the adenine nucleotide translocator: a target of nitric oxide, peroxynitrite, and 4-hydroxynonenal

The adenine nucleotide translocator: a target of nitric oxide,peroxynitrite, and 4-hydroxynonenal

Helena LA Vieira1, Anne-Sophie Belzacq2, Delphine Haouzi1, Francesca Bernassola3,Isabel Cohen1, Etienne Jacotot1, Karine F Ferri1, Chahrazed El Hamel1, Laura M Bartle4,Gerry Melino3, Catherine Brenner1,2, Victor Goldmacher4 and Guido Kroemer*,1

1Centre National de la Recherche Scienti®que, UMR1599, Institut Gustave Roussy, 39 rue Camille-Desmoulins, F-94805 Villejuif,France; 2CNRS-UMR6022, UniversiteÂ de Technologie de CompieÁgne, BP 20529, F-60205 CompieÁgne, France; 3BiochemistryLaboratory, University of Rome Tor Vergata, Rome 00133, Italy; 4Apoptosis Technology, Inc., 148 Sidney Street, Cambridge,Massachusetts MA 02139, USA

Nitric oxide (NO), peroxynitrite, and 4-hydroxynonenal(HNE) may be involved in the pathological demise ofcells via apoptosis. Apoptosis induced by these agents isinhibited by Bcl-2, suggesting the involvement ofmitochondria in the death pathway. In vitro, NO,peroxynitrite and HNE can cause direct permeabilizationof mitochondrial membranes, and this e�ect is inhibitedby cyclosporin A, indicating involvement of the perme-ability transition pore complex (PTPC) in the permea-bilization event. NO, peroxynitrite and HNE alsopermeabilize proteoliposomes containing the adeninenucleotide translocator (ANT), one of the key compo-nents of the PTPC, yet have no or little e�ects onprotein-free control liposomes. ANT-dependent, NO-,peroxynitrite- or HNE-induced permeabilization is atleast partially inhibited by recombinant Bcl-2 protein, aswell as the antioxidants trolox and butylated hydroxy-toluene. In vitro, none of the tested agents (NO,peroxynitrite, HNE, and tert-butylhydroperoxide) causespreferential carbonylation HNE adduction, or nitrotyr-osylation of ANT. However, all these agents inducedANT to undergo thiol oxidation/derivatization. Peroxy-nitrite and HNE also caused signi®cant lipid peroxida-tion, which was antagonized by butylated hydroxytoluenebut not by recombinant Bcl-2. Transfection-enforcedexpression of vMIA, a viral apoptosis inhibitor speci®-cally targeted to ANT, largely reduces the mitochondrialand nuclear signs of apoptosis induced by NO,peroxynitrite and HNE in intact cells. Taken togetherthese data suggest that NO, peroxynitrite, and HNEmay directly act on ANT to induce mitochondrialmembrane permeabilization and apoptosis. Oncogene(2001) 20, 4305 ± 4316.

Keywords: apoptosis; Bcl-2; vMIA; mitochondria;permeability transition

Introduction

Mitochondrial membrane permeabilization (MMP),which can a�ect both the inner and/or outermitochondrial membranes, precedes necrotic or apop-totic cell death and frequently occurs upstream of theapoptosis-speci®c activation of caspases. MMP resultsin the release cytochrome c, a caspase activator, Smac/DIABLO, and apoptosis-inducing factor (AIF), acaspase independent death e�ector, both of whichnormally are sequestered in the intermembrane space.MMP is, at least in part, regulated by the mitochon-drial permeability transition pore complex (PTPC),also called mitochondrial megachannel, a multiproteincomplex formed at the contact site between themitochondrial inner and outer membranes. The corecomponents of the PTPC are the adenine nucleotidetranslocator (ANT, in the inner membrane), cyclophi-lin D (in the matrix), and the voltage-dependent anionchannel (VDAC in the outer membrane) (Crompton etal., 1998; Green and Reed, 1998; Kroemer and Reed,2000; Marzo et al., 1998b; Wood®eld et al., 1998).

Five independent lines of evidence suggest theinvolvement of the PTPC in mitochondrial-signaling-mediated apoptosis. First, apoptosis is accompanied byan early permeabilization of mitochondrial membranes.Several PTPC-inhibitory agents prevent both MMPand subsequent cell death (Kroemer et al., 1998).Second, in cell-free systems, mitochondria or mito-chondrial proteins are rate-limiting for the activationof caspases and nucleases. Isolated mitochondriarelease apoptogenic proteins capable of activatingcaspases or endonucleases upon opening of the PTPCin vitro (Kluck et al., 1997; Liu et al., 1996; Susin et al.,1999; Zamzami et al., 1996). Third, the liposome-reconstituted PTPC is inhibited by recombinant Bcl-2or Bcl-XL, apoptosis-inhibitory proteins which areknown to prevent PTPC opening in cells and isolatedmitochondria (Marzo et al., 1998a,b). Bcl-2 alsoinhibits pore formation by ANT, both in proteolipo-somes (Marzo et al., 1998a) and in synthetic lipidbilayers (Brenner et al., 2000). Fourth, pro-apoptoticproteins such as Bax appear to facilitate MMP through

Oncogene (2001) 20, 4305 ± 4316ã 2001 Nature Publishing Group All rights reserved 0950 ± 9232/01 $15.00

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*Correspondence: G Kroemer, CNRS-UMR1599, Institut GustaveRoussy, Pavillon de Recherche 1, 39 rue Camille-Desmoulins, F-94805 Villejuif, France; E-mail: [email protected] 5 February 2001; revised 17 April 2001; accepted 30 April2001

the functional or physical interaction with the PTPCconstituents ANT (Brenner et al., 2000; Marzo et al.,1998a) and/or VDAC (Shimizu et al., 1999, 2000) tofacilitate MMP. Fifth, a number of endogenous, viral,or xenogenic e�ectors trigger the apoptotic signalingpathway by acting on the PTPC directly (Brenner andKroemer, 2000; Kroemer and Reed, 2000). Takentogether, these ®ndings suggest that PTPC opening issu�cient and, at least in some cases, necessary fortriggering apoptosis.

Among the various proteins contained in the PTPC,ANT and VDAC may be the ones responsible for theregulation of inner MMP and outer MMP, respec-tively. VDAC is the target of apoptosis regulatoryproteins such as hepatitis virus X protein (pro-apopotic) (Rahmani et al., 2000) and Neisseriamengintidis porin B (anti-apoptotic) (Massari et al.,2000). ANT, conversely, appears to be the target ofviral protein R (Vpr, pro-apoptotic) (Jacotot et al.,2000) and vMIA/UL37 from cytomegalovirus (anti-apoptotic) (Goldmacher et al., 1999). ANT has alsobeen identi®ed as a protein that can form non-speci®cchannels in response to Vpr (Jacotot et al., 2000),Ca2+ (Brustovetsky and Klingenberg, 1996), atractylo-side (Brenner et al., 2000), as well as the pro-oxydantsdiamide (which causes thiol crosslinking and enforcesANT dimerization) and tert-butylhydroperoxide (adonor of reactive oxygen species) (Costantini et al.,2000; Vieira et al., 2000). The ANT-mediatedpermeabilization of proteoliposome membranes isenhanced by Bax and inhibited by Bcl-2 (Brenner etal., 2000; Jacotot et al., 2000). Indeed, both Bax andBcl-2 interact with a domain of ANT (aa 54 ± 105)(Marzo et al., 1998a), which also contains the Vpr-binding peptide motif WXXF; aa 110 ± 114; ref. (BouHamdan et al., 1998; Jacotot et al., 2001), and which,by deletion mapping, has been determined to becritical for the apoptogenic e�ect of ANT (Bauer etal., 1999).

Nitric oxide (NO), peroxynitrite, and 4-hydroxyno-nenal (HNE) are pleiotropic e�ectors with multiplephysiological and pathogenic functions (Brune et al.,1999; Nicotera et al., 1999). NO is known to be acriticial mediator of pathological cell death in AIDS(Mossalaly et al., 1999), neurodegenerative diseases(Estevez et al., 1999), and ischemia reperfusion damage(Murphy, 1999). HNE is involved in the oxidativestress and cell death associated with neurodegenerativediseases (Mattson, 1998). Although anti-apoptotice�ects have been documented for NO in some systems(Melino et al., 1997), it appears that NO is, at least atpathologically relevant concentrations, mostly pro-apoptotic. Several mechanisms have been proposedfor this apoptogenic e�ect of NO: ceramide formation(Huwiler et al., 1999), induction of surface receptorsfor lethal ligands (e.g.; IFN-g receptors) (Allione et al.,1999), inhibition of the proteasome (Glockzin et al.,1999), activation of the p38 MAP kinase (Ghatan etal., 2000), and/or generation of peroxynitrite (viareaction with anion superoxide), a highly reactiveradical species (Groves, 1999). Peroxynitrite can

modify proteins via S-nitrosylation or tyrosylnitrosyla-tion. In addition, it acts as a pro-oxidant withpleiotropic e�ects on DNA, proteins, and membranes.HNE is a by-product of the peroxydation ofunsaturated fatty acids, in particular linoleic acid andarachidonic acid. It can derivatize glycine, histidine,and cysteine residues (Kristal et al., 1995; Szweda etal., 1993; Uchida and Stadtman, 1992), therebygenerating protein adducts. In addition, HNE causeslipid peroxidation. In several instances NO has beenshown to induce MMP, when added to intact cells(Hortelano et al., 1997, 1999; Ushmorov et al., 1999)or puri®ed mitochondria (Brookes et al., 2000;Ghafourifar et al., 1999; Hortelano et al., 1997).HNE also causes MMP, at least in vitro, in isolatedmitochondria (Kristal et al., 1995).

Although it appears that, as many other apoptosisinducers, NO, peroxynitrite, and HNE may causeMMP, their precise mode of action remains to bedetermined. To this end, we have investigated thee�ects of Bcl-2 and vMIA, mitochondrial cell deathsuppressors which prevent MMP and have been shownto physically interact with PTPC components (Gold-macher et al., 1999; Marzo et al., 1998b). In addition,we assessed the e�ects of NO, peroxynitrite, and HNEon puri®ed mitochondria and ANT proteoliposomes.We found that ANT reconstituted into synthetic lipidbilayers, can mediate membrane permeabilizationresponses to NO, peroxynitrite, and HNE. Theseresults suggest that ANT is a critical target ofapoptosis induction by NO, peroxynitrite, and HNE.

Results and Discussion

Bcl-2 prevents mitochondrial alterations induced bypro-oxidants and NO donors in intact cells

A variety of pro-apoptotic agents induce a dissipationof the DCm (quantitated with the ¯uorochromeDiOC6(3)), followed by the enhanced generation ofsuperoxide anion by the respiratory chain (measuredwith HE) (Zamzami et al., 1995). This applies also tothe apoptosis inducers peroxynitrite, HNE, and twodi�erent NO donors (SIN-1, which generates NO andROS or SNAP which only generates NO), which whenadded to Jurkat cells, cause a DCm reduction and anelevated generation of HE-detectable reactive oxygenspecies. Transfection-enforced expression of Bcl-2,which reportedly acts locally on mitochondrial mem-branes, largely reduces these mitochondrial changes(Figure 1). These data and analogous results obtainedwith Bcl-2-transfected HeLa and BJAB cells (notshown) suggest that mitochondria may play a role inthe apoptotic process triggered by NO donors,peroxynitrite and HNE.

Direct induction of MMP involves the PTPC

To investigate the mitochondrial e�ects of NOdonors, peroxynitrite and HNE on mitochondria,

Adenine nucleotide translocator in apoptosisHLA Vieira et al

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these agents were added to isolated mitochondria invitro, followed by assessment of large amplitudeswelling, which is known to trigger the mitochondrialrelease of soluble intermembrane proteins includingcytochrome c and AIF (Bernardi et al., 2001; Petit etal., 1998; Susin et al., 1999). As shown in Figure 2,NO donors, peroxynitrite, and HNE induced similarlevels of mitochondrial matrix swelling as did Ca2+

and the pro-oxidant t-BHP, which served as positivecontrols. Mitochondrial swelling was completely (inthe case of NO donors) or partially (in the case ofperoxynititre and HNE) suppressed by CsA, aprototype inhibitor of the PTPC (Crompton, 1999),suggesting the involvement of the PTPC. In addition,a complete inhibitory e�ect of the lipophilic anti-oxidant BHT was observed. SOD yielded ratherpartial inhibitory e�ects (with SNAP, peroxynitrite,and HNE) or no inhibitory e�ect at all (with SIN-1and t-BHP), whereas catalase mitigated swelling

induced by NO donors, peroxynitrite and HNE.Altogether these data suggest that the componentsassessed in this study can have direct mitochondriale�ects which involve the PTPC and are mediated, atleast in part, by oxidative reactions.

Direct permeabilization of ANT proteoliposomes

In view of the likely involvement of the PTPC inmitochondrial membrane permeabilization induced byNO donors, peroxynitrite, HNE and t-BHP (as positivecontrol), we tested whether these agents may a�ect thepermeability of ANT proteoliposomes. To this end,ANT-containing liposomes or protein-free controlliposomes were exposed to NO donors, peroxynitriteand HNE and membrane permeabilization wasassessed using a recently developed assay in whichthe accessibility of 4-methylumbelliferylphosphate (4-MUP) encapsulated into the liposomal lumen to

Figure 1 E�ect of NO-donors, peroxynitrite and HNE on Jurkat cells overexpressing Bcl-2 or vector-only transfected cells (Neo).(a) Representative FACS diagrams obtained with Neo or Bcl-2 cells cultured in the absence or presence of peroxynitrite (ONOO7,750 mM, 4 h) followed by staining with DiOC6(3) and HE to determine the mitochondrial transmembrane potential and thegeneration of reactive oxygen species, respectively. Numbers indicate the percentage of cells encountered in each quadrant. (b)Quantitation of the Bcl-2-mediated stabilization of mitochondrial parameters. Cells were cultured for 4 h in presence of 750 mMperoxynitrite, 25 mM t-BHP, or 20 mM HNE, or for 18 h in the presence of 800 mM SNAP or SIN-1; followed by staining withDiOC6(3) and HE. The percentage of cells with a low DCm (DiOC6(3)

low) which produce normal (HElow) or enhanced (HEhigh)levels of reactive oxygen species are given as the mean of ®ve independent experiments+s.e.m. Note that HEhigh cells are mostlyDiOC6(3)

low (a)

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alkaline phosphatase added to the extra-liposomalmilieu was assessed (Costantini et al., 2000). In thisexperimental set-up, the conversion of 4-MUP into the¯uorescent product 4-methylumbelliferone (4-MU)provides a quantitative measure of liposomal mem-brane permeabilization. NO donors and HNE weremore e�cient in permeabilizing ANT proteoliposomesthan plain liposomes, at any concentration (Figure 3).In contrast, peroxinitrite also permeabilized ANT-freecontrol liposomes. Nonetheless, at relatively lowconcentration (41.5 mM), this labile product wasslightly more e�cient (in ®ve independent experiments)in permeabilizing ANT proteoliposomes than controlliposomes (Figure 3). These data indicate that ANTfacilitates the permeabilization of membranes by NOdonors, peroxynitrite and HNE. In accord withobservations obtained on isolated mitochondria (Fig-ure 2, see above), the anti-oxidant trolox (which, asBHT, prevented lipid peroxidation) largely inhibitedthe permeabilization of ANT proteoliposomes (Figure4). Moreover, paralleling the results obtained on intactcells (Figure 1), recombinant Bcl-2 incorporated withANT into proteoliposomes reduced membrane permea-bilization induced by SNAP, SIN-1, and HNE (Figure4). In contrast, Bcl-2 only conferred a partial inhibitorye�ect on membrane permeabilization induced byperoxynitrite (Figure 4).

Covalent modifications of mitochondrial proteinsand/or lipids

Upon exposure to NO donors, peroxynitrite or HNE,ANT might undergo covalent derivatization, forinstance by carbonylation (detected by derivatizationwith dinitrophenylhydrazine, DNPH, which selectivelyreacts with carbonyl groups, yielding dinitrophenyl

[DNP]-haptenized proteins) (Yan and Sohal, 1998),nitrotyrosylation (detected by a nitrotyrosine-speci®cantibody), or adduction of HNE (detected by an HNE-speci®c antibody) (Kruman et al., 1997). To investigatethis possibility, puri®ed ANT was exposed to NOdonors, peroxynitrite or HNE, subjected to SDS gelelectrophoreses and immunodetection of DNP, nitro-tyrosyl residues, or HNE adducts (Figure 5). Puri®edANT appeared to be refractory to carbonylation andHNE adduction, however, it was susceptible to thecovalent modi®cation of tyrosine residues by peroxyni-trite (Figure 5). Previous studies indicated that someANT cysteine residues may be critical redox-sensitivesensors and that ANT thiol oxidation may favoropening of the PTPC (Costantini et al., 1996, 2000;Hashimoto et al., 1999). In view of the capacity of NOto react with thiols (S-nitrosylation) (Melino et al.,1997), we determined whether NO donors or peroxy-nitrite might covalently modify thiols of ANT (whichcontains three cysteins). As shown in Figure 6, NOdonors, peroxynitrite and HNE did cause an oxidation/derivatization of either soluble ANT, at doses thatappear to be functionally relevant (Figures 2 and 3).Peroxynitrite, HNE, and SIN-1 (but not SNAP)induced lipid peroxidation in proteolipsomes, whichwas inhibited by BHT but not by recombinant Bcl-2protein (Figure 7).

The ANT-interacting cell death suppressor vMIAprevents the mitochondrial and nuclear signs of apoptosisinduced by NO donors, peroxynitrite and HNE

vMIA is a cytomegalovirus-encoded cell deathprocessor which has been reported to speci®callyinteract with ANT (Goldmacher et al., 1999).Accordingly, in cells constitutively expressing vMIAtagged in its carboxy-terminus with the myc epitope,ANT but not another PTPC constitutent, VDAC co-immunoprecipitated with vMIAmyc. This result hasbeen obtained with two stably transfected cell lines,namely HeLa cancer cells and BJAB lymphoma cells(Figure 8). To test if ANT actually plays a critical rolein NO-, peroxynitrite- and HNE-induced apoptosis,the mitochondrial and nuclear e�ects of these agentswere comparatively assessed on cell lines expressingvMIA or vector-only (Neo) transfected control cells.As shown in Figure 9, BJAB-vMIA cells treated withNO, peroxynitrite or HNE exhibited a much lowerDCm loss and ROS production (determined byDiOC6(3))/HE staining and FACS analysis), ascompared to BJAB-Neo controls. Moreover, vMIAinhibited the loss of nuclear DNA induced by theseagents in BJAB cells. Similarly, vMIA protected HeLacells against DCm dissipation (quanti®ed by stainingwith JC-1 and ¯uorescence microscopy) and theHoechst 33324-detectable chromatin condensation(Figure 10). Concomitantly, vMIA inhibited themitochondrio-nuclear translocation of AIF and themitochondrio-cytosolic translocation of cytochrome cinduced by NO, peroxynitrite and HNE (Figure 11).Thus, vMIA prevents both the permeabilization of the

Figure 2 E�ect of NO-donors, peroxynitrite and HNE onisolated mitochondria. Puri®ed hepatocyte mitochondria werepre-incubated with bu�er only (control), CsA (1 mM), catalase(500 U/ml), SOD (400 U/ml), BHT (50 mM) for 2 min and thenincubated with 20 mM Ca2+ (negative control), Ca2+ (200 mM;positive control; 100% value), or 20 mM Ca2+ plus t-BHP(50 mM), SNAP (200 mM), SIN-1 (200 mM), ONOO7 (1.8 mM),or HNE (40 mM). Ten minutes after addition of the indicatedagents large amplitude swelling was quantitated as described inMaterials and methods. Results are mean values+s.d of threeindependent experiments


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inner mitochondrial membrane (which accounts forthe DCm loss) and the permeabilization of the outermitochondrial membrane (which allows for the releaseof cytochrome c and AIF). These results support thehypothesis that, at least in part, the apoptogenice�ects of NO, peroxynitrite and HNE are mediatedvia ANT.

Concluding remarks

The data presented in this work suggest that ANT is acritical target of apoptosis induction by NO, peroxy-nitrite and HNE, based on the observation that ANTreconstituted into proteoliposomes confers an en-hanced permeabilization response (as compared to

Figure 3 Comparative assessment of the e�ect of NO-donors, peroxynitrite and HNE on ANT proteoliposomes and protein-freecontrol liposomes. ANT proteoliposomes or plain liposomes were loaded with 4-MUP, washed, and incubated with the indicatedconcentration of permeabilizing agents for 1 h, followed by addition of alkaline phosphatase, which converts free 4-MUP (non-¯uorescent) into the ¯uorochrome 4-MU, yet does not gain access to 4-MUP which remains encapsulated in the liposomal lumen.One hundred per cent 4-MU ¯uorescence values were determined by addition of 200 mM of the ANT ligand atractyloside, as detailedin Materials and methods. Results are mean values of three independent experiments+s.d

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control liposomes devoid of ANT) to NO, peroxyni-trite and HNE (Figure 3), and this permeabilizationresponse is blunted by recombinant Bcl-2 (Figure 4). Inaddition, these agents induce mitochondrial andnuclear signs of apoptosis in a fashion that is inhibited

Figure 4 Inhibitory e�ects of anti-oxidants and Bcl-2 onpermeabilization of ANT proteolipomes by NO-donors, peroxy-nitrite and HNE. 4-MUP-preloaded ANT-proteoliposomes orANT-Bcl-2 proteoliposomes (molar ratio 1 : 1) were pre-incubatedwith bu�er only (control) or trolox (1 mM) for 20 min, thenexposed for 40 min to the t-BHP (25 mM), SNAP (100 mM), SIN-1(100 mM), ONOO7 (900 mM), or HNE (25 mM) and the degree ofmembrane permeabilization was assessed as in Figure 3. Resultsare mean values+s.d. (n=3)

Figure 5 Carbonylation, nitrotyrosylation, and HNE adductformation of ANT exposed to NO-donors, peroxynitrite andHNE. Puri®ed soluble ANT was exposed to the indicated agent(same conditions as in Figure 2), aliquoted and then subjected toimmunoblot detection for the determination of DNP adducts(indicative of protein carbonylation), nitro-tyrosylation, or HNEadducts using speci®c antibodies. As in internal control, theelectrophoretic mobility of puri®ed ANT was assessed byrevealing the blot with anti-ANT antibody (upper panel). Parallelexperiments performed on control proteins yielded positiveimmunodetection results with the DNP and HNE-reactiveantibodies (not shown)

Figure 6 Titration of thiols in puri®ed ANT exposed to NOdonors, peroxynitrite, or HNE. Quantitation of S-nitrosylationinduced by di�erent concentrations of NO donors (SNAP, SIN-1), peroxynitrite or HNE tested on ANT. Results are meanvalues+s.d. (n=3)

Figure 7 Quantitation of lipid peroxidation of ANT proteolipo-somes. Proteoliposomes containing ANT alone (control), pre-incubated with BHT (50 mM), or Bcl-2-ANT proteoliposomes(Bcl-2) were exposed to t-BHP, SNAP, SIN-1, ONOO7, or HNE(same doses as in Figure 4), and lipid peroxidation was measuredby the thiobarbiturate method

Figure 8 Association of ANT and lack of association of VDACwith vMIA. Lysates from BJAB/vMIA cells, HeLa/vMIA cells,or from vector only transfected control cells were immunopreci-pitated with the anti-myc antibody 9E10 covalently linked to A�-Prep-10 beads. This antibody recognizes a myc tag introducedinto the N-terminus of the vMIA construct. Bound proteins(lanes designated IP) were detected by immunoblot analysis toreveal the presence of vMIA, VDAC, and ANT, as described inMaterials and methods. Lanes designed L are samples of thetransfected cell lysate taken prior to immunoprecipitation


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by Bcl-2 (a protein known to stabilize mitochondrialmembranes, presumably via an e�ect on Bax, VDACand/or ANT) (Figure 1) and vMIA (a proteinspeci®cally interacting with ANT) (Figures 9 ± 11).

The detailed molecular mechanisms through whichNO, peroxynitrite and HNE act on ANT have beenaddressed in this work. NO donors fail to causecarbonylation or tyrosylnitrosylation of puri®ed ANTin vitro, and HNE fail to induce generation of ANT-HNE adducts. However, peroxynitrite causes detect-able tyrosylnitrosylation when added to puri®ed ANT(Figure 5). Alternatively, or in addition, NO donors,peroxynitrite and HNE oxidize/derivatize ANT thiols(Figure 6), a phenomenon that is known to convertANT into a non-speci®c pore (Costantini et al., 2000).

As, in the ANT proteoliposome system, peroxynitritehas two dissociable permeabilization e�ects, one that

does not depend on ANT (presumably mediated viadirect peroxidation of lipids) and another that requiresANT (and that is presumably inhibited by Bcl-2)(Figures 3 and 4), we tested the e�ect of NO,peroxynitrite and HNE in lipid peroxidation (Figure7). Similarly to peroxynitrite, high doses of SIN-1 orHNE (but not SNAP) cause lipid peroxidation,paralleling the fact that SIN-1, HNE and t-BHP (butnot SNAP) can have an ANT-independent membranepermeabilization e�ect (Figure 3). This may suggestthat in the case of peroxynitrite or in the case of highdoses of HNE and SIN-1, lipids can also be a target ofthese agents, probably acting as oxidants. Of note,SIN-1 (but not SNAP) is also a donor of smallamounts of ROS. This lipid peroxidation was notprevented by Bcl-2. This ®nding favors the hypothesisthat Bcl-2 prevents permeabilization of ANT proteo-

Figure 9 vMIA inhibition of NO-donors, peroxynitrite and HNE e�ects on DCm collapse and ROS generation. (a) RepresentativeFACS diagrams of BJAB vMIA or BJAB Neo control cells cultured for 18 h with SIN-1 (400 mM), followed by staining withDiOC6(3) and HE, as in Figure 1a. (b) Frequency of cells displaying a low DCm (DiOC6(3)

low) cells and enhanced ROS generation(HEhigh). BJAB cells transfected with the vector only (Neo) or with vMIA were cultured in the presence of peroxynitrite (700 mM,4 h), HNE (40 mM, 4 h), t-BHP (50 mM, 4 h), SNAP (800 mM, 18 h), or SIN-1 (800 mM, 18 h), followed by staining with DiOC6(3)and HE. Results are mean values+s.d. (n=3)

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liposomes via direct protein ± protein interactions withANT, and not via an anti-oxidant function of Bcl-2, asproposed previously (Hockenbery et al., 1993). Any-way, Bcl-2 could always prevent the upstream of anoxidation stress in intacted cells.

In intact cells, however, the peroxynitrite-inducedMMP and nuclear apoptosis was largely inhibited byBcl-2 (Figure 1) and vMIA (Figures 9 ± 11). This maysuggest that, in the context of the cellular environmentwith its multiple redox bu�ers, peroxynitrite morespeci®cally a�ects ANT than in proteoliposomes, and

lipid peroxidation might be less important. Alterna-tively, this may be interpreted to mean that, in intactcells, Bcl-2 and vMIA have also additional anti-apoptotic e�ects than those mediated via ANT.

In conclusion, it appears that several mediators ofpathological cell death, NO, peroxynitrite and HNEexert their pro-apoptotic e�ect, at least in part, throughANT. Intriguingly, NO was reported to be locallyproduced by mitochondria (Ghafourifar et al., 1999;Lopez-Figueroa et al., 2000). Future investigation willclarify whether this locally produced NO (and its

Figure 10 Protective e�ect of vMIA on nuclear apoptosis and DCm collapse induced by NO-donors, peroxynitrite and HNE.HeLa cells overexpressing vMIA and control cells transfected with the pcDNA 3.1 vector (Neo) were cultured for 22 h in thepresence of 150 mM t-BHP, 1.2 mM SNAP, 2 mM SIN-1, 1.8 mM peroxynitrite, or 60 mM HNE. Cells were stained with Hoechst33342 (blue ¯uorescence) and the DCm sensitive dye JC-1 (red ¯uorescence of mitochondria with high DCm, green ¯uorescence ofmitochondria with low DCm). (a) Representative ¯uorescence micrographs of HeLa Neo and HeLa vMIA cells. (b) Frequency ofcells presenting mitochondria with low DCm (green JC-1 ¯uorescence). (c) Frequency of cells with chromatin condensation andnuclear fragmentation


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Figure 11 Protective e�ect of vMIA on the translocation of AIF and cytochrome c translocation. HeLa cells were treated withNO-donors, peroxynitrite and HNE as in Figure 10, followed by ®xation, permeabilization, and immunostaining. (a) Cells werestained with AIF antibody (revealed by PE, red ¯uorescence) and the mitochondrial matrix protein hsp 60 (green ¯uorescence) aswell as counter-staining with Hoechst 33342 (blue ¯uorescence). As AIF and hsp 60 are colocalized in untreated control cells,mitochondria exhibit a yellow ¯uorescence (blend of green plus red). Di�use red cytoplasmic ¯uorescence and purple nuclear¯uorescence (blend of red+blue) are indicative of the AIF translocation. (b) Quantitation of AIF translocation induced by di�erentagents. (c) Cells were stained with speci®c antibodies for cytochrome c (detected by PE) and COX (revealed by FITC) as well asHoechst 33324. Note the di�use red ¯uorescence indicative of Cyt-c release from mitochondria, when control cells are treated withthe NO donor SIN-1. (d) Frequency of cytochrome c translocation. Results are mean values+s.e.m. of triplicates

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derivative peroxynitrite) induce any apoptosismodula-tory e�ects in addition to its regulatory e�ect onoxygen consumption by inhibition of cytochrome coxidase (Clementi et al., 1999).

Materials and methods

Cell lines and apoptosis induction

Jurkat cells transfected with the human Bcl-2 gene (Aillet etal., 1998), BJAB or HeLa cells transfected with thecytomegalovirus UL37 exon 1 gene coding for vMIA(Goldmacher et al., 1999), as well as control cells transfectedwith the pcDNA3.1 vector containing the neomycinresistance gene (Neo), were cultured in RPMI-1640 (Jurkat)or DMEM (BJAB or HeLa) medium supplemented with2 mM glutamine, 10% FCS, 1 mM pyruvate, 10 mM HEPESand 100 U/ml pencillin/streptomycin at 378C under 5% CO2,1 ± 56105 cell/ml were treated with various doses of tert-butylhydroperoxide (t-BHP, Sigma, St. Louis, MO, USA),peroxynitrite (ONOO7, Upstate Biotechnology) and 4-hydroxynonenal (HNE, OXIS, International Inc.) for 4 h at378C; and 3-morpholinosydnonimine (SIN-1, Sigma), S-nitroso-N-acetylpenicillamine (SNAP, Sigma, which releases60 nM free NO per 1 mM) overnight at 378C. Sinceperoxynitrite has a half life 51 s71 (Koppenol et al., 1990),it was added while throroughly mixing the culture medium.

Assessment of apoptosis-associated parameters

The following ¯uorochromes were employed to assessapoptosis-associated changes by cyto¯uorometry on a FACSVantage (Becton Dickinson), while gating the forward and theside scatters on viable cells: 3,3' dihexyloxacarbocyanineiodide (DiOC6(3), 20 nM) for DCm quanti®cation, hydro-ethidine (HE, 5 mM) for the determination of superoxideanion generation (Zamzami et al., 1995). Alternatively, cellscultured on a cover slip were stained with 5,5',6,6'-tetra-chloro-1,1', 3,3'-tetraethylbenzimidazolylcarbocyanine iodide(JC-1, 3 mM, Molecular Probes) and Hoechst 33342 (2 mM,Sigma), followed by ¯uorescence microscopic assessment ofapoptotic parameters, as described (Daugas et al., 2000; Ferriet al., 2000). A rabbit antiserum generated against aminoacids151 to 200 of AIF (Susin et al., 1999) was used onparaformaldehyde (4% w : v) and picric acid®xed (0.19 %v : v) cells and revealed with a goat anti-rabbit IgG conjugatedto phycoerythrine (PE) (Southern Biotechnology, Birming-ham, AL, USA). Cells were also stained for the detection ofcytochrome c (mAb 6H2.B4 from Pharmingen, detected by agoat anti-mouse IgG1 ¯uorescein isothiocyanate (PE) con-jugate; Southern Biotechnology), hsp60 (mAb H4149 fromSigma, revealed by a goat anti-mouse IgG1 FITC),cytochrome c oxidase (COX subunit IV, mAb 2038C12 fromPharmingen, detected by a goat anti-mouse IgG2a FITCconjugate), and/or chromatin (Hoechst 33342, 2 mM, 15 minof incubation at RT).

Isolation of mouse liver mitochondria and measurementof MTP opening

Mitochondria were puri®ed from Balb/c mouse livers on aPercoll1 gradient (Susin et al., 2000). One mg/ml ofmitochondrial protein were resuspended in 0.2 M sucrose,10 mM Tris-MOPS, pH 7.4, 5 mM succinate-Tris, 1 mM Pi,

2 mM rotenone and 10 mM EGTA-Tris (swelling bu�er). PTpore opening was monitored as the change of 908 lightscattering at 545 nm using a Hitachi F-4500 ¯uorescencespectrophomete. The decrease in light scattering induced byCa2+ (200 mM, 10 min) was considered as 100% value.

Purification of ANT and its reconstitution into liposomes

ANT was puri®ed from rat heart mitochondria, whichcontain the two ANT isoforms (ANT1, ANT2) (Dorner etal., 1999). After mechanical shearing, mitochondria weresuspended in 220 mM mannitol, 70 mM sucrose, 10 mM

HEPES, 200 mM EDTA, 100 mM DTT, 0.5 mg/ml subtilisin,pH 7.4, kept 8 min on ice and sedimented twice bydi�erential centrifugations (5 min, 500 g and 10 min,10 000 g). Mitochondrial proteins were solubilized by 6%[v : v] Triton X-100 (Boehring Mannheim) in 40 mM K2HPO4,40 mM KCl, 2 mM EDTA, pH 6.0, for 6 min at RT andsolubilized proteins were recovered by ultracentrifugation(30 min, 24 000 g, 48C). Then, the Triton X-100 extract wasapplied to a columns ®lled with 1 g of hydroxyapatite(BioGel HTP, BioRad), eluted with the previous bu�er anddiluted [v : v] with 20 mM MES (2-[N-morpholino]ethanesul-fonic acid), 200 mM EDTA, 0.5% Triton X-100, pH 6.0.Subsequently, the sample was separated on a Hitrap SPcolumn using a FPLC system (Pharmacia) and a linear NaClgradient (0 ± 1 M) (Marzo et al., 1998a). Puri®ed ANT and/orrecombinant human Bcl-2, produced and puri®ed asdescribed (JuÈ rgensmeier et al., 1998; Marzo et al., 1998a),were reconstituted in phosphatidylcholine/cardiolipin lipo-somes. Brie¯y, to prepare liposomes, 90 mg phosphatidylcho-line and 2 mg cardiolipin were mixed in 1 ml choroform, andthe solvent was evaporated under nitrogen. Dry lipids wereresuspended in 1 ml liposomes bu�er (125 mM sucrose+10 mM HEPES) containing 0.3% n-octyl-û-D-pyranoside andmixed by continuous vortexing for 40 min at RT. ANT(0.1 mg/ml) and/or Bcl-2 (0.1 mg/ml) were then mixed withliposomes [v : v] and incubated for 15 min at RT. Proteo-liposomes were ®nally dialyzed overnight at 48C.

Quantification of liposomal permeabilization

ANT proteoliposomes were sonicated in the presence of 4-umbelliferylphosphate (4-MUP, 1 mM, Sigma) and 10 mM

KCl (50 W, 22 s, Branson soni®er 250) on ice and washed onSephadex G-25 columns (PD-10, Pharmacia). Twenty-®ve ml-aliquots of liposomes were mixed [v : v] with variousconcentrations of pro-apoptotic inducers, incubated for 1 hat RT and diluted to 200 ml in liposomes bu�er. The inhibitorof mitochondrial membrane permeabilization cyclosporin A(CsA, 1 mM) or trolox (1 mM) were added to the liposomes15 min before pro-apoptotic inducers. After addition of 10 mlalkaline phosphatase (5 U/ml, Boehringer Mannheim) dilutedin liposomes bu�er+0.5 mM MgCl2, samples were incubatedfor 15 min at 378C and the enzymatic conversion of 4-MUPto 4-MU (4-methylumbelliferone) was stopped by addition of50 ml stop bu�er (10 mM HEPES-NaOH, 200 mM EDTA,pH 10.0). Fluorescence was subsequently determined using aPerkin Elmer spectro¯uorimeter (excitation 365 nm, emission450+5 nm). Atractyloside, a pro-apoptotic permeabilitytransition inducer, was used in each experiment as a standardto determine the 100% response. The percentage of 4-MUPrelease was calculated as the following : [(¯uorescence ofliposomes treated by the test substance ± ¯uorescence ofuntreated liposomes)/(¯uorescence of liposomes treated byatractyloside ± ¯uorescence of untreated liposomes)]6100.


4314

Oncogene

Protein modification assays

Puri®ed ANT (30 mg/ml) was treated with 10 mM 2,4-dinitrophenylhydrazine (DNPH, Sigma) for 30 min at RTand then subjected to SDS ±PAGE (12%). Western blot wasperformed with rabbit anti- 2,4-dinitrophenyl (DAKO) at1 : 000 dilution. Alternatively, puri®ed ANT was analysed bySDS±PAGE (12%) followed by Western blot immunodetec-tion with mouse anti-HNE (48) and rabbit anti-nitro-tyrosine(Upstate) at 2 mg/ml.

Titration of thiol groups

Cysteine residue was titrated by alkylation with iodoaceta-mide. Reaction mixtures containing 0.1 M Tris-acetatepH 7.5, 20 mM CaCl2, 2 mM 14C-iodoacetamide (59 mCi/mmol) and 0.5 mg of puri®ed ANT were incubated for 5 minat 238C. Aliquots were spotted on 3 MM ®lter paper. Filterswere washed in 20% TCA, 10% TCA, 5% TCA and absoluteethanol, dried and counted. To calculate the stoichiometry ofthe reaction, mol of 14C-iodoacetamide bound to the proteinswere estimated from the decay per min (d.p.m.) and correctedfor the e�ciency of the beta-counter. When the e�ect of NOwas studied, increasing concentrations of the NO-donorswere added to the reaction mixture and incubated for 5 min.Before the initiation of the reaction with 14C-iodoacetamide,the NO-donors were removed by gel ®ltration, using ChromaSpin-10 columns (Clontech, Palo Alto, CA, USA) accordingto the manufacturer's instructions.

Lipid peroxidation assay

The lipid peroxidation measurements were performed usingthe thiobarbituric acid assay as described (Buege and Aust,1978). Brie¯y, 0.25 ml of liposomes reconstituted with ANTand ANT+Bcl-2 were added to 0.5 ml TCA-TBA-HCl re-agent (15% [w : v] trichloroacetic acid, 0.375% [w : v] thio-barbituric acid; 0.25 N hydrochloric acid), mixed thoroughlyand heated in boiling water bath for 15 min. After cooling,the precipitate was eliminated by centrifugation at 1000 g for10 min. The absorbance was determined at 535 nm against ablank that contains all reagents minus the lipid. Lipidperoxidation was calculated as an increase over baselinelevels, determined for untreated liposomes.

Immunoprecipitation and protein blots

Cell extracts for Western blot analysis for detection ofvMIAmyc, VDAC and ANT were prepared by standardprocedures using 150 mM NaCl/5 mM EDTA/50 mM

Tris.HCl, PH 8.0/1% Triton-X-100 in the presence ofprotease inhibitors, and centrifuged at 10 000 g at 48C for10 min. For immunoprecipitation, the supernatants wereincubated with 9E10 anti-myc antibody covalently linked toA�-Prep 10 beads (Goldmacher et al., 1999), and washedwith the lysis bu�er. Protein samples were separated underreducing conditions by SDS ±PAGE and were analysed by astandard Western blot protocol using the ECL (enhancedchemoluminescence) detection system (Amersham). Thefollowing primary antibodies were used: 9E10 anti-mycantibody (Evan et al., 1985), to detect vMIAmyc; anti-human porin Ab-4 (Calbiochem), to detect VDAC, and andanti-ANT rabbit antiserum raised against puri®ed rat heartANT (Marzo et al., 1998a), to detect ANT. Secondaryantibodies used were HRP-labeled anti-mouse IgG (JackonsLabs) or anti-rabbit IgG (Southern Biotechnology).

AbbreviationsAIF, apoptosis-inducing factor; ANT, adenine nucleotidetranslocator; COX, cytochrome c oxidase; CsA, cyclo-sporin A; Cyt c, cytochrome c; DiOC6(3), 3,3'dihexyloxa-carbocyanine iodide; DCm, mitochondral transmembranepoteintal; BHT; butylated hydroxytoluene; HE, hydro-ethidine; HNE, 4-hydroxynonenal; 4-MU; 4-methylumbel-liferone; 4-MUP; 4-methylumbelliferylphosphate; ROS,reactive oxygen species; SIN-1, 3-morpholinosydnon-imine; SNAP, S-nitroso-N-acetylpenicillamine; PTPC, per-meability transition pore; SOD; superoxide dismutase; t-BHP, tert-butyhlhydroperoxide; VDAC, voltage-dependentanion channel; vMIA, viral mitochondria-localized inhi-bitor of apoptosis.

AcknowledgmentsWe thank Dr Nicole Israel (Pasteur Institute, Paris) forBcl-2 transfected Jurkat cells, Z Xie and JC Reed(Burnham Institute, La Jolla, CA, USA) for recombinantBcl-2, Dr MP Mattson (NIH, Bethesda, USA) for anti-HNE antibody, and D MeÂ tivier (CNRS, Villejuif, France)for cyto¯uorometric analyses. This work has been sup-ported by a special grant from the Ligue Nationale contrele Cancer, ComiteÂ Val de Marne de la Ligue contre leCancer, as well as grants from ANRS (to G Kroemer),FRM (to G Kroemer and C Brenner), and the EuropeanCommission Grant No. QL61-1999-00739 (to G Kroemerand G Melino). HLA Vieira receives a fellowship from theFundacË aÄ o para a CieÃ ncia e a Tecnologia PRAXIS XXI,Portugal; A-S Belzacq from ARC, E Jacotot from ANRS.

References

Aillet F, Masutani H, Elbim C, Raoul H, Chene L, NugeyreMT, Paya C, Barre-Sinoussi F, Gougerot-Pocidalo MAand Israel N. (1998). J. Virol., 72, 9698 ± 9705.

Allione A, Bernabei P, Bosticardo M, Ariotti S, Forni G andNovelli F. (1999). J. Immunol., 163, 4182 ± 4191.

Bauer MKA, Schubert A, Rocks O and Grimm S. (1999).J. Cell Biol., 147, 1493 ± 1501.

Bernardi P, Petronilli V, di Lisa F and Forte M. (2001).Trends Biochem. Sci., 26, 112 ± 117.

Bou Hamdan M, Xue Y, Baudat Y, Hu B, Sire J, PomerantzRJ and Duan LX. (1998). J. Biol. Chem., 273, 8009 ± 8016.

Brenner C, Cadiou H, Vieira HLA, Zamzami N, Marzo I,Xie Z, Leber B, Andrews D, Duclohier H, Reed JC andKroemer G. (2000). Oncogene, 19, 329 ± 336.

Brenner C and Kroemer G. (2000). Science, 289, 1150 ± 1151.Brookes PS, Salinas EP, Darley-Usmar K, Eiserich JP,

Freeman BA, Darmey-Usmar VM and Anderson PG.(2000). J. Biol. Chem., 275, 20474 ± 20479.

Brune B, von Knethen A and Sandau KB. (1999). Cell DeathDi�er., 6, 969 ± 975.

Brustovetsky N and Klingenberg M. (1996). Biochemistry,35, 8483 ± 8488.

Oncogene


4315

Buege JA and Aust SD. (1978). Meth. Enzymol., 52, 302 ±310.

Clementi E, Brown GC, Foxwell N and Moncada S. (1999).Proc. Natl. Acad. Sci., 96, 1559 ± 1562.

Costantini P, Belzacq A-S, Vieira HLA, Larochette N, dePablo M, Zamzami N, Susin SA, Brenner C and KroemerG. (2000). Oncogene, 19, 307 ± 314.

Costantini P, Chernyak BV, Petronilli V and Bernardi P.(1996). J. Biol. Chem., 271, 6746 ± 6751.

Crompton M. (1999). Biochem. J., 341, 233 ± 249.Crompton M, Virji S and Ward JM. (1998). Eur. J. Biochem.,

258, 729 ± 735.Daugas E, Susin SA, Zamzami N, Ferri K, Irinopoulos T,

Larochette N, Prevost MC, Leber B, Andrews D,Penninger J and Kroemer G. (2000). FASEB J., 14,729 ± 739.

Dorner A, Olesch M, Giessen S, Pauschinger M andSchulheiss HP. (1999). Biochim. Biophys. Acta, 1417,16 ± 24.

Estevez AG, Crow JP, Sampson JB, Reiter C, Zhuang Y,Richardson GJ, Tarpey MM, Barbeito L and Beckman JS.(1999). Science, 286, 2498 ± 2500.

Evan HI, Lewis GK, Ramsay G and Bishop JM. (1985).Mol.Cell. Biol., 5, 3610 ± 3616.

Ferri KF, Jacotot E, Blanco J, EsteÂ JA, Zamzami A, SusinSA, Brothers G, Reed JC, Penninger JM and Kroemer G.(2000). J. Exp. Med., 192, 1081 ± 1092.

Ghafourifar P, Schenk U, Klein SD and Richter C. (1999).J. Biol. Chem., 274, 31185 ± 31188.

Ghatan S, Larner S, Kinoshita Y, HetmanM, Patel L, Xia Z,Youle RJ and Morrison RS. (2000). J. Cell Biol., 150,335 ± 347.

Glockzin S, von Knethen A, Sche�ner M and Brune B.(1999). J. Biol. Chem., 274, 19581 ± 19586.

Goldmacher VS, Bartle LM, Skletskaya S, Dionne CA,Kedersha NL, Vater CA, Han JW, Lutz RJ, Watanabe S,McFarland EDC, Kie� ED, Mocarski ES and ChittendenT. (1999). Proc. Natl. Acad. Sci. USA, 96, 12536 ± 12541.

Green DR and Reed JC. (1998). Science, 281, 1309 ± 1312.Groves JT. (1999). Curr. Opin. Chem. Biol., 3, 226 ± 235.Hashimoto M, Majima E, Goto S, Shinohara Y and Terada

H. (1999). Biochemistry, 38, 1050 ± 1056.Hockenbery DM, Oltvai ZN, Yin XM, Milliman CL and

Korsmeyer SJ. (1993). Cell, 75, 241 ± 251.Hortelano S, Alvarez AM and Bosca L. (1999). FASEB J.,

13, 2311 ± 2317.Hortelano S, Dallaporta B, Zamzami N, Hirsch T, Susin SA,

Marzo I, Bosca L and Kroemer G. (1997). FEBS Lett.,410, 373 ± 377.

Huwiler A, Pfeilschifter J and van den Bosch H. (1999).J. Biol. Chem., 274, 7190 ± 7195.

Jacotot E, Ferri KF, El Hamel C, Brenner C, Druillenec S,Hoebeke J, Rustin P, MeÂ tivier D, Lenoir C, Geuskens M,Vieira HLA, Loe�er M, Belzacq A-S, Briand JP,Zamzami N, Edelman L, Xie ZH, Reed JC, Roques BPand Kroemer G. (2001). J. Exp. Med., 193, 509 ± 520.

Jacotot E, Ravagnan L, Loe�er M, Ferri KF, Vieira HLA,Zamzami N, Costantini P, Druillennec S, Hoebeke J,Brian JP, Irinopoulos T, Daugas E, Susin SA, Cointe D,Xie ZH, Reed JC, Roques BP and Kroemer G. (2000).J. Exp. Med., 191, 33 ± 45.

JuÈ rgensmeier JM, Xie Z, Deveraux Q, Ellerby L, Bredesen Dand Reed JC. (1998). Proc. Natl. Acad. Sci. USA, 95,4997 ± 5002.

Kluck RM, Bossy-Wetzel E, Green DR and Newmeyer DD.(1997). Science, 275, 1132 ± 1136.

Koppenol WH, Moreno JJ, Ryor WA, Ischiropoulos H andBeckman JS. (1990). Chem. Res. Toxicol., 5, 834 ± 842.

Kristal BS, Park BK and Yu BP. (1995). J. Biol. Chem., 271,6033 ± 6038.

Kroemer G, Dallaporta B and Resche-Rigon M. (1998).Annu. Rev. Physiol., 60, 619 ± 642.

Kroemer G and Reed JC. (2000). Nat. Med., 6, 513 ± 519.Kruman I, Bruce-Keller AJ, Bredesen D, Waeg G and

Mattson MP. (1997). J. Neurosci., 17, 5089 ± 5100.Liu XS, Kim CN, Yang J, Jemmerson R andWang X. (1996).

Cell, 86, 147 ± 157.Lopez-Figueroa MO, Caamano C, Morano MI, Ronn LC,

Akil H and Watson SJ. (2000). Biochem. Biophys. Res.Comm., 272, 129 ± 133.

Marzo I, Brenner C, Zamzami N, JuÈ rgensmeier J, Susin SA,Vieira HLA, PreÂ vost M-C, Xie Z, Matsuyama S, Reed JCand Kroemer G. (1998a). Science, 281, 2027 ± 2031.

Marzo I, Brenner C, Zamzami N, Susin SA, Beutner G,Brdiczka D, ReÂ my R, Xie ZH, Reed JC and Kroemer G.(1998b). J. Exp. Med., 187, 1261 ± 1271.

Massari P, Ho Y and Wetzler LM. (2000). Proc. Natl. Acad.Sci. USA, 97, 9070 ± 9075.

Mattson MP. (1998). Trends Neurosci., 21, 53 ± 57.Melino G, Bernassola F, Knight RA, Corasaniti MT, Nistico

G and Finazzi Agro A. (1997). Nature, 388, 432 ± 433.Mossalaly MD, Becherel PA and Debre P. (1999). Mol.

Med., 5, 812 ± 819.Murphy MP. (1999). Biochem. Biophys. Acta, 1411, 401 ±

414.Nicotera P, Bernassola F and Melino G. (1999). Cell Death

Di�er., 6, 931 ± 933.Petit PX, Goubern M, Diolez P, Susin SA, Zamzami N and

Kroemer G. (1998). FEBS Lett., 426, 111 ± 116.Rahmani Z, Huh KW, Lasher R and Siddiqui A. (2000).

J. Virol., 74, 2840 ± 2846.Shimizu S, Ide T, Yanagida T and Tsujimoti Y. (2000).

J. Biol. Chem., 275, 12321 ± 12325.Shimizu S, Narita M and Tsujimoto Y. (1999). Nature, 399,

483 ± 487.Susin SA, Larochette N, Geuskens M and Kroemer G.

(2000). Meth. Enzymol., 322, 205 ± 208.Susin SA, Lorenzo HK, Zamzami N, Marzo I, Snow BE,

Brothers GM, Mangion J, Jacotot E, Costantini P,Loe�er M, Larochette N, Goodlett DR, Aebersold R,Siderovski DP, Penninger JM and Kroemer G. (1999).Nature, 397, 441 ± 446.

Szweda LI, Uchida K, Tsai L and Stadtman ER. (1993).J. Biol. Chem., 268, 3342 ± 3347.

Uchida K and Stadtman ER. (1992). Proc. Natl. Acad. Sci.USA, 89, 4544 ± 4548.

Ushmorov A, Ratter F, Lehmann V, Droge W, SchirrmacherV and Umansky V. (1999). Blood, 93, 2342 ± 2352.

Vieira HL, Haouzi D, El Hamel C, Belzacq AS, Brenner Cand Kroemer G. (2000). Cell Death Di�er., 7, 1146 ± 1154.

Wood®eld K, Ruck A, Brdiczka D and Halestrap AP. (1998).Biochem. J., 336, 287 ± 290.

Yan LJ and Sohal RS. (1998). Proc. Natl. Acad. Sci. USA,95, 12896 ± 12901.

Zamzami N, Marchetti P, Castedo M, Decaudin D, MachoA, Hirsch T, Susin SA, Petit PX, Mignotte B and KroemerG. (1995). J. Exp. Med., 182, 367 ± 377.

Zamzami N, Susin SA, Marchetti P, Hirsch T, GoÂ mez-Monterrey I, Castedo M and Kroemer G. (1996). J. Exp.Med., 183, 1533 ± 1544.


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the adenine nucleotide translocator: a target of nitric oxide, peroxynitrite, and 4-hydroxynonenal

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