of June 10, 2018.This information is current as

Caspase-1 and -3Apoptosis Without Detectable Activation ofLymphocytes Induces Caspase-Dependent

B Activity in Human TκInhibition of NF-

Bukowski, Eric Hsi and James FinkeVladimir Kolenko, Tracy Bloom, Patricia Rayman, Ronald

http://www.jimmunol.org/content/163/2/5901999; 163:590-598; ;J Immunol 

Referenceshttp://www.jimmunol.org/content/163/2/590.full#ref-list-1

, 41 of which you can access for free at: cites 64 articlesThis article

        average*  

4 weeks from acceptance to publicationFast Publication! •    

Every submission reviewed by practicing scientistsNo Triage! •    

from submission to initial decisionRapid Reviews! 30 days* •    

Submit online. ?The JIWhy

Subscriptionhttp://jimmunol.org/subscription

is online at: The Journal of ImmunologyInformation about subscribing to

Permissionshttp://www.aai.org/About/Publications/JI/copyright.htmlSubmit copyright permission requests at:

Email Alertshttp://jimmunol.org/alertsReceive free email-alerts when new articles cite this article. Sign up at:

Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 1999 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

is published twice each month byThe Journal of Immunology

by guest on June 10, 2018http://w

ww

.jimm

unol.org/D

ownloaded from

by guest on June 10, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

Inhibition of NF- kB Activity in Human T LymphocytesInduces Caspase-Dependent Apoptosis Without DetectableActivation of Caspase-1 and -31

Vladimir Kolenko,* †2 Tracy Bloom,* Patricia Rayman,* Ronald Bukowski,† Eric Hsi,§ andJames Finke*†‡

NF-kB is involved in the transcriptional control of various genes that act as extrinsic and intrinsic survival factors for T cells. Ourfindings show that suppression of NF-kB activity with cell-permeable SN50 peptide, which masks the nuclear localization sequenceof NF-kB1 dimers and prevents their nuclear localization, induces apoptosis in resting normal human PBL. Inhibition of NF-kBresulted in the externalization of phosphatidylserine, induction of DNA breaks, and morphological changes consistent with apo-ptosis. DNA fragmentation was efficiently blocked by the caspase inhibitor Z-VAD-fmk and partially blocked by Ac-DEVD-fmk,suggesting that SN50-mediated apoptosis is caspase-dependent. Interestingly, apoptosis induced by NF-kB suppression, in contrastto that induced by TPEN (N,N,N*,N*-tetrakis [2-pyridylmethyl]ethylenediamine) or soluble Fas ligand (CD95), was observed in theabsence of active death effector proteases caspase-1-like (IL-1 converting enzyme), caspase-3-like (CPP32/Yama/apopain), andcaspase-6-like and without cleavage of caspase-3 substrates poly(ADP-ribose) polymerase and DNA fragmentation factor-45.These findings suggest either low level of activation is required or that different caspases are involved. Preactivation of T cellsresulting in NF-kB nuclear translocation protected cells from SN50-induced apoptosis. Our findings demonstrate an essential roleof NF-kB in survival of naive PBL. The Journal of Immunology,1999, 163: 590–598.

T he transcription factor NF-kB plays a critical role in thedevelopment and maintenance of T cell-mediated im-mune responses. NF-kB consists of multiple members of

the Rel family of proteins that include NF-kB1 (p105/p50), NF-kB2 (pl00/p52), RelA (p65), RelB, and c-Rel (1, 2). Rel proteinsform hetero- and homodimeric complexes that differ in their trans-activating activity (1). In T lymphocytes, the RelA/p50 het-erodimer is known to initiate transactivation, whereas homodimersof p50 appear to be suppressive (1, 2). The NF-kB dimers arepresent in cytoplasm in an inactive form bound to inhibitory sub-units, IkBs (3–7). Upon activation IkB is phosphorylated, whichmarks the inhibitor for ubiquitination and degradation by the pro-teasome-dependent pathway (8–11). This process allows translo-cation of active NF-kB complexes into the nucleus, where theybind to specific DNA motifs in the promoter/enhancer regions oftarget genes and activate transcription (8–13).

There is growing evidence that NF-kB regulates the suscepti-bility of certain cell types to apoptosis through the transcriptionalcontrol of protective genes (1, 14, 15). Knockout transgenic micelacking the RelA component of NF-kB complex displayed embry-onic lethality and liver cell apoptosis (16). Inhibition of NF-kBnuclear translocation also enhanced apoptosis induced by ionizingradiation or the chemotherapeutic drug, danorubicin (17). NF-kBalso appears to regulate the susceptibility of lymphoid cells to

apoptosis. Addition of various inhibitors of NF-kB/Rel activationto normal murine B lymphocytes or to B cell lymphomas resultedin apoptosis (18, 19). A protective role for c-Rel in preventing thisprocess was confirmed using microinjection of its specific inhibi-tor, IkBa, or injection of Ab to the c-Rel subunit (18, 19). Addi-tional findings suggest that ligation of CD95 (Fas/APO1) de-pressed NF-kB activation by causing the degradation of the NF-kBsubunit RelA, a process that may enhance the decay of an immuneresponse (20).

Recent studies have identified the involvement of multiplecaspases in the proteolytic cascade of apoptosis (21–26). Variousstimuli that induce apoptosis, including UV, Fas Ag, drug treat-ment, growth factor withdrawal, and virus infection, have beenshown to activate caspases that specifically cleave proteins at theC terminus of aspartic acid residues (27–30). Caspases are syn-thesized as zymogens that require proteolytic cleavage to generateactive enzyme subunits. These activating cleavage events are con-ducted by other caspases and are thought to represent a majorregulatory step in the apoptosis pathway (21–26). Activatedcaspases cleave several target proteins that include poly(ADP-ri-bose) polymerase (PARP)3 (31, 32), retinoblastoma protein (33,34), cytoskeletal proteins (35–38), Bcl-2 (39), and Bcl-xL (40). Arecent study has also identified RelA as a substrate for activatedcaspase-3 (20). Cleavage of the 45-kDa subunit of DNA fragmen-tation factor (DFF-45) by activated caspases leads to fragmenta-tion of genomic DNA into nucleosomal fragments, one hallmark ofapoptosis (41).

The role NF-kB plays in the regulation of apoptosis in T lym-phocytes has not been well defined. There is evidence that inhibi-tion of NF-kB activation following transient transfection with a

Departments of *Immunology,†Hematology-Oncology,‡Urology, and§AnatomicPathology, Cleveland Clinic Foundation, Cleveland, OH 44195

Received for publication June 16, 1998. Accepted for publication April 26, 1999.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby markedadvertisementin accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by U.S. Public Health Service Grant CA56937.2 Address correspondence and reprint requests to Dr. Vladimir Kolenko, The Cleve-land Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195. E-mail address:kolenkv@ces.mtp.ccf.org

3 Abbreviations used in this paper: PARP, poly(ADP-ribose) polymerase; DFF, DNAfragmentation factor; ICE, IL-1 converting enzyme; TPEN,N,N,N9,N9,-tetrakis (2-pyridylmethyl)ethylenediamine; NLS, nuclear localization sequence; L, ligand;TPCK, N-tosyl-L-phenylalanine chloromethyl ketone.

Copyright © 1999 by The American Association of Immunologists 0022-1767/99/$02.00

by guest on June 10, 2018http://w

ww

.jimm

unol.org/D

ownloaded from

mutant form of IkBa made the Jurkat T cell line susceptible toTNF-a-mediated apoptosis (42). Here, we show that ,in restinghuman peripheral blood-derived T cells, the inhibition of NF-kBactivation results in apoptosis. The cell-permeable peptide SN50was found not to inhibit the stimulus-dependent degradation of theinhibitor IkBa, but rather to block the nuclear translocation ofNF-kB (43). This inhibitory peptide induced exposure of phospha-tidylserine on the cell surface, an early event in apoptosis, and theformation of specific DNA breaks, as defined by DNA ladderingand TUNEL assay. SN50-mediated apoptosis is caspase-depen-dent, since DNA fragmentation was efficiently blocked by thecaspase inhibitor Z-VAD-fmk and partially blocked by DEVD-fmk. However, apoptosis occurred in the absence of detectableactive caspase-1-like (IL-1 converting enzyme (ICE)), caspase-3-like (CPP32/Yama/apopain), and caspase-6-like proteases andwithout detectable proteolysis of PARP and DFF-45, suggestingthat either low level of activation is required or different caspasesare involved. Preactivation with either IL-2 or PMA/ionomycininduced NF-kB activation and prevented apoptosis following ex-posure to SN50. However, continued exposure to SN50 did induceapoptosis in preactivated T cells, which coincided with suppres-sion of NF-kB.

Materials and MethodsAbs and reagents

SN50 and SN50 M peptides and Ab to PARP were obtained from BiomolResearch Laboratories (Plymouth Meeting, PA). Abs used in Western blot-ting for NF-kB1 (p50), RelA (p65), IkBa, DFF-45, and caspase-1 wereobtained from Santa Cruz Biotechnology (Santa Cruz, CA). Abs to Bcl-2,Bax, and caspase-3 were purchased from Transduction Laboratories (Lex-ington, KY). Secondary HRP-conjugated sheep anti-mouse and donkeyanti-rabbit Abs were purchased from Amersham (Arlington Heights, IL).The caspase inhibitors Z-VAD-fmk and DEVD-fmk were purchased fromCalbiochem (La Jolla, CA). Reagents used in magnetic T cell separationwere obtained from StemCell Technologies (Vancouver, Canada). Recom-binant human IL-2 was provided by Chiron Therapeutics (Emeryville,CA). PMA, ionomycin, andN,N,N9,N9-tetrakis [2-pyridylmethyl]ethyl-enediamine (TPEN) were obtained from Sigma (St. Louis, MO). Mediumused for the culture of T cells was RPMI 1640 (BioWhittaker, Walkers-ville, MD) supplemented with 10% FBS (HyClone, Logan, UT),L-glu-tamine (2 mM), gentamicin (50 mg/L), sodium pyruvate (1 mM), and non-essential amino acids (0.1 mM) (Life Technologies, Long Island, NY).

Isolation of peripheral blood-derived T lymphocytes

PBL from healthy volunteers were isolated and purified as previously de-scribed (44, 45). In brief, PBL were subjected to Ficoll-Hypaque (Phar-macia, Uppsala, Sweden) density gradient centrifugation and then depletedof macrophages, B cells, and NK cells by negative selection using themagnetic cell separation procedure (StemCell Technologies). The T cellisolation procedure yielded cells that were.98% positive for CD3, asdetermined by immunocytometry.

Immunocytometry

All analyses were performed for 3,000 or 10,000 event list mode filesacquired through a forward vs orthogonal scatter gate. Matched isotypiccontrols were used for each particular subclass of Ig and system employed.

Analyses were performed on the FACScan (Becton Dickinson, FranklinLakes, NJ). Live gating of the forward and orthogonal scatter channels wasemployed to exclude debris and to selectively acquire lymphocytes events.All values presented are based on percent lymphocytes as determined bylight scatter. Individual fluorescent populations were determined throughthe use of acquisition and contouring/quadrant analysis software(CellQuest; Becton Dickinson).

Cell lysis and Western blot analysis

Whole cell lysates were prepared as described previously (46) in buffercontaining protease inhibitors, aprotinin (5mg/ml), leupeptin (2mg/ml),and PMSF (1 mM). Samples were placed on ice for 20 min with occasionalvortexing, followed by centrifugation at 14,000 rpm for 15 min at 4°C.

To prepare cytoplasmic and nuclear extracts, PBL (13 107 cells) wereharvested and washed with PBS at 4°C and then centrifuged at 1500 rpm

for 5 min. The cell pellet was resuspended in 150ml of buffer A (10mmol/L HEPES (pH 7.9), 10 mmol/L KCl, 0.1 mmol/L EGTA, 0.1mmol/L EDTA, 1 mmol/L DTT, 1 mM PMSF, 5mg/ml aprotinin, 2mg/mlleupeptin, 100mg/ml pefabloc, and 100mg/ml chymostatin). The cellswere incubated on ice for 15 min, and then 10ml of 10% Nonidet P-40solution (Sigma) was added and cells were vigorously mixed for 20 sbefore centrifugation. The cytoplasmic extract was aliquoted and the nu-clear pellet rinsed with hypotonic buffer A. Pelleted nuclei were resus-pended in 60ml of buffer C (25% glycerol, 20 mmol/L HEPES (pH 7.9),0.42 mol/L NaCl, 0.1 mmol/L EGTA, 1 mmol/L EDTA, 1 mmol/L DTT,1 mM PMSF, 5mg/ml aprotinin, 2mg/ml leupeptin, 100mg/ml Pefabloc,and 100mg/ml chymostatin) and rotated at 4°C for 20 min. The nuclearfraction was centrifuged at 14,000 rpm for 10 min at 4°C. Protein concen-tration was measured with a commercial kit (Pierce, Rockford, IL).

Equivalent amounts of protein from whole cell lysates, cytoplasmic, andnuclear fractions (10mg) were mixed with equal volume of 23 Laemmlisample buffer, boiled, and resolved by electrophoresis in 7.5% and 14%SDS-PAGE. The proteins were transferred from the gel to a nitrocellulosemembrane using an electroblotting apparatus (Bio-Rad, Richmond, CA)(15 V, 3 mA/cm2 for 27 min). Membranes were incubated in blockingsolution containing 5% nonfat dry milk, in TBS overnight to inhibit non-specific binding. The membranes were then incubated with specific Ab (1mg/ml) for 1 h. After washing in Tris/0.1% Tween 20 for 30 min, mem-branes were incubated for another 30 min with HRP-conjugated secondaryAb. The membranes were then washed and developed with enhancedchemiluminescence (ECL Western Blotting Kit; Amersham).

FIGURE 1. SN50 peptide induces apoptosis in PBL.A, Highly enrichednaive T cells were cultured in medium alone or treated with indicatedconcentrations of SN50 peptide for 24 h. The percentage of apoptotic cellswas determined by TUNEL staining as indicated inMaterials and Meth-ods.B, DNA from T cells cultured in either medium or SN50 (75mg/ml)for 24 h was isolated as described inMaterial and Methodsand subjectedto 1.5% agarose gel electrophoresis. The DNA was visualized by ethidiumbromide staining.C, T cells were either untreated or cultured with 75mg/ml of SN50 or SN50 M peptides for 24 h. Percentage of apoptotic cellswas calculated as indicated inA.

591The Journal of Immunology

by guest on June 10, 2018http://w

ww

.jimm

unol.org/D

ownloaded from

EMSA

Nuclear extracts were prepared from T cells before and after stimulationwith PMA/ionomycin (0, 0.5, and 2 h). Binding reactions were performedusing 8mg of nuclear protein preincubated on ice for 10 min in a 20-mltotal reaction volume containing 20 mM HEPES (pH 7.9), 80 mM NaCl,0.1 mM EDTA, 1 mM DTT, 8% glycerol, and 2mg of poly(dI-dC) (Phar-macia). The reaction mixture was then incubated with the radiolabeledoligonucleotide for 20 min at room temperature. Samples were analyzed byelectrophoresis in a 6% nondenaturing polyacrylamide gel with 0.25 TBEbuffer (22.2 mM Tris, 22.2 mM boric acid, 0.5 mM EDTA). Gels werevacuum-dried and exposed to film at280°C.

Oligonucleotide containing a tandem repeat corresponding to thekBelement of the IL-2R gene was used as the probe. Radiolabeled double-stranded probe was prepared by annealing a coding strand template to acomplimentary 10 base primer and filling in the overhang using DNApolymerase I in the presence of [a-32P]dCTP. The sequence was 59-CAACGGCAGGGGAATCTCCCTCTCCTT-39, and the underlined por-tion represents thekB binding motif. Radiolabeled oligonucleotide probeswere prepared that correspond to NF-AT (59-CGCCCAAAGAGGAAAATTTGTTTCATA-39) and AP-1 (59-CGCTTGATGACTCAGCCGGAA-39) (Santa Cruz Biotechnology).

Measurement of caspases activity

Caspase and caspase-3 activity was measured using fluorometric tetrapep-tide substrates. The assays were performed in 96-well plates by incubating20 mg of cell lysates with 180ml of reaction buffer (100 mmol/L HEPES(pH 7.5), 20% v/v glycerol, 5 mmol/L DTT, and 0.5 mmol/L EDTA) con-taining 50mM YVAD-AMC, VEID-AMC, or DEVD-AMC (PharMingen,San Diego, CA). Release of 7-amino-4-methyl-coumarin (AMC) was mon-itored after 1 h ofincubation at 37°C on a microplate fluorometer withexcitation and emission wavelengths of 380 and 460 nm, respectively.

Measurement of apoptosis

Early apoptotic changes were identified by apoptosis detection kit withfluorescein-conjugated annexin V, which binds to exposed phosphatidyl-serine on the surface of apoptotic cells (47) according to protocol providedwith the kit (R&D Systems, Minneapolis, MN).

DNA fragmentation was detected using The Phoenix Flow Systems(San Diego, CA) APO-BRDU kit according to the protocol provided withthe kit. Briefly, PBL were harvested, washed in PBS, and 13 106 cellswere resuspended in 1% paraformaldehyde for 15 min on ice, washed twicewith ice cold PBS, and fixed in 70% cold ethanol overnight. The fixed cellswere washed twice in wash buffer, incubated with DNA labeling solution,followed by incubation with fluorescein-PRB-1 Ab solution and analysisby flow cytometry in the presence of propidium iodide/RNase solution.

DNA laddering as another measure of DNA fragmentation was deter-mined by horizontal agarose gel electrophoresis using a previously pub-lished method (48).

Apoptosis was also determined by conventional light microscopy. Spe-cifically, cytospin samples were assessed for the cellular and nuclearchanges characteristically associated with apoptotic cell death (cell shrink-age, chromatin condensation, and karyorrhexis).

ResultsThe SN50 peptide induces apoptosis in PBL

SN50 is composed of a nuclear localization sequence (NLS) forNF-kB1(p50) linked to the hydrophobic region of the signal pep-tide of Kaposi fibroblast growth factor, a cell-permeable carrier(43). SN50 blocks the intracellular recognition mechanism for theNLS on p50/p50 homodimers and heterodimers (p50/RelA) thatinhibits their nuclear translocation in Jurkat T cell line (43, 49). Todetermine whether NF-kB activity might regulate the survival ofhuman peripheral blood-derived T cells, we incubated resting Tcells with varying concentrations of SN50 peptide for 24 h beforeperforming the TUNEL assay. Concentration-dependent inductionof DNA fragmentation by SN50 peptide is presented in Fig. 1A andillustrates that apoptosis is observed with a concentration of 25mg/ml, although 75mg/ml is most active. The level of apoptosiscoincided with the degree of NF-kB inhibition mediated by dif-ferent concentrations of SN50 peptide (Figs. 1 and 7).

FIGURE 2. Time course of SN50 peptide induced apoptosis in naive Tcells. T lymphocytes were cultured for the indicated time with or without75 mg/ml of SN50 peptide. The percentage of apoptotic cells was deter-mined by TUNEL staining as indicated inMaterial and Methods.

FIGURE 3. SN50 induced morphological changes in T cells that areconsistent with apoptosis. Naive T cells were cultured in medium (upperpanel), SN50 (75mg/ml)(middle panel), or TPEN (15mM) (bottom panel)for 24 h before preparing cytospins and staining with hematoxylin andeosin. Arrows indicate either fragmented (black) or condensed (white)nuclei.

592 INHIBITION OF NF-kB INDUCES APOPTOSIS OF T CELLS

by guest on June 10, 2018http://w

ww

.jimm

unol.org/D

ownloaded from

While the results described above suggested that inhibition ofNF-kB nuclear translocation leads to induction of apoptosis, pos-sible nonspecific effects of SN50 peptide were examined. To showthat apoptotic activity of SN50 peptide is based on its effect onNF-kB nuclear translocation, T lymphocytes were incubated with75 mg/ml of control SN50 M peptide, which has mutations in tworesidues within the nuclear localization sequence and does not pre-vent NF-kB nuclear translocation (43) (Fig. 7). In contrast toSN50, the mutant peptide did not induce apoptosis (Fig. 1C).

To define the time frame required for SN50-mediated apoptosis,naive T cells were treated with 75mg/ml of SN50 peptide andharvested at various time points as indicated (Fig. 2). The induc-tion of DNA fragmentation by SN50 peptide in T lymphocytes wasnoticeable after 6 h ofincubation. The percentage of apoptoticcells continued to increase for up to 24 h following the addition ofSN50 peptide.

Additional experiments verified that SN50 does indeed induceDNA fragmentation in PBL. In one set, DNA fragmentation wasconfirmed by the demonstration of characteristic DNA laddering inagarose gel after T cells were cocultured for 24 h with the inhib-itory peptide (75mg/ml) (Fig. 1B). Morphological studies werealso performed on T cells after exposure to either SN50 or TPEN,a Zn chelator that is known to induce apoptosis (50). As shown inFig. 3, similar changes in morphology were noted when T cellswere treated with either SN50 or TPEN. In both cases, changescharacteristic for apoptotic cells were seen, and this included frag-mentation of the nuclei as well as nuclear condensation.

SN50 treatment induces phosphatidylserine externalization innaive T lymphocytes

Previous studies demonstrated that phosphatidylserine is exportedto the outer plasma membrane leaflet of apoptotic cells to serve asa trigger for recognition of apoptotic cells by phagocytes (51–53).Phosphatidylserine externalization is an early and widespreadevent occurring during apoptosis of various cell types. This pro-cess can be measured using annexin V, a protein with a high af-finity for this lipid (47). Here, we determined whether the apopto-sis induced in resting T cells by SN50 involved the externalizationof phosphatidylserine. Using annexin V staining, low levels ofphosphatidylserine were present on T cells cultured in medium. Incontrast, incubation of PBL with SN50 peptide resulted in in-creased level of phosphatidylserine externalization (Fig. 4). Thesefindings show that prevention of NF-kB nuclear localization innaive T cells can induce two critical events involved in apoptosis,phosphatidylserine externalization and DNA breaks.

Apoptosis induced by SN50 is caspase-dependent but occurs inthe absence of detectable caspase-1-, caspase-3-, and caspase-6-like activity

Recent studies have identified members of the family of caspaseproteases (formerly ICE/CED-3 proteases) as key participants inapoptosis that act upstream of endonucleases (22, 23). It is welldocumented that a variety of apoptotic agents induce sequentialactivation of caspases in different cell types (21–30). It has alsobeen shown that activation of ICE/CED-3 family proteases is re-quired for phosphatidylserine externalization during CD95-in-duced apoptosis (54). To assess the potential involvement ofcaspase family members in apoptosis induced by inhibition ofNF-kB nuclear translocation, we tested whether the caspase inhib-itors Z-VAD-fmk and Ac-DEVD-fmk would prevent DNA breaksinduced by SN50. Z-VAD-fmk is considered a general caspaseinhibitor, while the DEVD sequence inhibits primarily caspase-3,although caspase-6, -7, -8, and -10 are also affected (55). Six ex-periments were performed, and a representative experiment is pre-sented in Fig. 5A. Pretreatment with Z-VAD-fmk efficientlyblocked SN50-mediated apoptosis (mean 876 8.9% SD reduc-tion, n 5 6), whereas Ac-DEVD-fmk partially blocked DNAbreaks (mean 576 29.6% SD reduction,n 5 6). As a control forboth inhibitors, we tested their ability to block Fas (CD95)-medi-ated apoptosis of the Fas-sensitive Jurkat T cell line. Treatmentwith soluble Fas ligand (FasL) (CD95L) (100 ng/ml) for 24 hinduced apoptosis in Jurkats (48% apoptotic cells); however, Z-VAD-fmk as well as Ac-DEVD-fmk were effective at blocking

FIGURE 4. Annexin V binding in SN50-treated T cells. T lymphocyteswere incubated either in the presence or absence of 75mg/ml SN50 peptidefor 24 h followed by staining with FITC-conjugated annexin V and anal-ysis by flow cytometry as described inMaterials and Methods.

FIGURE 5. Induction of DNA breaks by SN50 peptide treatment isblocked by caspase inhibitors.A, Naive T cells were incubated with andwithout SN50 in the presence or absence of the caspase inhibitors Z-VAD-fmk and Ac-DEVD-fmk. After 24 h, cells were assessed by the TUNELassay.B, Jurkat T cells were incubated alone or with soluble FasL (CD95L)for 24 h in either the presence or absence of the caspase inhibitors men-tioned inA. Cells were then assayed for DNA breaks by TUNEL.

593The Journal of Immunology

by guest on June 10, 2018http://w

ww

.jimm

unol.org/D

ownloaded from

Fas-mediated killing (Fig. 5B). The differential ability of Ac-DEVD-fmk to block apoptosis mediated by SN50 and FasL sug-gests that there may be differences in the caspases involved in thedeath pathways induced by these two agents.

To determine which caspases are activated as a result of sup-pressing NF-kB, we examined lysates from SN50-treated T cellsfor their ability to cleave YVAD-AMC, DEVD-AMC, and VEID-AMC fluorogenic substrates. Enzymatic activity was monitoredafter 6 and 24 h of T cell treatment with 75mg/ml SN50 peptide.As shown in Fig. 6A, there was no cleavage of these substrates bylysates of T lymphocytes treated with SN50. Immunoblotting withthe same lysates demonstrated that there was no change in theexpression levels of either caspase-1 or caspase-3 before and afterstimulation, and there was no evidence of cleavage products thatare present following the activation of these two proteins (data notshown). In parallel, cleavage of PARP protein, which is one of thewell-established intracellular substrates for caspase-3 (31, 32), wasstudied by immunoblotting (Fig. 6B). No proteolytic cleavage ofPARP protein was detected in T cells treated with 75mg/ml SN50peptide. There was also no detectable caspase-3-dependent cleav-age of DFF-45 (Fig. 6C). However, with the same cells, caspaseactivation was demonstrated after 24 h of treatment with TPEN, asevident by cleavage of all three fluorogenic substrates. Moreover,there was detectable cleavage of PARP and DFF-45 in TPEN-treated cells (Fig. 6). The activation of caspase enzymatic activitywas also easily detected in Jurkats cells stimulated with FasL (V.Kolenko, unpublished observations). These experiments furthersuggest that apoptosis of naive T lymphocytes mediated by inhi-bition of NF-kB nuclear translocation may involve distinct set ofcaspases than those induced by TPEN and FasL.

Bcl-2, Bcl-xL, and Bax proteins are involved in the regulation ofcell susceptibility to apoptosis where Bcl-2 or Bcl-xL inhibits apo-ptosis, while Bax promotes apoptosis (40, 56, 57). We examined

the levels of Bcl-2, Bcl-xL, and Bax proteins in naive T cells cul-tured with or without SN50 peptide to determine whether the lev-els are modulated in a way that suggests a role for these proteinsin regulating SN50-mediated apoptosis. Recent studies haveshown that resting PBL express high levels of Bcl-2 and Bax pro-teins with no detectable expression of Bcl-x (56, 58). In agreementwith this observation, we found high levels of Bcl-2 and Bax pro-tein in resting T lymphocytes that did not vary after 24 h of celltreatment with 75mg/ml SN50 peptide (data not shown). At thistime, .60% of the T cell population treated with SN50 had un-dergone apoptosis as determined by TUNEL assay. No detectablelevel of either Bcl-xL or Bcl-xS proteins was observed in naive Tlymphocytes cultured in medium alone or in the presence of SN50peptide.

Induction of apoptosis in T cells is attributable to suppression ofkB binding activity

Here, we document that the SN50 peptide inhibits the nuclear lo-calization of NF-kB in peripheral blood-derived T cells. Incuba-tion for 1 h with SN50 (50 and 75mg/ml) reduced the backgroundlevel of kB binding observed in resting T cells (Fig. 7). It alsoinhibited the increase inkB binding activity following 2 h of stim-ulation with PMA/ionomycin. SN50 M that had 2 of 10 NLS res-idues mutated did not inhibit DNA binding activity. Immunoblot-ting (Fig. 7B) confirmed that SN50 treatment, in a concentration-dependent manner, prevented nuclear localization of RelA and p50after stimulation, however it had no effect on the cytoplasmic lev-els of these proteins. At 75mg/ml, SN50 also eliminated the back-ground level of Rel proteins that are present in the nuclei of restingcells. These experiments also demonstrated that SN50 mediates itseffect on NF-kB dimers following normal degradation of the in-hibitor IkBa (Fig. 7C).

FIGURE 6. Suppression of p50/RelA nuclear localization by SN50 peptide does not induce detectable caspase-1-, caspase-3-, or caspase-6-like activityin naive T cells.A, Normal T cells were incubated in either medium alone or in the presence of 75mg/ml SN50 peptide for the indicated time. Cell lysateswere assayed with fluorogenic substrates (YVAD-AMC, DEVD-AMC, VEID-AMC) as described inMaterials and Methods. Aliquots of the same cellswere subjected to 7.5% SDS-PAGE, blotted, and probed with anti-PARP (B) and anti-DFF-45 (C) Abs.

594 INHIBITION OF NF-kB INDUCES APOPTOSIS OF T CELLS

by guest on June 10, 2018http://w

ww

.jimm

unol.org/D

ownloaded from

We wanted to know if the induction of apoptosis in normalresting T cells was linked to the defect in NF-kB activation. There-fore, we initially tested whether SN50 was selective at blockingNF-kB activation without altering nuclear localization of othertranscription factors. At 75mg/ml, SN50 inhibited binding of nu-clear extracts to AP-1, NF-AT, as well as thekB probe, which isconsistent with a recent report where 210mg/ml of SN50 pre-vented nuclear localization of multiple transcription factors (49).However, at 37.5mg/ml (n 5 3), SN50 appeared selective in thatkB binding activity was blocked, whereas AP-1 and NF-AT bind-ing activity was unaffected (Fig. 8). In the same experiments,where only NF-kB activation was suppressed, there was consistentinduction of apoptosis, suggesting that defective NF-kB is respon-sible for the initiation of apoptosis in SN50-treated T lymphocytes.

We also determined whether another agent known to suppressNF-kB activation would also induce DNA breaks in T cells andthus reproduce the results observed with SN50 peptide. We usedN-tosyl-L-phenylalanine chloromethyl ketone (TPCK), since it hasbeen shown to inhibitkB binding activity (18). Pretreatment withTPCK (100mM) prevented PMA/ionomycin induction of NF-kB(Fig. 9A). TPCK appears to function by interfering with proteo-some activity, which blocks the stimulus-dependent phosphoryla-tion and degradation of IkBa (18) (Fig. 9B). We show here thatsuppression of NF-kB activation by TPCK can result in the induc-tion of DNA breaks (TUNEL assay) (Fig. 9C) within 24 h, sug-gesting that inhibition ofkB binding activity through distinctmechanisms can induce the death pathway in naive T cells. Similarresults were also obtained with a new BAY 11–7082 inhibitor thatselectively blocks IkBa degradation (59).

Preactivation promotes survival of T lymphocytes treated withSN50 peptide

We also determined whether preactivation of NF-kB by externalstimuli would alter the sensitivity of T cells to SN50-mediated

apoptosis. Within 15 min of T cell activation, there is a significantincrease in the nuclear localization of RelA, c-Rel, and p50 dimers(55). This translocation of Rel proteins coincides with an increasein kB-specific DNA binding activity, where the peak activity oc-curs within 2 h of stimulation (60). Preactivation of T cells withPMA/ionomycin for 2 h completely blocked DNA fragmentationinduced by a 24-h exposure to SN50 (Fig. 10). In the same cellsthat were not preactivated with PMA/ionomycin, SN50 inducedsignificant apoptosis (48%). Similar results were observed withIL-2, which is also known to activate NF-kB. These results showthat prior activation and nuclear translocation of NF-kB can pro-tect cells from apoptosis mediated by SN50. However, the protec-tive effect of preactivation is eventually reversed by continued ex-posure to SN50 after 3 days through the inhibition of furtherNF-kB activation (our unpublished observations).

DiscussionThe involvement of NF-kB in regulating the apoptotic responsehas been suggested previously by several groups (1, 14–19). In-hibition of NF-kB nuclear translocation increased the susceptibil-ity of cells to undergo apoptosis induced by TNF-a, ionizing ra-diation, and cancer chemotherapeutic drugs (17). The suppressionof NF-kB activation by protease inhibitors that block IkBa deg-radation induced apoptosis in murine splenic B cells (18, 19). Sim-ilar findings were also reported following the microinjection ofeither GST-IkBa fusion protein or an Ab to c-Rel (18, 19). Incontrast, ectopic expression of c-Rel was found to ablate the in-duction of apoptosis induced in B cells following suppression ofNF-kB activation by inhibitors of IkBa degradation (18, 19).

FIGURE 8. SN50 inhibited NF-kB binding activity without affectingAP-1 and NF-AT DNA binding activity.A, T cells were treated with andwithout SN50 (37.5mg/ml) before stimulating for 2 h with PMA/ionomy-cin. Nuclear extracts were then subjected to EMSA using labeled probesthat correspond to AP-1, NF-kB, and NF-AT sequences as described inMaterial and Methods.B, The same cells as described inA were examinedafter 24 h for DNA breaks by TUNEL.

FIGURE 7. SN50 peptide inhibits NF-kB activation in the presence ofnormal degradation of the inhibitor, IkBa. A, Highly enriched naive T cellswere cultured in medium alone or treated with indicated concentrations ofSN50 peptide for 1 h before stimulation with PMA (20 ng/ml)/ionomycin(75 mg/ml). kB binding activity was detected in nuclear extracts by per-forming EMSA with a labeledkB probe.B, Aliquots of the same cells weresubjected to Western blotting to demonstrate dose-dependent suppressionof nuclear translocation of NF-kB/RelA by SN50 peptide.C, Immunoblot-ting for IkBa in cytoplasmic extract shown inB.

595The Journal of Immunology

by guest on June 10, 2018http://w

ww

.jimm

unol.org/D

ownloaded from

These findings point to a critical role of NF-kB family members inthe protection of cells against various forms of apoptotic cell death.

The data presented here shows that blocking nuclear transloca-tion of NF-kB dimers by the cell-permeable peptide, SN50, in-duced apoptosis in normal peripheral blood-derived T lympho-cytes. In contrast, the inactive control SN50 M mutant peptide hadno inhibitory effect on eitherkB binding activity or cell viability.Whether suppression of NF-kB activation is responsible for theinitiation of apoptosis in peripheral T cells is supported by theobservation that, at the SN50 concentration of 37.5mg/ml, induc-tion of apoptosis coincided with selective suppression ofkB bind-ing activity as evident by normal nuclear localization of other tran-scription factors, such as AP-1 and NF-AT. This conclusion is alsosupported by the fact that suppression of NF-kB activation byanother mechanism (TPCK) also induced DNA breaks.

Given the finding that SN50-mediated apoptosis was observedin resting T cells leaves open the possibility that low levels ofconstitutivekB binding activity are required to maintain cellularviability in this lymphoid population. As noted by Western blottingand by gel mobility shifts assays (Figs. 7 and 8) (60), there are lowlevels of NF-kB1 and RelA expression in the nucleus without anyexternal stimulation. The fact that SN50 blocked nuclear translo-cation of p50 and RelA and subsequently induced apoptosis isconsistent with the possibility that the constitutive expression ofNF-kB in resting T cells promotes survival.

Susceptibility of T cells to apoptosis appears to be linked to thestate of their activation. This conclusion is supported by previouslypublished data that shows mitogen activation of T cells enhancestheir resistance tog-irradiation (56). In agreement with this idea,our study shows that preactivation of naive T cells with eitherPMA/ionomycin or IL-2 completely blocked DNA fragmentationinduced by SN50 peptide. Cells needed to be preactivated for atleast 2 h before their exposure to SN50 peptide to become resistantto SN50-mediated apoptosis. The kinetics of induction of resis-tance paralleled the appearance ofkB DNA binding activity (ourunpublished observations). It may be that NF-kB activation resultsin transcriptional up-regulation of genes encoding proteins in-volved in protection against apoptosis. The protective product maybe distinct from Bcl-2, since SN50 inhibited NF-kB activation andinduced apoptosis even in the presence of significant levels ofBcl-2. Whether overexpression of anti-apoptotic proteins, such asBcl-xL, can protect T cells from apoptosis induced by blockingNF-kB nuclear translocation is currently under investigation.

Activation of caspase proteases was previously shown to be re-quired for the induction of apoptosis in different cell types (21–30).Our findings with the caspase inhibitors Z-VAD-fmk and Ac-DEVD-fmk are consistent with the possibility that SN50-mediatedapoptosis in T cells is caspase-dependent. However, there appearsto be a difference in either the level or types of caspases inducedas a consequence of NF-kB suppression by SN50 peptide whencompared with other inducers of apoptosis, such as TPEN or FasL.In contrast to FasL or TPEN, the SN50 peptide did not inducedetectable enzymatic activity of caspase-1-, caspase-3-, orcaspase-6-like proteases using fluorogenic peptide substrates,YVAD-AMC, DEVD-AMC, and VEID-AMC, respectively. Al-though both caspase-1 and caspase-3 were constitutively expressedin precursor forms, we did not detect processed subunits of theseproteins in lysates from T cells treated with SN50 peptide, furthersuggesting they were not activated by SN50. In support of thisconclusion is the observation that no proteolytic cleavage of eitherPARP or DFF-45, well-established substrates for caspase-3, wasdetected in T cells treated with SN50 peptide. These data suggestthat caspase-1, caspase-3, and caspase-6 may not be the primarycaspases required for apoptosis induced by inhibition of NF-kBnuclear translocation in T cells. Additional studies are needed toidentify the caspase pathway involved in SN50-mediatedapoptosis.

In certain pathological conditions, such as cancer, down-regu-lation of NF-kB activity may represent a mechanism for inhibitingthe development of T cell immune responses. Impaired activationof NF-kB has been reported in T cells derived from tumor-bearingmice and cancer patients (60, 61). Furthermore, products present inthe tumor environment, such as IL-10, gangliosides, and prosta-glandin E2, are known to inhibit NF-kB activation and down-reg-ulate immune responses (62–64). The blocking of NF-kB trans-location may make T cells more susceptible to apoptosis. Theevidence presented here suggests that the prevention of nuclearexpression of NF-kB dimers can induce apoptosis in Tlymphocytes.

FIGURE 9. Suppression of NF-kB activation by TPCK can induceDNA breaks in naive T cells.A, T cells were incubated in medium ormedium supplemented with TPCK (100mM) for 1 h before stimulationwith PMA/ionomycin for the times indicated. Nuclear extracts were pre-pared and assessed forkB binding activity by EMSA.B, With the samesamples used inA, cytoplasmic extracts were analyzed by immunoblottingfor IkBa expression.C, The same cells as described inA were examinedafter 24 h for DNA breaks by TUNEL.

FIGURE 10. Activation promotes survival of T lymphocytes treatedwith SN50 peptide. T cells were either untreated, activated with PMA (20ng/ml)/ionomycin (0.75mg/ml), or stimulated with IL-2 (1000 IU/ml) for2 h before the addition of 75mg/ml SN50 peptide. The percentage ofapoptotic cells was determined after 24 h by TUNEL staining.

596 INHIBITION OF NF-kB INDUCES APOPTOSIS OF T CELLS

by guest on June 10, 2018http://w

ww

.jimm

unol.org/D

ownloaded from

References1. Baeuerle, P. A., and T. Henkel. 1994. Function and activation of NF-kB in the

immune system.Annu. Rev. Immunol. 12:141.2. Baeuerle, P. A., and D. Baltimore. 1996. NF-kB: ten years after.Cell 87:13.3. Molitor, J. A., W. H. Walker, D. Doerre, D. W. Ballard, and W. C. Greene. 1990.

NF-kB: a family of inducible and differentially expressed enhancer-binding pro-teins in human T cells.Proc. Natl. Acad. Sci. USA 87:10028.

4. Arima, N., W. A. Kuziel, T. A. Grdina, and W. C. Greene. 1992. IL-2-inducedsignal transduction involves the activation of nuclear NF-kB expression.J. Im-munol. 149:83.

5. Granelli-Piperno, A., and P. Nolon. 1991. Nuclear transcription factors that bindto elements of the IL-2 promoter: induction requirements in primary human Tcells.J. Immunol. 147:2734.

6. Costello, R., C. Lipcey, M. Algarte, C. Cerdan, P. A. Baeuerle, D. Olive, andJ. Imbert. 1993. Activation of primary human T-lymphocytes through CD2 plusCD28 adhesion molecules induces long-term nuclear expression of NF-kB. CellGrowth Differ. 4:329.

7. Lowenthal, J. W., D. W. Ballard, E. Bohnlein, and W. C. Greene. 1989. Tumornecrosis factora induces proteins that bind specifically tokB-like enhancer el-ements and regulate interleukin-2 receptora-chain gene expression in primaryhuman T lymphocytes.Proc. Natl. Acad. Sci. USA 86:2331.

8. Beg, A. A., T. S. Finco, P. V. Nantermet, and A. S. Baldwin, Jr. 1993. Tumornecrosis factor and interleukin-l lead to phosphorylation and loss of IkBa: amechanism for NF-kB activation.Mol. Cell. Biol. 13:3301.

9. Palombella, V., O. Rando, A. Goldberg, and T. Maniatis. 1994. The ubiquitin-proteasome pathway is required for processing the NF-kB1 precursor protein andthe activation of NF-kB. Cell 78:773.

10. Brown, K., S. Gerstberger, L. Carlson, G. Franzoso, and U. Siebenlist. 1995.Control of IkB-a proteolysis by site-specific, signal-induced phosphorylation.Science 267:1485.

11. Thompson, J. E., R. J. Phillips, H. Erdjumant-Bromage, P. Tempst, and S. Ghosh.1995. IkB-b regulated the persistent response in a biphasic activation of NF-kB.Cell 80:573.

12. Brockman, J. A., D. C. Scherer, T. A. McKinsey, S. M. Hall, X. Qi, W. Y. Lee,and D. W. Ballard. 1995. Coupling of signal response domain in IkBa to multiplepathways for NF-kB activation.Mol. Cell. Biol. 15: 2809.

13. Traenckner, E. B., H. L. Pahl, T. Henkel, K. N. Schmidt, S. Wilk, andP. A. Baeuerle. 1995. Phosphorylation of human IkBa on serines 32 and 36controls IkBa proteolysis and NF-kB activation in response to diverse stimuli.EMBO J. 14:2876.

14. La Rosa, F. A., J. P. Pierce, and G. E. Sonenshein. 1994. Differential regulationof the c-myc oncogene promoter by the NF-kB rel family of transcription factors.Mol. Cell. Biol. 14:1039.

15. Wu, H., and G. Lozano. 1994. NF-kB activation of p53. A potential mechanismfor suppressing cell growth in response to stress.J. Biol. Chem. 269:20067.

16. Beg, A. A., W. Sha, R. Bronson, S. Ghosh, and D. Baltimore. 1995. Embryoniclethality and liver degeneration in mice lacking the RelA component of NF-kB.Nature 376:167.

17. Wang, C.-Y., M. W. Mayo, and A. S. Baldwin, Jr. 1996. TNF- and cancer therapyinduced apoptosis: potentiation by inhibition of NF-kB. Science 274:784.

18. Wu, M., H. Lee, R. E. Bellas, S. L. Schauer, M. Arsura, D. Katz, M. J. FitzGerald,T. L. Rothstein, D. H. Sherr, and G. E. Sonenshein. 1996. Inhibition of NF-kB/Rel induces apoptosis of murine B cells.EMB0 J. 15:4682.

19. Arsura, M., M. Wu, and G. E. Sonenshein. 1996. TGFb1 inhibits NF-kB/Relactivity inducing apoptosis of B cells: transcriptional activation of IkB-a. Immu-nity 5:31.

20. Ravi, R., A. Bedi, E. J. Fuchs, and A. Bedi. 1998. CD95 (Fas)-induced caspase-mediated proteolysis of NF-kB. Cancer Res. 58:882.

21. Alnemri, E. S., D. J. Livingston, D. W. Nicholson, G. Salvesen,N. A. Thornberry, W. W. Wong, and J. Yuan. 1996. Human ICE/CED-3 proteasenomenclature.Cell 87:171.

22. Chinnaiyan, A. M., K. Orth, K. O’Rourke, H. Duan, G. G. Poirier, andV. M. Dixit. 1996. Molecular ordering of cell death pathway.J. Biol. Chem.271:4573.

23. Orth, K., K. O’Rourke, G. S. Salvesen, and V. M. Dixit. 1996. Molecular order-ing of apoptotic mammalian CED-3/ICE-like proteases.J. Biol. Chem.271:20977.

24. Enari, M., R. V. Talanian, W. W. Wong, and S. Nagata. 1996. Sequential acti-vation of ICE-like and CPP32-like proteases during Fas-mediated apoptosis.Na-ture 380:723.

25. Salvesen, G. S., and V. M. Dixit. 1997. Caspases: intracellular signaling by pro-teolysis.Cell 91:443.

26. Nagata, S. 1997. Apoptosis by death factor.Cell 88:355.27. Martin, S. J., D. D. Newmeyer, S. Mathias, D. M. Farschon, H.-G. Wang,

J. C. Reed, R. N. Kolesnic, and D. R. Green. 1995. Cell-free reconstitution ofFas-, UV radiation and ceramide-induced apoptosis.EMBO J. 14:5191.

28. Fraser, A., and G. Evan. 1996. A license to kill.Cell 85:781.29. Takahashi, A., and W. C. Earnshaw. 1996. ICE-related proteases in apoptosis.

Curr. Opin. Genet. Dev. 6:50.30. Nava, V. E., A. Rosen, M. A. Veliuona, R. J. Clem, B. Levine, and

J. M. Hardwick. 1998. Sindbis virus induces apoptosis through a caspase-depen-dent, CrmA-sensitive pathway.J. Virol. 72:452.

31. Nicholson, D. W., A. Ali, N. A. Thornberry, J. P. Vaillancourt, C. K. Ding,M. Gallant, Y. Gareau, P. R. Griffin, M. Labelle, and Y. A. Lazebnik. 1995.Identification and inhibition of the ICE/CED-3 protease necessary for mammalianapoptosis.Nature 376:37.

32. Tewari, M., L. Quan, K. O’Rourke, S. Desnoyers, Z. Zeng, D. R. Beidler,G. G. Poirier, G. S. Salvesen, and V. M. Dixit. 1995. Yama/CPP32b, a mam-malian homolog of CED-3, is a CrmA-inhibitable protease that cleaves the deathsubstrate poly(ADP-ribose) polymerase.Cell 81:801.

33. An, B., J. R. Jin, P. Lin, and Q. P. Dou. 1996. Failure to activate interleukin-1bconverting enzyme like proteases and to cleave retinoblastoma protein in drug-resistant cells.FEBS Lett. 399:158.

34. Janicke, R. U., P. A. Walker, X. Y. Lin, and A. G. Porter. 1996. Specific cleavageof the retinoblastoma protein by an ICE-like protease in apoptosis.EMBO J.15:6969.

35. Martin, S. J., G. A. O’Brien, W. K. Nishioka, A. J. McGahon, A. Mahboubi,T. C. Saido, and D. R. Green. 1995. Proteolysis of fodrin (non-erythroid spectrin)during apoptosis.J. Biol. Chem. 270:6425.

36. Cryns, V. L., H. Bergeron, H. Zhu, J. Li, and J. Yuan. 1996. Specific cleavage ofa-fodrin during Fas- and tumor necrosis factor-induced apoptosis is mediated byan interleukin-1b-converting enzyme/Ced-3 protease distinct from the poly-(ADP-ribose) polymerase protease.J. Biol. Chem. 271:31277.

37. Lazebnik, Y. A., A. Takahashi, R. D. Moir, R. D. Goldman, G. G. Poirier,S. H. Kaufmann, and W. C. Earnshaw. 1995. Studies of the lamin proteinasereveal multiple parallel biochemical pathways during apoptotic execution.Proc.Natl. Acad. Sci. USA 92:9042.

38. Rao, L., D. Perez, and E. White. 1996. Lamin proteolysis facilitates nuclearevents during apoptosis.J. Cell Biol. 135:1441.

39. Cheng, E. H., D. G. Kirsch, R. J. Clem, R. Ravi, M. B. Kastan, A. Bedi, K. Ueno,and J. M. Hardwick. 1997. Conversion of Bcl-2 to a Bax-like death effector bycaspases.Science 278:1966.

40. Clem, R. J., E. H. Cheng, C. L. Karp, D. G. Kirsch, K. Ueno, A. Takahashi,M. B. Kastan, D. E. Griffin, W. C. Earnshaw, M. A. Veliuona, andJ. M. Hardwick. 1998. Modulation of cell death by Bcl-xL through caspase in-teraction.Proc. Natl. Acad. Sci. USA 95:554.

41. Liu, X., H. Zou, C. Slaughter, and H. Wang. 1997. DFF, a heterodimeric proteinthat functions downstream of caspase-3 to trigger DNA fragmentation duringapoptosis.Cell 89:175.

42. Jeremias, I., C. Kupatt, B. Baumann, I. Herr, T. Wirth, and K. M. Debatin. 1998.Inhibition of nuclear factorkB activation attenuates apoptosis resistance in lym-phoid cells.Blood 91:4624.

43. Lin, Y. Z., S. Y. Yao, R. A. Veach, T. R. Torgerson, and J. Hawiger. 1995.Inhibition of nuclear translocation of transcription factor NF-kB by a syntheticpeptide containing a cell membrane-permeable motif and nuclear lacalizationsequence.J. Biol. Chem. 270:14255.

44. Finke, J. H., A. H. Zea, J. Stanley, D. L. Longo, H. Mizoguchi, R. R. Tubbs,R. H. Wiltrout, J. J. O’Shea, S. Kudoh, E. Klein, R. M. Bukowski, andA. C. Ochoa. 1993. Loss of T-cell receptorz chain and p56lck in T-cell infiltratinghuman renal cell carcinoma.Cancer Res. 53:5613.

45. Wang, Q., J. Stanley, S. Kudoh, J. Myles, V. M. Kolenko, T. Yi, R. R. Tubbs,R. M. Bukowski, and J. H. Finke. 1995. T Cells infiltrating non-Hodgkin’s B celllymphomas show altered tyrosine phosphorylation pattern even though T cellreceptor/CD3-associated kinases are present.J. Immunol. 155:1382.

46. Kolenko, V., Q. Wang, M. C. Riedy, J. O’Shea, J. Ritz,. M. K. Cathcart,P. Rayman, R. Tubbs, M. Edinger, A. Novick, R. Bukowski, and J. Finke. 1997.Tumor-induced suppression of T lymphocyte proliferation coincides with inhi-bition of Jak3 expression and IL-2 receptor signaling: role of soluble productsfrom human renal cell carcinomas.J. Immunol. 159:3057.

47. Koopman, G., C. P. M. Reutelingsperger, G. A. M. Kuijten, R. M. J. Keehnen,S. T. Pals, and M. H. J. Oers. 1994. Annexin V for flow cytometric detection ofphosphatidylserine expression on B cells undergoing apoptosis.Blood 84:1415.

48. Zamzami, N., P. Marchetti, M. Castedo, C. Zanin, J.-L. Vayssiere, P. X. Petit, andG. Kroemer. 1995. Reduction in mitochondrial potential constitutes an early ir-reversible step of programmed lymphocyte death in vivo.J. Exp. Med. 181:1661.

49. Torgerson, T., A. D. Colosia, J. P. Donahue, Y.-Z. Lin, and J. Hawiger. 1998.Regulation of NF-kB, AP-1, NFAT, and STAT1 nuclear import in T lymphocytesby noninvasive delivery of peptide carrying the nuclear localization sequence ofNF-kB p50.J. Immunol. 161:6084.

50. Treves, S., P. L. Trentini, M. Ascanelli, G. Bucci, and F. DiVirgilio. 1994. Ap-optosis is dependent on intracellular zinc and independent on intracellular cal-cium in lymphocytes.Exp. Cell Res. 211:339.

51. Fadok, V. A., D. R. Voelker, P. A. Campbell, J. J. Cohen, D. L. Bratton, andP. M. Henson. 1992. Exposure of phosphatidylserine on the surface of apoptoticlymphocytes triggers specific recognition and removal by macrophages.J. Im-munol. 148:2207.

52. Fadok, V. A., J. S. Savill, S. Haslett, D. L. Bratton, D. E. Doherty,P. A. Campbell, and P. M. Henson. 1992. Different populations of macrophagesuse either the vitronectin receptor or the phosphatidylserine receptor to recognizeand remove apoptotic cells.J. Immunol. 149:4029.

53. Martin, S. J., C. P. Reutelingsperger, A. J. McGahon, J. A. Rader,R. C. van Schie, D. M. LaFace, and D. R. Green. 1995. Early redistribution ofplasma membrane phosphatidylserine is a general feature of apoptosis regardlessof the initiating stimulus: inhibition by overexpression of Bcl-2 and Abl.J. Exp.Med. 182:1545.

54. Martin, S. J., D. M. Finucane, G. P. Amarante-Mendes, G. A. O’Brien, andD. R. Green. 1996. Phosphatidylserine externalization during CD95-inducedapoptosis of cells and cytoplasts requires ICE/CED-3 protease activity.J. Biol.Chem. 271:28753.

597The Journal of Immunology

by guest on June 10, 2018http://w

ww

.jimm

unol.org/D

ownloaded from

55. Garsia-Calvo, M., E. P. Peterson, B. Leiting, R. Ruel, D. W. Nicholson, andN. A. Thornberry. 1998. Inhibition of human caspases by peptide-based andmacromolecular inhibitors.J. Biol. Chem. 273:32608.

56. Boise, L. H., A. J. Minn, P. J. Noel, C. H. June, M. A. Accavitti, T. Lindsten, andC. B. Thompson. 1995. CD28 costimulation can promote T cell survival byenhancing the expression of Bcl-xL. Immunity 3:87.

57. Akbar, A. N., N. J. Borthwick, R. G. Wickremasinghe, P. Panayiotidis, D. Pilling,M. Bofill, S. Krajewski, J. C. Reed, and M. Salmon. 1996. Interleukin-2 receptorcommong-chain signaling cytokines regulate activated T cell apoptosis in re-sponse to growth factor withdrawal: selective induction of anti-apoptotic (bcl-2,bcl-xL) but not pro-apoptotic (bax, bcl-xS) gene expression.Eur. J. Immunol.26:294.

58. Broome, H. E., C. M. Dargan, S. Krajewski, and J. C. Reed. 1995. Expression ofBcl-2, Bcl-x, and Bax after T cell activation and IL-2 withdrawal.J. Immunol.155:2311.

59. Pierce, J. W., R. Schoenleber, G. Jesmok, J. Best, S. A. Moore, T. Collins, andM. E. Gerritsen. 1997. Novel inhibitors of cytokine-induced IkBa phosphoryla-tion and endothelial cell adhesion molecule expression show anti-inflammatoryeffects in vivo.J. Biol. Chem. 272:21096.

60. Ling, W., P. Rayman, R. Uzzo, P. Clark, H. J. Kim, R. Tubbs, A. Novick,R. Bukowski, T. Hamilton, and J. Finke. 1998. Impaired activation of NF-kB inT cells from a subset of renal cell carcinoma patients is mediated by inhibition ofphosphorylation and degradation of the inhibitor, IkBa. Blood 92:1.

61. Correa, M. R., A. C. Ochoa, P. Ghosh, H. Mizoguchi, L. Harvey, andD. L. Longo. 1997. Sequential development of structural and functional alter-ations in T cells from tumor-bearing mice.J. Immunol. 158:5292.

62. Romano, M. F., A. Lamberti, A. Petrella, R. Bisogni, P. F. Tassone,S. Formisano, S. Venuta, and M. C. Turco. 1996. IL-10 inhibits nuclear factor-kB/Rel nuclear activity in CD3-stimulated human peripheral T lymphocytes.J. Im-munol. 156:2119.

63. Chen, D., and E. V. Rothenberg. 1994. Interleukin 2 transcription factors asmolecular targets of cAMP inhibition: delayed inhibition kinetics and combina-torial transcription roles.J. Exp. Med. 179:931.

64. Irani, D. N., K. I. Lin, and D. E. Griffin. 1996. Brain-derived gangliosides reg-ulate the cytokine production and proliferation of activated T cells.J. Immunol.157:4333.

598 INHIBITION OF NF-kB INDUCES APOPTOSIS OF T CELLS

by guest on June 10, 2018http://w

ww

.jimm

unol.org/D

ownloaded from