of oxygen (ROS) and nitrogen (RNS) (6, 7). Although,the role of ROS in apoptosis has been extensively studied (6, 7),contradictory results including both inhibitory and stimulatoryeffects of ROS on apoptosis have been reported. For example,induction of apoptosis by activation of the Fas receptor hasbeen suggested to be dependent on the generation of H2O2formation (20, 22–26), while other studies report an inhibitoryrole of H2O2 on Fas-mediated cell death (27–29).

In the present work, we evaluate the relationship betweenGSH depletion, ROS generation, and their respective roles inapoptosis at the single cell level. We present evidence thatclearly uncouples for the first time the role of GSH depletion inapoptosis from the generation of ROS. Furthermore, we dem-onstrate that changes inGSH content are necessary for apopto-sis to occur. In contrast, increases in ROS did not modulateapoptotic cell death. Our data suggest an important role forchanges in intracellular GSH content in the activation of pro-grammed cell death independent from increases in ROSformation.

EXPERIMENTAL PROCEDURES

Reagents—RPMI 1640, penicillin/streptomycin, heat-inac-tivated fetal calf serum, sodium pyruvate and were fromInvitrogen. MK571 was purchased from BioMol (Plymouth,PA). N-Acetyl-L-cysteine (NAC), deferoxamine mesylate(also known as desferrioxamine or desferroxamine), ebselen,diphenylene-iodonium (DPI), catalase (bovine liver), Mn-TBAP (Mn(III)tetrakis(4-benzoic acid) porphyrin chloride),MnTE-2-PyP (manganese(III)-5,10,15,20-tetrakis(N-ethylpyri-dinium-2-yl) porphyrin pentachloride), and trolox were fromCalbiochem/EMD Biosciences Inc. (San Diego, CA). Mono-chlorobimane (mBCl), dihydrorhodamine 123 (DHR), 3�-(p-hydroxyphenyl) fluorescein (HPF), dihydroethidium (DHE),4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-inda-cene-3-propio-nyl ethylenediamine hydrochloride (BODIPY� FLEDA), cis-parinaric acid (CsPA), 4-amino-5-methylamino-2’,7’-difluoro-fluorescein (DAF-FM diacetate), carboxy-PTIO, TOTO-3, and Singlet Oxygen Sensor Green werefrom Molecular Probes Inc. (Eugene, OR). Cytofix/cytoperm kit,and the monoclonal antibodies FITC-conjugated anti-cleavedcaspase 3, and PE-conjugated anti-poly(ADP-ribose) polymerase(PARP) cleaved site-specific were from BD Biosciences (SanDiego, CA). Anti-cleaved caspase 3, anti-caspase-3, anti-caspase-6, anti-caspase-7, anti-PARP, and anti-�-fodrin antibod-ies were from Cell Signaling Technology Inc (Beverly, MA). Allother reagents were from Sigma/Aldrich.Cell Culture andMedia—Jurkat cells, E6.1 clone (human leu-

kemia) were obtained from American Tissue Culture Collec-tion (Manassas, VA). Cells were cultured in RPMI 1640medium containing 10%heat-inactivated fetal calf serum, 2mMglutamine, 31 mg/liter penicillin, and 50 mg/liter streptomycinat 37 °C, 7% CO2 atmosphere. Apoptosis was induced by incu-bating cells (5–7 � 105 cells/ml) at 37 °C, 7% CO2 for the timeindicated with either human recombinant FasL (Kamiya Bio-medical Co., Seattle, WA), cycloheximide, etoposide or treat-ment with ultraviolet C light (UVC) light in a UV Stratalinker1800 (Stratagene La Jolla, CA). In media containing high GSH

(�GSH) or NAC (�NAC), NaCl was substituted with 25 mML-glutathione or 10 mMNAC,maintaining the same osmolarityof the media. Media osmolarity was measured on a Wescor5500 vapor pressure osmometer (Logan, UT).Fluorescence-activated Cell Sorting (FACS)—Apoptotic

parameters were analyzed by FACS, using a BD LSR II flowcytometer and BD FACSDiva Software (Becton Dickinson, SanJose, CA) for data analysis. Samples were analyzed and in allcases 1� 104 cells were recorded. Fluorophores were diluted inMe2SO or dimethylformamide (DMF) and preloaded just priorto FACS analysis and incubated at 37 °C, 7% CO2 in cell culturemedium, unless otherwise indicated. Control cells were incu-bated with vehicles, which never exceed 0.1% in the final con-centration. Propidium iodide (PI) was added to a final concen-tration of 10�g/ml just prior to FACS analysis and analyzed forFL-2 (488 excitation, 575/26 emission). Sequential analysis ofthe distinct fluorophores was used and cells with increased PIfluorescence (loss of membrane integrity) were excluded dur-ing the analysis unless otherwise stated. Cell debris or frag-ments were gated out from the analysis on a forward scatter/side scatter plot. The appearance of discrete cell populationswas identified as described previously (13). Briefly, gates wereset on dot plots of the respective dye-fluorescence versus for-ward scatter, and then represented accordingly. Changes in thebiochemical parameters during apoptosis are observed aschanges in the normal distribution of the population of cellswith differences in the mean fluorescence intensity for the dis-tinct fluorescent reporters used, compared with control cells inthe absence of stimuli and were determined as follows.Changes in Intracellular Glutathione Content, GSHi—For

intracellular GSH determinations, cells were preloaded for 15min with 10 �M mBCl, which forms blue fluorescent adductswith intracellular glutathione (30). Immediately prior to flowcytometry examination, PI was added. For mBCl, cells wereexcited with a Violet (UV) 405 nm laser, and emission wasacquired with a 440/40 filter. Changes in the GSHi are reflectedby the appearance of populations of cells with differences inmBCl fluorescence, reflecting changes in GSHi. Populationswere gated as previously described (13). Briefly, contour plots ofmBCl fluorescence versus forward scatter were used to identifypopulations of cells with different GSHi levels induced afterFasL treatment. Three different cell populationswere identifiedand represented independently.Detection of Reactive Oxygen Species (ROS)—Hydrogen

peroxide formation was detected using nonfluorescent DHR,which after its oxidation by H2O2 in the presence of peroxi-dases, or Fe2� results in cationic rhodamine (31). Hydroxylradical (�OH�) formation was analyzed using HPF, which isessentially nonfluorescent until it reacts with �OH yielding abright green fluorescent compound (32). Nitric oxide (NO)detection was performed using DAF-FM. After its cleavageby intracellular esterases, DAF is nonfluorescent until itreacts with the spontaneous oxidation product of nitricoxide, nitrosonium cation, to form a fluorescent heterocyclebenzotrizole that is trapped in the cytosol. Cells were pre-loaded for 30 min with 5 �M of either DHR, HPF,or DAF-FM and analyzed for FL1 (488 excitation, 530/30emission).

Uncoupling GSH Depletion and ROS in FasL-induced Apoptosis

OCTOBER 19, 2007 • VOLUME 282 • NUMBER 42 JOURNAL OF BIOLOGICAL CHEMISTRY 30453


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Generation of lipid peroxides (LPO) was assessed usingBODIPY� FL EDA, which upon interaction with peroxyl radi-cals looses its fluorescence; and also by fluorescence quenchingof the fatty acid analog CsPA (33). Cells were equilibrated witheither 5 �M BODIPY� FL EDA or 1.5 �M CsPA for 30 min.Then, cells were washed and resuspended in fresh mediumbefore apoptosis induction. BODIPY� FL EDA was analyzed inFL-1 and CsPA was analyzed at 325/355 nm excitation and440/40 emission. Superoxide anion (�O2

�) formation wasdetected using DHE, which after its oxidization to ethidium by�O2

�, it intercalates within the DNA and exhibits bright red flu-orescence (34). Cells were preloaded for 30minwith 5�MDHE,and changes in its fluorescence were detected in FL-2. For DHEanalysis, TOTO-3 dye was added to the samples instead of PIand analyzed at 633 excitation and 660/20 emission to assessedloss of membrane integrity.Caspase 3 and PARP Cleavage Analysis by FACS—Simulta-

neous analysis of the cleavage of caspase 3 and PARP was per-formed as previously described (13). Briefly, cells were washedwith phosphate-buffered saline (PBS), then fixed and perme-abilized using the Cytofix/Cytoperm kit for 30 min at roomtemperature in the dark according to the manufacturer’s spec-ifications. Cells were then pelleted and washed with the Perm/Washbuffer and stained for 1 h at room temperature in the darkwith FITC-conjugated anti-cleaved caspase 3 or PE-conjugatedanti-PARP cleavage site-specific. Following incubation, cellswere washed with the PermWash buffer and analyzed by flowcytometry. PE and FITC fluorescence were examined for FL-1and FL-2, respectively.Analysis of Plasma Membrane Lipid Symmetry—Changes in

the phosphatidylserine symmetry were determined usingannexin V conjugated to FITC (Trevigen, Gaithersburg, MD).Briefly, 5 � 105 control or treated cells, initially washed in PBS,were incubated with 1 �l of annexin-FITC and PI for 15 min atroom temperature according to the manufacturer’s instruc-tions. Annexin-FITC/PI-stained samples were diluted in 400�lof 1� annexin binding buffer and examined immediately byFACS. FITC fluorescence was analyzed on FL1. Early apoptoticcells are defined as having annexin positive, propidium iodidenegative staining. Late apoptotic and non-viable cells are bothannexin and propidium iodide-positive.Differential Interference Contrast (DIC)Microscopy—AZeiss

LSM 510 microscope (One Zeiss Drive, Thornwood, NY) wasused to obtain DIC images. Cells were prestained for 30 min at37 °C and 5% CO2 with 5 �g/ml of Hoechst 33342 and thenwashed with fresh culture media. Hoechst-stained cells wereexamined with a Plan-Apo 63 � oil N.A. � 1.4 objective lens toobtain simultaneous DIC and UV images. Hoechst was excitedat 390 nmwith a TitaniumSapphire 750 nm laser, and emissionwas collected with a BP 465 filter. Images were analyzed withLSM 5 image browser software.Extracellular GSH andGSSGDeterminations—Extracellular

GSH determinations were performed as described in Ref. 13with minor modifications to improve GSH recovery. Experi-ments were performed inHanks’ balanced salt solution (SIGMA/Aldrich). Cells were incubated with 250�M L-(�S,5S)-�-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (acivicin) for 1 h,to inhibit the catabolism of released GSH by the �-glutamyl-

transferase (GGT). Then, cells (2 � 107 cells/ml) were inducedto undergo apoptosis with FasL. This allowed an improvedrecovery of accumulated extracellular GSH. Acivicin was pres-ent throughout the experiment. Samples were centrifuged, andaliquots were taken for extracellular determinations. Pelletswere resuspended in ice-cold phosphate-buffered saline,homogenized, and kept frozen until use for protein determina-tions. The extracellular GSH and GSSG concentrations weremeasured enzymatically using the GSH/GSSG-412 kit (OxisResearch, Portland, OR). Samples were prepared in 5% meta-phosphoric acid to remove proteins, with or without 1-methyl-2-vinyl-pyridium trifluoromethane sulfonate, a GSH-specificscavenger. Ellman’s reagent (5�,5-dithiobis-2-nitrobenzoicacid) reacts with GSH to form a product with an absorptionmaximum at 412 nm. GSSG was determined using glutathionereductase and �-nicotinamide adenine dinucleotide phosphate(NADPH) to reduce GSSG to GSH followed by reaction withEllman’s reagent. Results are normalized to protein concentra-tion for each sample.[3H]GSHUptakeAssays—Apoptosiswas induced in the pres-

ence of 1�Ci of [3H]GSH ([glycine-2-3H]glutathione, 1.53TBq/mmol, Perkin Elmer, Boston,MA). The experiments were donein the presence of 250 �M acivicin and 1 mM BSO (DL-buthi-onine-(S,R)-sulfoximine) to inhibit both GSH catabolism andde novo synthesis, respectively. At the indicated time, cells wereseparated from the medium by filtering through a glass micro-fiber filter (Whatman). Filters were washed with ice-cold PBS.Then, the filter was dried and placed in a scintillation vialcontaining 10 ml of Ecolite (ICN Radiochemicals, CostaMesa, CA). The radioactivity was counted in a LS6500 scin-tillation counter (Beckman Coulter, Fullerton, CA). Resultsare expressed as the radioactivity incorporated (dpm) nor-malized to protein concentration.Protein Extraction andWestern Immunoblotting—Cells were

pelleted, washed once with ice-cold PBS, and lysed in buffercontaining 20 mM Tris-HCl, 150 mM NaCl, 1 mM Na2EDTA, 1mM EGTA, 1% Triton X-100, and protease inhibitors (Com-plete Mini protease mixture, Roche). Samples were sonicated,centrifuged, and the pellets discarded. Then, samples wereassayed in a Beckman DU650 spectrophotometer for proteinconcentration using Bio-Rad protein assay, and cell extractswere normalized to equal protein concentration. Loadingbuffer containing glycerol, sodium dodecyl sulfate (SDS), andbromphenol blue was added, and samples were denatured at99 °C for 5 min. Protein extracts, 50 �g/sample, were separatedby SDS-polyacrylamide gel electrophoresis (PAGE) on 4–20%gradient polyacrylamide Tris/glycine gels (Novex, Invitrogen,CA) and transferred to nitrocellulose. Membranes wereblocked in Tris-buffered saline (TBS) containing 0.1% Tweenand 10%nonfat drymilk for 1 h. Antibodies were diluted inTBScontaining 0.1% Tween and 5% nonfat dry milk. Blots wereincubated with the corresponding primary antibody (1:1000)overnight. Then, blots were incubated for 1 h with the corre-sponding horseradish peroxidase-linked secondary antibodies(Amersham Biosciences) diluted 1:10,000 in TBS containing0.05% Tween, and 0.5% nonfat dry milk. Blots were then visu-alized on film with the ECL chemiluminescent system (Amer-sham Biosciences). Blots were subsequently stripped using




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65 mM Tris-HCl, pH 6.7, 100 mM�-mercaptoethanol, 2% SDS buffer,and reprobed with distinct antibod-ies. To verify equal protein loading,blots were also reprobed with anti-�-tubulin antibody.Statistical Analysis—When indi-

cated, significance of differences inmean values were calculated usingthe two-tailed Student’s t test. Thenumber of experiments is indicatedin each case in the legends of thefigures.

RESULTS

FasL-induced GSH Loss Is In-dependent of, but Necessary for, theGeneration of ROS—Glutathionedepletion is a common phenome-non during apoptosis (11–14).Moststudies suggest that GSH depletionduring apoptosis is an indicator forROS formation and oxidative stress.Such findings have led to the wide-spread assumption that GSH deple-tion is a byproduct of the apoptoticprocess; and that ROS formationand GSH depletion in apoptosis arecoupled together as cause and con-sequence phenomena (6, 7, 35).However, we and others (11–13, 15,16) have demonstrated that GSHloss is mediated by the activation ofa specific efflux pump. Triggeringapoptosis induces the formation ofROS and RNS (6, 7, 35). Thus, wesought to investigate the relation-ship between GSH depletion andROS formation in FasL-inducedapoptosis in Jurkat cells. Because ofthe stochastic nature of apoptosis,multiparametric flow cytometry(FACS) was used because it allowsus to identify the appearance of dis-crete populations of cells at differ-ent stages in apoptosis and to ana-lyze them at the single cell level.Such analysis is not possible withconventional bulk biochemicalassays. Fig. 1 (histograms) showsthat FasL induced a dose-dependentgeneration of several ROS formsincluding H2O2 (Fig. 1A), superox-ide anion �O2

� (Fig. 1B), hydroxylradical �OH (Fig. 1C). We alsodetected lipid peroxide (LPO) for-mation (Fig. 1D), observed as a lossof BODIPY FL-EDA-fluorescence

FIGURE 1. Reactive oxygen species generation correlates with changes in intracellular glutathione, GSHi.Reactive oxygen species formation was assessed by FACS using DHR for H2O2 detection; DHE for superoxideanion (�O2

�); HPF for hydroxyl radical (�OH�); and BODIPY� FL EDA, for lipid peroxides (LPO). Changes in intra-cellular glutathione concentration GSHi were determined by FACS using the thiol binding dye monochlo-robimane, mBCl. For the induction of apoptosis, Jurkat cells were incubated with FasL for 4 h at theconcentration indicated. Data are expressed as changes in ROS-sensitive dye fluorescence represented byfrequency histograms (upper panels in A–D) or in contour plots versus changes in mBCl fluorescence (GSHi) (lowerpanels in A–D). In contour plots, the quadrant center gray lines was set at the mean fluorescence intensity for mBCland the corresponding ROS dye, as a reference to indicate basal levels of GSHi and ROS. Populations were gated andrepresented according to the differences in ROS levels. Plots are representative of n � 3 independent experiments.




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because of its oxidation. Simultaneous analysis of changes inGSH content and ROS demonstrate that ROS formation wasconcomitant with GSH depletion (Fig. 1, A–D, contour plots).To detect the generation of H2O2 we used DHR, which hasproved to be a more stable dye for H2O2 detection (32, 36).AlthoughDHRcan also be oxidized by peroxynitrite (OONO�)(31), ebselen (10–100 �M), a cell permeable scavenger ofOONO�, did not inhibit DHR oxidation during FasL-inducedapoptosis, suggesting that changes in DHR reflect changes inH2O2 content.We did not detect singlet oxygen (1O2) (assessedwith the Singlet Oxygen Sensor Green dye), or nitric oxide(NO) (analyzed using the nitric oxide dye DAF-FM) formationduring FasL-induced apoptosis (data not shown). Reactive oxy-gen species generation occurred in all cases, prior to the loss ofmembrane integrity assessed by changes in PI fluorescence.We next analyzed the role of ROS on themodulation of GSH

depletion by the use of a variety of antioxidants (Fig. 2, A–C).Hydrogen peroxide formation induced by FasL was preventedin the presence of pyruvate (Fig. 2A, upper panels), which actsas a potent intracellular scavenger for H2O2 (37–39). Superox-ide anion formation was drastically reduced by the cell perme-ablemanganic-porphyrinsMnTE-2-PyP (Fig. 2B,upper panels)and MnTBAP, which act as a superoxide dismutase (SOD)mimics, but not by the inhibitor of the NADPH oxidase, DPI

(50 �M) (data not shown). In addition, dimethyl sulfoxide(Me2SO or DMSO), which quenches �OH to form the stablenonradical compound methanosulfinic acid, significantlydecreased �OH� formation (Fig. 2C, upper panels). Finally, sup-plementation of cells with trolox, a cell-permeable vitamin Eanalog, which is known to be a potent lipophilic scavenger oflipid peroxides, prevented LPO formation (Fig. 2D, upper pan-els). Similar results were also observed when we used CsPA toanalyze LPO formation (data not shown). Reduced glutathionehas been reported to scavenge directly or indirectly many ofthese and other ROS forms. However, scavenging of the ROSgenerated upon Fas activation by these antioxidants, did notsignificantly affect GSH depletion (Fig. 2, A–D, lower panels)corroborating thatGSHdepletion is not related to its oxidation.As reported previously, supplementation with trolox, signifi-cantly reduced cellular GSH content (40) (Fig. 2D).The studies described above led us to consider: 1) whether

GSHdepletionwas required for ROS formation; and 2)whetherROS subsequently mediated the reported regulatory effects ofGSH depletion in apoptosis. Our previous study demonstratedthat GSH loss during FasL-induced apoptosis is prevented bydecreasing the electrochemical gradient of GSH across theplasma membrane with high extracellular GSH medium (13).High extracellular GSHmedium significantly reduced the gen-

FIGURE 2. Reactive oxygen species scavenging by antioxidants does not affect FasL-induced GSH loss. Reactive oxygen species formation and changesin GSHi were assessed by FACS. Apoptosis was induced in Jurkat cells by incubation with FasL (50 ng/ml FasL) for 4 h. The effect of the antioxidants 10 mM

sodium pyruvate (for H2O2) (A), 2% Me2SO (DMSO) (�OH�) (B), 250 �M MnTE-2-PyP (�O2�) (C), and 5 mM trolox (LPO) (D), was individually assessed. Jurkat

cells were preincubated in RPMI for 1 h at 37 °C with the agents dissolved in either Me2SO or ethanol, and antioxidants remained throughout theexperiment. In all cases, control conditions include vehicles at the same concentration. Populations were gated according to the differences in ROSlevels. Data are expressed as frequency histograms of either changes in the fluorescence of the ROS-sensitive dyes (upper panels A–C) or mBCl (lowerpanels A–C). In A–C lower panels, GSH depletion is depicted as the appearance of a population of cells at the left of the gray solid lines. The plots arerepresentative of n � 3 independent experiments.




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eration of all ROS formed during FasL stimuli (Fig. 3). A com-parable degree of prevention of ROS formationwas observed inthe presence of NAC, a thiol dipeptide that replenishes intra-cellular GSH content. These data strongly indicate that GSHdepletion is necessary for ROS generation and that this deple-tion precedes cellular oxidative stress. We have previouslydemonstrated thatMK571 is a potent stimulator of GSH deple-tion during apoptosis (13). Fig. 4 shows that stimulation ofGSHloss by MK571 was paralleled by an increase in oxidative stress

measured by an increase in the lev-els of �O2

�. These data suggest thatGSH depletion regulates oxidativestress during apoptosis.Reactive Oxygen Species Genera-

tion Is a Bystander PhenomenonThat Does Not Regulate FasL-in-duced Apoptosis in Jurkat Cells—We have recently demonstratedthat GSH depletion is necessary forthe activation of the executionphase of apoptosis (13). The role ofROS in apoptosis has been largelyimplicated by the use of agents thatby themselves stimulate ROS for-mation. However, the involvementof ROS in apoptosis induced byphysiological stimuli remains con-troversial (41). Because we observedthat GSH depletion preceded ROSformation, we sought to determineif ROSmodulates apoptosis inducedby FasL. Fig. 5A shows that FasL-in-duced apoptosis,measured by phos-phatidyl externalization and loss ofplasma membrane integrity (cellviability), was not affected in the

presence of any of the antioxidants demonstrated to inhibitROS formation. Similarly, FasL induced cleavage of caspase 3and PARP simultaneously analyzed by FACS, as well as othermorphological parameters of apoptosis including nuclear con-densation, plasmamembrane blebbing, and cellular fragmenta-tion were not affected by the non-thiol antioxidants tested (Fig.5, B andC). To evaluate the possibility that quenching one ROSis compensated by other ROS formed, and so their possible roleon GSH loss and apoptosis is underestimated, we assessed theeffect of an antioxidant mixture (ANT-CK), which containedpyruvate, MnTE-2-PyP, Me2SO, and trolox at the concentra-tions used to individually quench each ROS, on apoptosisinduced by Fas activation. The ANT-CK did not prevented theappearance of any of the apoptotic markers examined (Fig. 5).We also analyzed the effect of other antioxidants for distinctROS and RNS. Singlet oxygen is another highly reactive freeradical, which acts as a common intermediate in a range ofbiological systems exposed to oxidative stress. Moreover, sin-glet oxygen has been shown to be generated in other biologicalprocesses that do not involve light (42). The RNS, NO, andOONO� have also been reported to act as important modula-tors of apoptosis (43, 44). Neither the antioxidants for NO andOONO�, carboxy-PTIO (100 �M) and ebselen (10–100 �M),nor the 1O2 scavenger, sodium azide (NaN3, 10 mM), had aneffect on FasL-induced GSH loss and apoptosis when usedalone or in combination with the ANT-CK (data not shown).Thus, FasL-induced apoptosis in Jurkat cells is clearly inde-pendent from increases in ROS formation.Previous studies have reported a protective effect of several

other antioxidants such as catalase and deferoxamine on apo-ptosis induced by Fas activation (22, 25, 45). However, little

FIGURE 3. Glutathione depletion is necessary for ROS formation. Jurkat cells were incubated with FasL (50ng/ml FasL) for 4 h in the presence or absence of high extracellular GSH medium (25 mM) or NAC (10 mM). Mediawas switched at the time of FasL stimuli. High glutathione (�GSH) and N-acetyl-cysteine (�NAC) medium wasmade by substitution of NaCl, maintaining the isomolarity of the media. Reactive oxygen species formationwas assessed by FACS as explained under “Experimental Procedures” and gated and expressed as in Fig. 2. Plotsare representative of at least n � 3 independent experiments.

FIGURE 4. Stimulation of GSH depletion enhances ROS formation. Super-oxide anion (�O2

�) formation (used as a marker of oxidative stress) andchanges in GSHi were assessed by FACS as in Fig. 1. Apoptosis was induced inJurkat cells by incubation with FasL (50 ng/ml FasL) for 4 h in the presence orabsence of 50 �M MK571. Data are expressed as changes in DHE-fluorescence(�O2

�) represented by frequency histograms (upper panels), or in contour plotsversus changes mBCl fluorescence (GSHi) (lower panels). In contour plots, thequadrant center was set at the mean fluorescence intensity for mBCl and thecorresponding ROS dye, as a reference to indicate basal levels of GSHi andROS. Populations were gated and represented according to the differences inDHE fluorescence or �O2

� content, as in Fig. 1B. Plots are representative of n �3 independent experiments.




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FIGURE 5. Reactive oxygen species do not modulate FasL-induced apoptosis. FasL-induced apoptosis was assessed by (A) phosphatidylserineexternalization and loss of plasma membrane integrity (cell viability); (B) simultaneous detection of cleaved caspase 3 and PARP by FACS; as well as by(C) changes in morphological hallmarks of apoptosis which include nuclear condensation, plasma membrane blebbing, and cellular fragmentation.Apoptosis was induced by 50 ng/ml FasL over 4 h. The effect of sodium pyruvate (10 mM), Me2SO (DMSO) (2%), MnTE-2-PyP (250 �M), trolox (5 mM) wasindividually assessed or in conjunction (ANT-CK). In A, early externalization of phosphatidylserine is shown as an increase in the number of cells that hadan increase in annexin V-FITC fluorescence (population b), prior to the loss of membrane integrity or high PI fluorescence with respect to control cells(population a). Loss of plasma membrane integrity or cell viability in later stages of apoptosis is reflected as an increase in both PI and FITC fluorescence(population c). The contour plots are representative of a single experiment representative of n � 3. In B, contour plots in control panels show thedistribution of cells with background fluorescence for FITC-conjugated anti-cleaved caspase 3, or PE-conjugated anti-cleaved PARP antibodies (popu-lation a, black). Simultaneous cleavage of caspase 3 and PARP during apoptosis is reflected as a coincident increase in the fluorescence for FITC and PE(population d, gray). Other less represented populations indicate cells which are positive for either cleaved caspase 3 (population b, dark gray) or cleavedPARP (population c, light gray) antibodies respectively, which never reach more than 10% of the total sample. Plots are representative of at least n � 3independent experiments. In C, DIC images were obtained to analyze changes in cell morphology during apoptosis. Nuclei condensation is observed asa picnotic and brighter nuclei of cells stained with Hoechst 3342 (see black arrows for examples). Examples of cells fragmented or with plasmamembrane blebs are pointed by white arrows. Images are representative of at least three independent experiments.




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effectiveness of these antioxidants was observed in our experi-mental model. Fig. 6, A and B shows that catalase and deferox-amine do not scavenge the increase in H2O2 and �OH� forma-tion induced by FasL. Moreover, they did not prevent GSHdepletion and apoptosis under the same conditions (Fig. 6,A–D). The lack of effect of these antioxidants in our system,particularly on the scavenging of ROS, ismore consistent with alimited cell permeability of these agents. Catalase (a 240-kDaenzyme that catalyzes H2O2 to water and oxygen) does notpermeate the cells unless plasma membrane integrity is com-promised. Similarly, deferoxamine is a lysosomotropic ironchelator, which has been demonstrated to be impermeable tothe plasmamembrane and if internalized, has been shown to beuptaken by endocytosis and exclusively localized in the cells atthe lysosome compartment (46–48). This discrepancy likelyrelies on the different cell types used (22, 25, 45). Medan et al.(25) reported that when they used macrophages, catalase anddeferoxamine efficiently scavenge ROS and prevent apoptosisinduced by Fas activation. This is probably ascribed to the phag-ocytic potential of macrophages that might efficiently accumu-late these antioxidants. In contrast, in our studies, we usedpyruvate that has been reported be actively uptaken by mono-

carboxylate transporters by cellsand to act as a potent intracellularscavenger for H2O2 (37–39). Simi-larly, dimethyl sulfoxide freely per-meates the plasma membrane anddirectly quenches �OH�. Otherantioxidants tested here, such assuperoxide dismutase mimics(MnTBAP) and vitamin E (trolox),to scavenge �O2

� and LPO, were alsoused in their cell-permeable form toensure effective ROS scavenging asshown in Fig. 2.Glutathione Transport Regulates

Apoptosis Induced by MultipleStimuli—Weandothers (11–13, 15)have recently shown that the reduc-tion in theGSH content during apo-ptosis in Jurkat cells is mediatedthrough the activation of an effluxtransport mechanism rather thandue to its oxidation to GSSG or tothe loss of membrane integrity. Werecently showed that this transportis stimulated by distinct agentsincludingMK571 and is inhibited byhigh extracellular GSH concentra-tions. Fig. 7A shows that indeed,FasL-induced GSH depletion isstimulated by MK571 and pre-vented by high GSH medium. Thesame stimulatory effects are ob-served by other OA� substratesincluding taurocholic acid andestrone sulfate (data not shown).We have previously demonstrated

that MK571 is a more potent stimulator of GSH depletion dur-ing apoptosis (13). In accordance with our previous work (13),FasL-induced extracellular GSH accumulation was signifi-cantly stimulated by MK571 (Fig. 7B). A slight, but significantaccumulation of oxidizedGSH in its disulfide form (GSSG) wasalso detected extracellularly, which might be ascribed to GSHoxidation after its release.We next evaluated whether GSH efflux and depletion is a

necessary for the progression of apoptosis induced by differentapoptotic stimuli known to activate cell death via distinct sig-naling pathways. Fig. 8A shows that FasL-induced apoptosisobserved as phosphatidylserine externalization (Fig. 8A), nucleicondensation, plasmamembrane blebbing, and cell fragmenta-tion (Fig. 8B), was prevented by inhibition of GSH depletion byhigh extracellular GSH. In contrast, stimulation of GSH deple-tion with MK571 accelerated the progression of cell death.Additionally, high extracellular GSH medium prevented apo-ptosis induced by other stimuli including stress (UVC radia-tion), impairment of protein synthesis (cycloheximide), andinhibition of topoisomerase II (etoposide) (Fig. 8C). Togetherthese results suggest that the regulation of cell death progres-sion by changes in GSH content is a common component of

FIGURE 6. Catalase and deferoxamine are ineffective against ROS formation and apoptosis. Apoptosiswas induced in Jurkat cells by incubation with FasL (50 ng/ml FasL) for 4 h in the presence or absence of 1000milliunits/ml catalase (bovine liver) or 1 mM desferoxamine mesylate. Jurkat cells were preincubated in RPMI for1 h at 37 °C with the agents, and antioxidants remained throughout the experiment. In all cases, controlconditions include vehicles at the same concentration. A and B, simultaneous analysis of changes in GSHcontent and generation of H2O2 or �OH� was performed by FACS. Data are expressed as in Fig. 2. FasL-inducedapoptosis was assessed by (C) phosphatidylserine externalization and loss plasma membrane integrity;(D) simultaneous detection of cleaved caspase 3 and PARP; and (E) DIC images depicting nuclear conden-sation, plasma membrane blebbing, and cellular fragmentation. Results are expressed as in Fig. 5. Plotsand images are representative of n � three independent experiments.




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apoptosis induced by a variety of signals that activate bothintrinsic and extrinsic pathways of cell death.N-Acetyl-cysteine is a derivative of the sulfur-containing

amino acid cysteine and an intermediary (along with glutamicacid and glycine) in the conversion of cysteine to GSH. NAC isthought to be transported into the cell directly where it ishydrolyzed to cysteine before it serves as a precursor for GSHformation. In the presence of NAC, FasL-induced GSH deple-tion was prevented (Fig. 9A). Replenishing of the intracellularGSH pools with the thiol dipeptide NAC also protected Jurkatcell from apoptosis induced by FasL analyzed by phosphatidyl-serine externalization (Fig. 9B); nuclei condensation, plasmamembrane blebbing, and cell fragmentation (Fig. 9D); andcleavage of execution caspases 3, 6, and 7 and of their substratesPARP and �-fodrin (Fig. 9, C and E). Moreover, the enhance-ment of apoptosis by MK571-induced stimulation of GSHdepletion was fully prevented by NAC (Fig. 9).It is still controversial how extracellular GSH influences cel-

lular redox state. In contrast to NAC, GSH is thought to betaken up poorly by cells, and thus it only serves to provide con-stituent amino acids. Indeed, GSH has to be broken down intoits constituent amino acids by the �-glutamyl transferase (GGT)transported into the cell, and then resynthesized de novo by theactivity of the �-glutamylcysteine synthetase (�-GCS).We nextstudied the mechanism for extracellular GSH to maintain the

level of intracellular GSH and reduce Fas-mediated apoptosis.To asses the role of de novo synthesis of GSH in this process, weevaluated the protective effect of high extracellular GSH in thepresence of BSO, an inhibitor of the �-GCS. A 24-h treatmentwith BSO significantly depletes cells of intracellular GSH(�GSH) (Fig. 10A). When GSH-depleted cells were preincu-bated with high GSH medium the intracellular GSH pool issignificantly replenished after 4 h, even with persistent inhibi-tion of de novo GSH synthesis pathway with BSO. Fig. 10Bshows that high extracellular GSH stimulates an active uptakeof radiolabeled [3H]GSH in the presence of BSO and acivicin.These results suggest that high extracellular GSH is able toreplenish the intracellular GSH pool without the involvementof de novo synthesis; thus, suggesting an influx of extracellularGSH. Finally, high extracellular GSH was still able to prevent

FIGURE 7. Glutathione loss is mediated by a plasma membrane transport,which is stimulated by MK571 and inhibited by high extracellular GSHmedium. A, changes in GSHi were determined by FACS. Effect of 4 h of treat-ment with 25 ng/ml FasL on the GSHi of Jurkat cells. Populations were gatedaccording to their GSHi levels on an mBCl fluorescence versus forward scatterplot as explained under “Experimental Procedures.” Plots are representativeof at least four independent experiments. B, extracellular determinations ofreduced (GSH) and oxidized (GSSG) glutathione. Cells (2 � 107 cells/ml) wereincubated (1 h) with acivicin (250 �M), and then, stimulated with FasL (100ng/ml) for 2 h with or without MK571 (50 �M). Samples were centrifuged, andaliquots of the media were taken to determine changes in extracellular (e)levels of GSH and GSSG. Results are normalized to protein concentration foreach sample and are means of n � 4 � ES. *, p � 0.05, against correspondingcontrol values; **, p � 0.05 against corresponding FasL values.

FIGURE 8. Glutathione transport regulates apoptosis induced by distinctstimuli. For the induction of apoptosis, Jurkat cells were incubated with 25ng/ml FasL for the time indicated. Data are expressed as frequency histo-grams of mBCl fluorescence. MK571 (50 �M) and high extracellular GSHmedium (�GSH) were added at the time of FasL stimuli. In A, apoptosis wasassessed by phosphatidylserine externalization using annexin-FITC stainingas explained under “Experimental Procedures.” Plots are representative of atleast n � 4 independent experiments, and in B, DIC images were obtained toanalyze changes in cell morphology during apoptosis. Images are represent-ative of at least three independent experiments (see Fig. 5). In C, the effect ofhigh extracellular GSH on apoptosis induced by 8 h exposure of FasL (25ng/ml), UVC radiation (15 mJ/cm2), cycloheximide (30 �M), and etoposide(100 �M) was studied. Apoptosis was assessed by the % of cells with cleavedcaspase 3 assessed by FACS as in Fig. 5. Data are expressed as mean � S.E. ofn � 3. *, p � 0.005 against treatments in the absence of high extracellular GSH(�GSH).




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FasL-inducedGSH depletion (Fig. 10C) and apoptosis induced byFasL, analyzed by phosphatidylserine externalization (Fig. 10D);cleavage of execution caspases 3 and PARP (Fig. 10E); and nucleichromatin condensation, plasma membrane blebbing, and cellfragmentation (Fig. 10E), even when GSH de novo synthesis isinhibited. These results suggest that high GSH concentrationsmight protect against apoptosis by reversingGSHefflux transportand not by replenishing GSH pools via de novo synthesis.

DISCUSSION

The redox environment of the cells has been suggested to beimportant in the control of apoptosis. Small thiols, includingglutathione are viewed as protective antioxidants acting as freeradical scavengers in response to oxidative damage; thus, beingimportant players in the redox balance of the cell. Glutathionedepletion has been shown to occur in apoptosis induced by awide variety of stimuli, and has been shown mediated by itsextrusion and not its oxidation (11–17). Glutathione depletionhas been shown to directly modulate both the permeability

transition pore formation, and the activation of executioncaspases (13, 49–54). In vitro studies have also shown that areduction in the GSHi is necessary for the formation of theapoptosome (23). We recently demonstrated that GSH deple-tion during FasL-induced apoptosis ismediated by its extrusionthrough the activation of a plasma membrane GSH transportmechanism. In addition we demonstrated that GSH efflux isnecessary for the activation of the execution phase of FasL-induced apoptosis (13). However, the precise mechanism bywhich GSH loss regulates apoptosis remains unclear. BecauseGSH depletion and ROS generation are both phenomenalargely thought to be correlated, we sought to clarify the rela-tionship betweenROSandGSHdepletion during FasL-inducedapoptosis. Here, we present evidence that shows not only thatGSHdepletion is independent of ROS generation, but that ROSformation is most likely a bystander phenomenon in the pro-gression of FasL-induced apoptosis in lymphoid cells. We pro-pose that thiol regulation of apoptosis is largely independent ofROS formation. Finally, GSHdepletionwas observed to be nec-

FIGURE 9. N-acetyl-L-cysteine protects against FasL-induced GSH depletion and apoptosis. Apoptosis was induced in Jurkat cells by incubation with 25 (A)and 50 (B–E) ng/ml FasL for 4 h. MK571 (50 �M) and high extracellular NAC medium (�NAC) were added at the time of FasL stimuli. A, changes in GSHi weredetermined by FACS. Populations were gated according to their GSHi levels on an mBCl fluorescence versus forward scatter plot as explained under “Experi-mental Procedures.” FasL-induced apoptosis was assessed by (B) phosphatidylserine externalization and loss of plasma membrane integrity; (C) simultaneousdetection of cleaved caspase 3 and PARP; (D) DIC images depicting nuclear condensation, plasma membrane blebbing, and cellular fragmentation; and(E) immunoblot detection of full-length and cleaved forms of execution caspases 3, 6, and 7, as well as of their substrates PARP and �-fodrin. Results areexpressed as in Fig. 5. Plots, images, and blots are representative of n � 3 independent experiments.




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essary for the progression of apoptosis induced by several dif-ferent stimuli, which act by different signaling pathways toinduce cell death.We previously reported that GSH efflux in FasL-4 induced

apoptosis is mediated, by a transporter with characteristics of aGSH/OA� exchanger (13). Members of the SLCO/OATPtransporter family have been suggested to function as bidirec-tional transporters of glutathione in exchange for an organicanion (OA�) substrate (55–62) acting then, as GSH exchang-ers. In addition, they are reported to transport a wide range ofOA� in exchange for GSH (56, 59). Glutathione efflux by theseproteins has been reported to be trans-stimulated by extracel-

lular OA� substrates (i.e. its transport is accelerated by thepresence of extracellular substrates that increase the exchangetransport rate). The driving force for the net GSH exportthrough GSH/OA� exchangers is determined by the electro-chemical gradient of GSH across the plasma membrane (11,59). Glutathione is 90% freely distributed in the cytosol where itis present in concentrations up to 10 mM in animal cells,whereas in plasma, concentrations are 0.01 mM, and in cellculture medium they are around 0.003 mM. Moreover, becauseGSH is negatively charged at physiological pH, there is a largeintracellular negative potential of �30 to �60 mV that facili-tates its extrusion. Increasing the extracellular GSH concentra-

FIGURE 10. High extracellular GSH protects against FasL-induced GSH depletion and apoptosis independent of de novo synthesis. The role of denovo GSH synthesis in the protective effects of high extracellular GSH was evaluated using BSO (1 mM) an inhibitor of the �GCS. In A, cells were treatedwith BSO for 24 h (�GSH). This, significantly depleted cells of GSHi compared with control cells (control panel). When GSH-depleted cells were preincu-bated for 4 h prior to FACS analysis with high GSH medium (�GSH), the GSHi pool was replenished even in the presence of BSO. In B, Jurkat cells werepreincubated with 1 �Ci of 3H-GSH in the presence of 250 �M acivicin and BSO, with or without high extracellular GSH. Data are expressed as the amountof radioactivity uptaken by the cells (dpm), normalized by protein concentration per sample. *, p � 0.05, against -GSH values. In C–F, apoptosis wasinduced in Jurkat cells by incubation with 50 ng/ml FasL for 4 h. High extracellular GSH medium (�GSH) was added at the time of FasL stimuli. C, changesin GSHi were determined by FACS. Populations were gated according to their GSHi levels on an mBCl fluorescence versus forward scatter plot asexplained under “Experimental Procedures.” After 4 h, BSO starts depleting cells of GSHi, which is prevented by high extracellular GSH medium. Forreference, grey solid lines in A and C depict the medium fluorescence intensity of mBCl in control cells, which reflects basal levels of GSH; grey dashed linesin A depict the medium fluorescence intensity of mBCl in BSO-treated cells, which reflects depleted levels of intracellular GSH. FasL-induced apoptosiswas assessed by (D) phosphatidylserine externalization and loss of plasma membrane integrity; (E) simultaneous detection of cleaved caspase 3 andPARP; and (F) DIC images depicting nuclear condensation, plasma membrane blebbing, and cellular fragmentation. Results are expressed as in Fig. 5.BSO in B–F was preincubated for 1 h and was present throughout the experiments. Plots and images are representative of n � 3 independentexperiments.




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tion decreases the driving force of GSH extrusion and reducesnet GSH efflux mediated by GSH/OA� exchangers. Thus, highextracellular GSH should stimulate GSH/OA� exchange ofGSHi; however, net reduction of GSHi is prevented by itsexchange for extracellular GSH.Bi-directional GSH/OA� has been reported in different cell

types including human cell lines (56, 57, 60–64). AlthoughOATP (human)/Oatp (rat, mouse) proteins were initiallyreported to mediate this exchange transport (57, 58, 62), arecent study has suggested that GSH/OA� exchange is notmediated by this family of transporters (65, 66). We have pre-viously detected the presence ofmRNA for 7 differentmembersof the SLCO/OATP family of transporters in Jurkat cells (13).We have initially proposed that an exchange transporter medi-ates GSH efflux during apoptosis based on several characteris-tics: 1) stimulation by a wide variety of extracellular OA� sub-strates including MK571; 2) reduction by high extracellularGSH medium independent of de novo synthesis; thus, suggest-ing that high extracellular GSH medium reverses GSH effluxduring apoptosis by reducing its electrochemical gradientacross the plasma membrane; and 3) activation of an OA�

influx that parallels GSH efflux (13).Other studies have suggested that ABCC/MRP transporters

may be involved in GSH efflux during apoptosis (16, 67, 68). Incontrast to our observations, Ballatori and co-workers recentlyreported that inhibitors of MRP transport including MK571prevent GSH loss and apoptosis in Jurkat cells. This discrep-ancy between the two studies is likely explained by the fact thatthe Ballatori and co-workers’ experiments were performedunder a glucose-depleted condition,which is an apoptotic stim-ulus by itself. In addition, their bulk assays used to asses bothapoptosis and changes in GSHi cannot discriminate betweenapoptotic and necrotic cells. Because apoptosis is a stochasticcellular process, we have analyzed these events at the single celllevel by flow cytometry. Whether GSH efflux during apoptosisis mediated by OATP, MRP, or any other still unidentifiedtransporter remains to be clarified.The progression of apoptosis has been largely reported to

correlate with the generation of ROS and RNS (7). Glutathionecontributes to key antioxidant metabolic pathways by actingeither as a proton donor, or as the cofactor for nucleophilicconjugation and thus, it has been shown to scavenge a widerange of ROS and RNS. While GSH scavenges �O2

�, �OH, 1O2,and NO directly, it catalytically detoxifies cells from hydroper-oxides (H2O2), OONO�, and lipid peroxides by the action ofglutathione peroxidases. Because glutathione is the majorintracellular antioxidant defense within the cells, we proposethat its depletion might be a prerequisite for the generation ofROS that could potentially modulate the apoptotic machinery.We show that inhibition of GSH depletion by either high extra-cellular GSH or NAC abolishes the increase in ROS formation.Moreover, we observed a tight link betweenGSH transport andROS formation because ROS generation was stimulated byMK571 a potent stimulator of GSH efflux during FasL-inducedapoptosis (13).The role of ROS in the progression of the cell death program

induced by FasL is controversial. While previous studies sug-gested a role of ROS in apoptosis (22–24), contradictory results

have also been described (27, 28, 69–71). Previous reports havestudied the role of one particular ROS in apoptosis; however,the formation or even scavenging of one ROS can potentiallylead to the generation of others by different reactions. FasL-mediated apoptosis has been reported to induce the generationof H2O2 (20, 22, 24–26, 45, 72, 73), �O2

� (22, 23, 73–78). OH�

(25), NO (79), and lipid peroxides (80, 81). During apoptosis,�O2

� has been specifically shown to be formed due to a switchfrom 4-electron reduction of O2 to a 1-electron reduction (7,82). Superoxide anion can be subsequently converted to H2O2or1O2, by non-enzymatic reactions. Furthermore, �O2

� can bederived enzymatically to H2O2 from its dismutation by SOD.Reaction of H2O2 with �O2

� in the presence of iron can furtherproduce �OH by Fenton-type reactions (83). The hydroxyl rad-ical can trigger a chain of peroxidation reactions by subtractingallylic hydrogen atoms from proximal unsaturated lipids (84),which is highly toxic to the cell because a single ROS can gen-erate a number of toxicants because of the autocatalytic prop-agation of lipid peroxidation reactions. We observed an exam-ple of this interrelationship of one ROS generation with otherswhen in the presence of the SOD mimic MnTE-e-PyP, whichprevents �O2

� formation upon Fas stimulation, H2O2 formationwas enhanced as a byproduct of �O2

� dismutation (data notshown). We took an integrative approach by analyzing the roleof several reactive species in apoptosis, by the use of severalantioxidants alone or in conjunction. Scavenging of H2O2, �O2

�,�OH, and LPO formed upon Fas stimulation, neither regulateGSH loss, nor protect from the progression of apoptosis.More-over, the presence of non-thiol antioxidants stimulated FasL-induced apoptosis which is in accordance with other reportsthat suggested an inhibitory role of distinct ROS during celldeath (27, 28, 69–71). To eliminate the possibility that the roleof ROS in apoptosis was masked or underestimated by a possi-ble compensation of other ROS, we analyzed the combinedeffect of several antioxidants as amixture. The antioxidantmix-ture tested did not protect against apoptosis upon FasL stimuli.Other antioxidants for 1O2, NO, and ONOO� when adminis-tered alone or in conjunction with others also had no effect onapoptosis. These results suggest that the increases in the forma-tion of reactive species during FasL-induced apoptosis do notregulate GSH loss or cell death.Many reports have proposed a role for ROS in death recep-

tor-mediated apoptosis, particularly during Fas-mediated apo-ptosis (20, 22–26, 45, 72–78, 85, 86). Antagonistic roles for ROShave on the other hand, also been reported (27–29, 69, 87–92).The role of ROS in Fas-induced apoptosis has been suggestedpreviously by the protective effect of the extracellular additionof thiols (GSH or NAC) on cell death progression, although theseeffects have been largely ascribed to the well known capacitiesof thiol compounds as antioxidants (22, 23, 72, 78, 93, 94). Wehave observed here that high extracellular GSH andNAC, bothof which prevent GSH depletion, protect Jurkat cells fromundergoing apoptosis induced by death receptor activation(FasL), stress (UVC radiation) and drugs (cycloheximide andetoposide). In contrast, other equally effective antioxidants thatprevented ROS formation upon FasL stimuli, used alone or inconjunction, hadno effect on apoptosis. Thus,we conclude thatinhibition of apoptosis by using any experimental approach




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that prevents GSH depletion or replenishes cellular GSH levelscannot be used as an indicator of a role of reactive species inapoptosis, but rather as evidence of a regulatory role of changesin GSH content. This hypothesis is supported by previousobservations showing that apoptosis can occur under anaerobicconditions, i.e. in the absence of ROS formation (41); or in cellsdeficient in respiration and mitochondrial oxidative phospho-rylation (95), which is the source of ROSduring apoptosis (7, 82,96). Moreover, in peripheral T-lymphocytes protection fromapoptosis by thiol-compounds is observed in the absence of anyROS formation (93). These observations are important in thesense that they indicate, a regulatory role for changes in GSHcontent during apoptosis independent from ROS, and theyexplain contradictory results on the protective effects of thioland non-thiol antioxidants in apoptosis.Thus, we show that the generation of ROS and oxidative

stress upon FasL stimuli do not regulate the progression of apo-ptotic cell death.However, the exactmechanism involved in themodulation of apoptosis by GSH depletion remains unclear.Although the role of GSH in distinct cell processes has beenmainly ascribed to its potent antioxidant properties, GSHmight modulate apoptosis by other distinct mechanisms.Thiol/disulfide exchange reactions at the level of cysteine resi-dues in proteins involved in the apoptotic cascade have beenrecently suggested to regulate the progression of apoptosis (93,97). Thus, GSH loss might be regulating apoptosis involvepost-translational modifications of apoptotic enzymes bythiol exchange reactions. Thiol exchange reactions mightalso be involved in the regulation of ion channels andchanges in the ionic homeostasis of the cell (4, 5, 98) that areknown to regulate the activation of caspases and endonucle-ases (4, 5, 98). More studies are necessary to elucidate theexact mechanism or mechanisms involved in the regulationof apoptosis by GSH depletion.

Acknowledgments—We thank Dr. John B. Pritchard and Dr. RonaldP. Mason for the internal review and comments on this manu-script. We thank Jeff Reece and Jeff Tucker for technical support inthe DIC microscopy, and Maria Sifre for assistance with the FACSexperiments.

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Rodrigo Franco, Mihalis I. Panayiotidis and John A. Cidlowskiof Reactive Oxygen Species Formation

Glutathione Depletion Is Necessary for Apoptosis in Lymphoid Cells Independent

doi: 10.1074/jbc.M703091200 originally published online August 27, 20072007, 282:30452-30465.J. Biol. Chem.

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GlutathioneDepletionIsNecessaryforApoptosisin ... · hasprimarilybeenthoughttoreflectthegenerationofreactive species of oxygen (ROS) and nitrogen (RNS) (6, 7). Although, theroleofROSinapoptosishasbeenextensivelystudied