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The Journal of Immunology, 1999, 163: 590-598.
Copyright © 1999 by The American Association of Immunologists

Inhibition of NF-{kappa}B Activity in Human T Lymphocytes Induces Caspase-Dependent Apoptosis Without Detectable Activation of Caspase-1 and -31

Vladimir Kolenko*,{dagger},2, Tracy Bloom*, Patricia Rayman*, Ronald Bukowski{dagger}, Eric Hsi§ and James Finke*,{dagger},{ddagger}

Departments of * Immunology, {dagger} Hematology-Oncology, {ddagger} Urology, and § Anatomic Pathology, Cleveland Clinic Foundation, Cleveland, OH 44195


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
NF-{kappa}B is involved in the transcriptional control of various genes that act as extrinsic and intrinsic survival factors for T cells. Our findings show that suppression of NF-{kappa}B activity with cell-permeable SN50 peptide, which masks the nuclear localization sequence of NF-{kappa}B1 dimers and prevents their nuclear localization, induces apoptosis in resting normal human PBL. Inhibition of NF-{kappa}B resulted in the externalization of phosphatidylserine, induction of DNA breaks, and morphological changes consistent with apoptosis. 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-{kappa}B suppression, in contrast to that induced by TPEN (N,N,N',N'-tetrakis [2-pyridylmethyl]ethylenediamine) or soluble Fas ligand (CD95), was observed in the absence of active death effector proteases caspase-1-like (IL-1 converting enzyme), caspase-3-like (CPP32/Yama/apopain), and caspase-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 cells resulting in NF-{kappa}B nuclear translocation protected cells from SN50-induced apoptosis. Our findings demonstrate an essential role of NF-{kappa}B in survival of naive PBL.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The transcription factor NF-{kappa}B plays a critical role in the development and maintenance of T cell-mediated immune responses. NF-{kappa}B consists of multiple members of the Rel family of proteins that include NF-{kappa}B1 (p105/p50), NF-{kappa}B2 (pl00/p52), RelA (p65), RelB, and c-Rel (1, 2). Rel proteins form hetero- and homodimeric complexes that differ in their transactivating activity (1). In T lymphocytes, the RelA/p50 heterodimer is known to initiate transactivation, whereas homodimers of p50 appear to be suppressive (1, 2). The NF-{kappa}B dimers are present in cytoplasm in an inactive form bound to inhibitory subunits, I{kappa}Bs (3, 4, 5, 6, 7). Upon activation I{kappa}B is phosphorylated, which marks the inhibitor for ubiquitination and degradation by the proteasome-dependent pathway (8, 9, 10, 11). This process allows translocation of active NF-{kappa}B complexes into the nucleus, where they bind to specific DNA motifs in the promoter/enhancer regions of target genes and activate transcription (8, 9, 10, 11, 12, 13).

There is growing evidence that NF-{kappa}B regulates the susceptibility of certain cell types to apoptosis through the transcriptional control of protective genes (1, 14, 15). Knockout transgenic mice lacking the RelA component of NF-{kappa}B complex displayed embryonic lethality and liver cell apoptosis (16). Inhibition of NF-{kappa}B nuclear translocation also enhanced apoptosis induced by ionizing radiation or the chemotherapeutic drug, danorubicin (17). NF-{kappa}B also appears to regulate the susceptibility of lymphoid cells to apoptosis. Addition of various inhibitors of NF-{kappa}B/Rel activation to normal murine B lymphocytes or to B cell lymphomas resulted in apoptosis (18, 19). A protective role for c-Rel in preventing this process was confirmed using microinjection of its specific inhibitor, I{kappa}B{alpha}, or injection of Ab to the c-Rel subunit (18, 19). Additional findings suggest that ligation of CD95 (Fas/APO1) depressed NF-{kappa}B activation by causing the degradation of the NF-{kappa}B subunit RelA, a process that may enhance the decay of an immune response (20).

Recent studies have identified the involvement of multiple caspases in the proteolytic cascade of apoptosis (21, 22, 23, 24, 25, 26). Various stimuli that induce apoptosis, including UV, Fas Ag, drug treatment, growth factor withdrawal, and virus infection, have been shown to activate caspases that specifically cleave proteins at the C terminus of aspartic acid residues (27, 28, 29, 30). Caspases are synthesized as zymogens that require proteolytic cleavage to generate active enzyme subunits. These activating cleavage events are conducted by other caspases and are thought to represent a major regulatory step in the apoptosis pathway (21, 22, 23, 24, 25, 26). Activated caspases cleave several target proteins that include poly(ADP-ribose) polymerase (PARP)3 (31, 32), retinoblastoma protein (33, 34), cytoskeletal proteins (35, 36, 37, 38), Bcl-2 (39), and Bcl-xL (40). A recent study has also identified RelA as a substrate for activated caspase-3 (20). Cleavage of the 45-kDa subunit of DNA fragmentation factor (DFF-45) by activated caspases leads to fragmentation of genomic DNA into nucleosomal fragments, one hallmark of apoptosis (41).

The role NF-{kappa}B plays in the regulation of apoptosis in T lymphocytes has not been well defined. There is evidence that inhibition of NF-{kappa}B activation following transient transfection with a mutant form of I{kappa}B{alpha} made the Jurkat T cell line susceptible to TNF-{alpha}-mediated apoptosis (42). Here, we show that ,in resting human peripheral blood-derived T cells, the inhibition of NF-{kappa}B activation results in apoptosis. The cell-permeable peptide SN50 was found not to inhibit the stimulus-dependent degradation of the inhibitor I{kappa}B{alpha}, but rather to block the nuclear translocation of NF-{kappa}B (43). This inhibitory peptide induced exposure of phosphatidylserine on the cell surface, an early event in apoptosis, and the formation of specific DNA breaks, as defined by DNA laddering and TUNEL assay. SN50-mediated apoptosis is caspase-dependent, since DNA fragmentation was efficiently blocked by the caspase inhibitor Z-VAD-fmk and partially blocked by DEVD-fmk. However, apoptosis occurred in the absence of detectable active caspase-1-like (IL-1 converting enzyme (ICE)), caspase-3-like (CPP32/Yama/apopain), and caspase-6-like proteases and without detectable proteolysis of PARP and DFF-45, suggesting that either low level of activation is required or different caspases are involved. Preactivation with either IL-2 or PMA/ionomycin induced NF-{kappa}B activation and prevented apoptosis following exposure to SN50. However, continued exposure to SN50 did induce apoptosis in preactivated T cells, which coincided with suppression of NF-{kappa}B.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Abs and reagents

SN50 and SN50 M peptides and Ab to PARP were obtained from Biomol Research Laboratories (Plymouth Meeting, PA). Abs used in Western blotting for NF-{kappa}B1 (p50), RelA (p65), I{kappa}B{alpha}, DFF-45, and caspase-1 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Abs to Bcl-2, Bax, and caspase-3 were purchased from Transduction Laboratories (Lexington, KY). Secondary HRP-conjugated sheep anti-mouse and donkey anti-rabbit Abs were purchased from Amersham (Arlington Heights, IL). The caspase inhibitors Z-VAD-fmk and DEVD-fmk were purchased from Calbiochem (La Jolla, CA). Reagents used in magnetic T cell separation were obtained from StemCell Technologies (Vancouver, Canada). Recombinant human IL-2 was provided by Chiron Therapeutics (Emeryville, CA). PMA, ionomycin, and N,N,N',N'-tetrakis [2-pyridylmethyl]ethylenediamine (TPEN) were obtained from Sigma (St. Louis, MO). Medium used for the culture of T cells was RPMI 1640 (BioWhittaker, Walkersville, MD) supplemented with 10% FBS (HyClone, Logan, UT), L-glutamine (2 mM), gentamicin (50 mg/L), sodium pyruvate (1 mM), and nonessential 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 described (44, 45). In brief, PBL were subjected to Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) density gradient centrifugation and then depleted of macrophages, B cells, and NK cells by negative selection using the magnetic cell separation procedure (StemCell Technologies). The T cell isolation procedure yielded cells that were >98% positive for CD3, as determined by immunocytometry.

Immunocytometry

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

Analyses were performed on the FACScan (Becton Dickinson, Franklin Lakes, NJ). Live gating of the forward and orthogonal scatter channels was employed to exclude debris and to selectively acquire lymphocytes events. All values presented are based on percent lymphocytes as determined by light scatter. Individual fluorescent populations were determined through the 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 buffer containing protease inhibitors, aprotinin (5 µg/ml), leupeptin (2 µg/ml), and PMSF (1 mM). Samples were placed on ice for 20 min with occasional vortexing, followed by centrifugation at 14,000 rpm for 15 min at 4°C.

To prepare cytoplasmic and nuclear extracts, PBL (1 x 107 cells) were harvested and washed with PBS at 4°C and then centrifuged at 1500 rpm for 5 min. The cell pellet was resuspended in 150 µl of buffer A (10 mmol/L HEPES (pH 7.9), 10 mmol/L KCl, 0.1 mmol/L EGTA, 0.1 mmol/L EDTA, 1 mmol/L DTT, 1 mM PMSF, 5 µg/ml aprotinin, 2 µg/ml leupeptin, 100 µg/ml pefabloc, and 100 µg/ml chymostatin). The cells were incubated on ice for 15 min, and then 10 µl of 10% Nonidet P-40 solution (Sigma) was added and cells were vigorously mixed for 20 s before centrifugation. The cytoplasmic extract was aliquoted and the nuclear pellet rinsed with hypotonic buffer A. Pelleted nuclei were resuspended in 60 µl 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, 5 µg/ml aprotinin, 2 µg/ml leupeptin, 100 µg/ml Pefabloc, and 100 µg/ml chymostatin) and rotated at 4°C for 20 min. The nuclear fraction was centrifuged at 14,000 rpm for 10 min at 4°C. Protein concentration was measured with a commercial kit (Pierce, Rockford, IL).

Equivalent amounts of protein from whole cell lysates, cytoplasmic, and nuclear fractions (10 µg) were mixed with equal volume of 2x Laemmli sample buffer, boiled, and resolved by electrophoresis in 7.5% and 14% SDS-PAGE. The proteins were transferred from the gel to a nitrocellulose membrane using an electroblotting apparatus (Bio-Rad, Richmond, CA) (15 V, 3 mA/cm2 for 27 min). Membranes were incubated in blocking solution containing 5% nonfat dry milk, in TBS overnight to inhibit nonspecific binding. The membranes were then incubated with specific Ab (1 µg/ml) for 1 h. After washing in Tris/0.1% Tween 20 for 30 min, membranes were incubated for another 30 min with HRP-conjugated secondary Ab. The membranes were then washed and developed with enhanced chemiluminescence (ECL Western Blotting Kit; Amersham).

EMSA

Nuclear extracts were prepared from T cells before and after stimulation with PMA/ionomycin (0, 0.5, and 2 h). Binding reactions were performed using 8 µg of nuclear protein preincubated on ice for 10 min in a 20-µl total reaction volume containing 20 mM HEPES (pH 7.9), 80 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 8% glycerol, and 2 µg of poly(dI-dC) (Pharmacia). The reaction mixture was then incubated with the radiolabeled oligonucleotide for 20 min at room temperature. Samples were analyzed by electrophoresis in a 6% nondenaturing polyacrylamide gel with 0.25 TBE buffer (22.2 mM Tris, 22.2 mM boric acid, 0.5 mM EDTA). Gels were vacuum-dried and exposed to film at -80°C.

Oligonucleotide containing a tandem repeat corresponding to the {kappa}B element of the IL-2R gene was used as the probe. Radiolabeled double-stranded probe was prepared by annealing a coding strand template to a complimentary 10 base primer and filling in the overhang using DNA polymerase I in the presence of [{alpha}-32P]dCTP. The sequence was 5'-CAACGGCAGGGGAATCTCCCTCTCCTT-3', and the underlined portion represents the {kappa}B binding motif. Radiolabeled oligonucleotide probes were prepared that correspond to NF-AT (5'-CGCCCAAAGAGGAAAATTTGTTTCATA-3') and AP-1 (5'-CGCTTGATGACTCAGCCGGAA-3') (Santa Cruz Biotechnology).

Measurement of caspases activity

Caspase and caspase-3 activity was measured using fluorometric tetrapeptide substrates. The assays were performed in 96-well plates by incubating 20 µg of cell lysates with 180 µl of reaction buffer (100 mmol/L HEPES (pH 7.5), 20% v/v glycerol, 5 mmol/L DTT, and 0.5 mmol/L EDTA) containing 50 µM YVAD-AMC, VEID-AMC, or DEVD-AMC (PharMingen, San Diego, CA). Release of 7-amino-4-methyl-coumarin (AMC) was monitored after 1 h of incubation at 37°C on a microplate fluorometer with excitation and emission wavelengths of 380 and 460 nm, respectively.

Measurement of apoptosis

Early apoptotic changes were identified by apoptosis detection kit with fluorescein-conjugated annexin V, which binds to exposed phosphatidylserine on the surface of apoptotic cells (47) according to protocol provided with 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 with the kit. Briefly, PBL were harvested, washed in PBS, and 1 x 106 cells were resuspended in 1% paraformaldehyde for 15 min on ice, washed twice with ice cold PBS, and fixed in 70% cold ethanol overnight. The fixed cells were washed twice in wash buffer, incubated with DNA labeling solution, followed by incubation with fluorescein-PRB-1 Ab solution and analysis by flow cytometry in the presence of propidium iodide/RNase solution.

DNA laddering as another measure of DNA fragmentation was determined by horizontal agarose gel electrophoresis using a previously published method (48).

Apoptosis was also determined by conventional light microscopy. Specifically, cytospin samples were assessed for the cellular and nuclear changes characteristically associated with apoptotic cell death (cell shrinkage, chromatin condensation, and karyorrhexis).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The SN50 peptide induces apoptosis in PBL

SN50 is composed of a nuclear localization sequence (NLS) for NF-{kappa}B1(p50) linked to the hydrophobic region of the signal peptide of Kaposi fibroblast growth factor, a cell-permeable carrier (43). SN50 blocks the intracellular recognition mechanism for the NLS on p50/p50 homodimers and heterodimers (p50/RelA) that inhibits their nuclear translocation in Jurkat T cell line (43, 49). To determine whether NF-{kappa}B activity might regulate the survival of human peripheral blood-derived T cells, we incubated resting T cells with varying concentrations of SN50 peptide for 24 h before performing the TUNEL assay. Concentration-dependent induction of DNA fragmentation by SN50 peptide is presented in Fig. 1GoA and illustrates that apoptosis is observed with a concentration of 25 µg/ml, although 75 µg/ml is most active. The level of apoptosis coincided with the degree of NF-{kappa}B inhibition mediated by different concentrations of SN50 peptide (Figs. 1Go and 7Go).



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  		FIGURE 1. SN50 peptide induces apoptosis in PBL. A, Highly enriched naive T cells were cultured in medium alone or treated with indicated concentrations of SN50 peptide for 24 h. The percentage of apoptotic cells was determined by TUNEL staining as indicated in Materials and Methods. B, DNA from T cells cultured in either medium or SN50 (75 µg/ml) for 24 h was isolated as described in Material and Methods and subjected to 1.5% agarose gel electrophoresis. The DNA was visualized by ethidium bromide staining. C, T cells were either untreated or cultured with 75 µg/ml of SN50 or SN50 M peptides for 24 h. Percentage of apoptotic cells was calculated as indicated in A.

 


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  		FIGURE 7. SN50 peptide inhibits NF-{kappa}B activation in the presence of normal degradation of the inhibitor, I{kappa}B{alpha}. A, Highly enriched naive T cells were cultured in medium alone or treated with indicated concentrations of SN50 peptide for 1 h before stimulation with PMA (20 ng/ml)/ionomycin (75 µg/ml). {kappa}B binding activity was detected in nuclear extracts by performing EMSA with a labeled {kappa}B probe. B, Aliquots of the same cells were subjected to Western blotting to demonstrate dose-dependent suppression of nuclear translocation of NF-{kappa}B/RelA by SN50 peptide. C, Immunoblotting for I{kappa}B{alpha} in cytoplasmic extract shown in B.

 
While the results described above suggested that inhibition of NF-{kappa}B nuclear translocation leads to induction of apoptosis, possible nonspecific effects of SN50 peptide were examined. To show that apoptotic activity of SN50 peptide is based on its effect on NF-{kappa}B nuclear translocation, T lymphocytes were incubated with 75 µg/ml of control SN50 M peptide, which has mutations in two residues within the nuclear localization sequence and does not prevent NF-{kappa}B nuclear translocation (43) (Fig. 7Go). In contrast to SN50, the mutant peptide did not induce apoptosis (Fig. 1GoC).

To define the time frame required for SN50-mediated apoptosis, naive T cells were treated with 75 µg/ml of SN50 peptide and harvested at various time points as indicated (Fig. 2Go). The induction of DNA fragmentation by SN50 peptide in T lymphocytes was noticeable after 6 h of incubation. The percentage of apoptotic cells continued to increase for up to 24 h following the addition of SN50 peptide.



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  		FIGURE 2. Time course of SN50 peptide induced apoptosis in naive T cells. T lymphocytes were cultured for the indicated time with or without 75 µg/ml of SN50 peptide. The percentage of apoptotic cells was determined by TUNEL staining as indicated in Material and Methods.

 
Additional experiments verified that SN50 does indeed induce DNA fragmentation in PBL. In one set, DNA fragmentation was confirmed by the demonstration of characteristic DNA laddering in agarose gel after T cells were cocultured for 24 h with the inhibitory peptide (75 µg/ml) (Fig. 1GoB). Morphological studies were also performed on T cells after exposure to either SN50 or TPEN, a Zn chelator that is known to induce apoptosis (50). As shown in Fig. 3Go, similar changes in morphology were noted when T cells were treated with either SN50 or TPEN. In both cases, changes characteristic for apoptotic cells were seen, and this included fragmentation of the nuclei as well as nuclear condensation.



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

 
SN50 treatment induces phosphatidylserine externalization in naive T lymphocytes

Previous studies demonstrated that phosphatidylserine is exported to the outer plasma membrane leaflet of apoptotic cells to serve as a trigger for recognition of apoptotic cells by phagocytes (51, 52, 53). Phosphatidylserine externalization is an early and widespread event occurring during apoptosis of various cell types. This process can be measured using annexin V, a protein with a high affinity for this lipid (47). Here, we determined whether the apoptosis induced in resting T cells by SN50 involved the externalization of phosphatidylserine. Using annexin V staining, low levels of phosphatidylserine were present on T cells cultured in medium. In contrast, incubation of PBL with SN50 peptide resulted in increased level of phosphatidylserine externalization (Fig. 4Go). These findings show that prevention of NF-{kappa}B nuclear localization in naive T cells can induce two critical events involved in apoptosis, phosphatidylserine externalization and DNA breaks.



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  		FIGURE 4. Annexin V binding in SN50-treated T cells. T lymphocytes were incubated either in the presence or absence of 75 µg/ml SN50 peptide for 24 h followed by staining with FITC-conjugated annexin V and analysis by flow cytometry as described in Materials and Methods.

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

Recent studies have identified members of the family of caspase proteases (formerly ICE/CED-3 proteases) as key participants in apoptosis that act upstream of endonucleases (22, 23). It is well documented that a variety of apoptotic agents induce sequential activation of caspases in different cell types (21, 22, 23, 24, 25, 26, 27, 28, 29, 30). It has also been shown that activation of ICE/CED-3 family proteases is required for phosphatidylserine externalization during CD95-induced apoptosis (54). To assess the potential involvement of caspase family members in apoptosis induced by inhibition of NF-{kappa}B nuclear translocation, we tested whether the caspase inhibitors Z-VAD-fmk and Ac-DEVD-fmk would prevent DNA breaks induced by SN50. Z-VAD-fmk is considered a general caspase inhibitor, while the DEVD sequence inhibits primarily caspase-3, although caspase-6, -7, -8, and -10 are also affected (55). Six experiments were performed, and a representative experiment is presented in Fig. 5GoA. Pretreatment with Z-VAD-fmk efficiently blocked SN50-mediated apoptosis (mean 87 ± 8.9% SD reduction, n = 6), whereas Ac-DEVD-fmk partially blocked DNA breaks (mean 57 ± 29.6% SD reduction, n = 6). As a control for both inhibitors, we tested their ability to block Fas (CD95)-mediated apoptosis of the Fas-sensitive Jurkat T cell line. Treatment with soluble Fas ligand (FasL) (CD95L) (100 ng/ml) for 24 h induced apoptosis in Jurkats (48% apoptotic cells); however, Z-VAD-fmk as well as Ac-DEVD-fmk were effective at blocking Fas-mediated killing (Fig. 5GoB). The differential ability of Ac-DEVD-fmk to block apoptosis mediated by SN50 and FasL suggests that there may be differences in the caspases involved in the death pathways induced by these two agents.



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  		FIGURE 5. Induction of DNA breaks by SN50 peptide treatment is blocked by caspase inhibitors. A, Naive T cells were incubated with and without 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 TUNEL assay. 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 mentioned in A. Cells were then assayed for DNA breaks by TUNEL.

 
To determine which caspases are activated as a result of suppressing NF-{kappa}B, we examined lysates from SN50-treated T cells for their ability to cleave YVAD-AMC, DEVD-AMC, and VEID-AMC fluorogenic substrates. Enzymatic activity was monitored after 6 and 24 h of T cell treatment with 75 µg/ml SN50 peptide. As shown in Fig. 6GoA, there was no cleavage of these substrates by lysates of T lymphocytes treated with SN50. Immunoblotting with the same lysates demonstrated that there was no change in the expression levels of either caspase-1 or caspase-3 before and after stimulation, and there was no evidence of cleavage products that are present following the activation of these two proteins (data not shown). In parallel, cleavage of PARP protein, which is one of the well-established intracellular substrates for caspase-3 (31, 32), was studied by immunoblotting (Fig. 6GoB). No proteolytic cleavage of PARP protein was detected in T cells treated with 75 µg/ml SN50 peptide. There was also no detectable caspase-3-dependent cleavage of DFF-45 (Fig. 6GoC). However, with the same cells, caspase activation was demonstrated after 24 h of treatment with TPEN, as evident by cleavage of all three fluorogenic substrates. Moreover, there was detectable cleavage of PARP and DFF-45 in TPEN-treated cells (Fig. 6Go). The activation of caspase enzymatic activity was also easily detected in Jurkats cells stimulated with FasL (V. Kolenko, unpublished observations). These experiments further suggest that apoptosis of naive T lymphocytes mediated by inhibition of NF-{kappa}B nuclear translocation may involve distinct set of caspases than those induced by TPEN and FasL.



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

 
Bcl-2, Bcl-xL, and Bax proteins are involved in the regulation of cell susceptibility to apoptosis where Bcl-2 or Bcl-xL inhibits apoptosis, while Bax promotes apoptosis (40, 56, 57). We examined the levels of Bcl-2, Bcl-xL, and Bax proteins in naive T cells cultured with or without SN50 peptide to determine whether the levels are modulated in a way that suggests a role for these proteins in regulating SN50-mediated apoptosis. Recent studies have shown that resting PBL express high levels of Bcl-2 and Bax proteins with no detectable expression of Bcl-x (56, 58). In agreement with this observation, we found high levels of Bcl-2 and Bax protein in resting T lymphocytes that did not vary after 24 h of cell treatment with 75 µg/ml SN50 peptide (data not shown). At this time, >60% of the T cell population treated with SN50 had undergone apoptosis as determined by TUNEL assay. No detectable level of either Bcl-xL or Bcl-xS proteins was observed in naive T lymphocytes cultured in medium alone or in the presence of SN50 peptide.

Induction of apoptosis in T cells is attributable to suppression of {kappa}B binding activity

Here, we document that the SN50 peptide inhibits the nuclear localization of NF-{kappa}B in peripheral blood-derived T cells. Incubation for 1 h with SN50 (50 and 75 µg/ml) reduced the background level of {kappa}B binding observed in resting T cells (Fig. 7Go). It also inhibited the increase in {kappa}B binding activity following 2 h of stimulation with PMA/ionomycin. SN50 M that had 2 of 10 NLS residues mutated did not inhibit DNA binding activity. Immunoblotting (Fig. 7GoB) confirmed that SN50 treatment, in a concentration-dependent manner, prevented nuclear localization of RelA and p50 after stimulation, however it had no effect on the cytoplasmic levels of these proteins. At 75 µg/ml, SN50 also eliminated the background level of Rel proteins that are present in the nuclei of resting cells. These experiments also demonstrated that SN50 mediates its effect on NF-{kappa}B dimers following normal degradation of the inhibitor I{kappa}B{alpha} (Fig. 7GoC).

We wanted to know if the induction of apoptosis in normal resting T cells was linked to the defect in NF-{kappa}B activation. Therefore, we initially tested whether SN50 was selective at blocking NF-{kappa}B activation without altering nuclear localization of other transcription factors. At 75 µg/ml, SN50 inhibited binding of nuclear extracts to AP-1, NF-AT, as well as the {kappa}B probe, which is consistent with a recent report where 210 µg/ml of SN50 prevented nuclear localization of multiple transcription factors (49). However, at 37.5 µg/ml (n = 3), SN50 appeared selective in that {kappa}B binding activity was blocked, whereas AP-1 and NF-AT binding activity was unaffected (Fig. 8Go). In the same experiments, where only NF-{kappa}B activation was suppressed, there was consistent induction of apoptosis, suggesting that defective NF-{kappa}B is responsible for the initiation of apoptosis in SN50-treated T lymphocytes.



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  		FIGURE 8. SN50 inhibited NF-{kappa}B binding activity without affecting AP-1 and NF-AT DNA binding activity. A, T cells were treated with and without SN50 (37.5 µg/ml) before stimulating for 2 h with PMA/ionomycin. Nuclear extracts were then subjected to EMSA using labeled probes that correspond to AP-1, NF-{kappa}B, and NF-AT sequences as described in Material and Methods. B, The same cells as described in A were examined after 24 h for DNA breaks by TUNEL.

 
We also determined whether another agent known to suppress NF-{kappa}B activation would also induce DNA breaks in T cells and thus reproduce the results observed with SN50 peptide. We used N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), since it has been shown to inhibit {kappa}B binding activity (18). Pretreatment with TPCK (100 µM) prevented PMA/ionomycin induction of NF-{kappa}B (Fig. 9GoA). TPCK appears to function by interfering with proteosome activity, which blocks the stimulus-dependent phosphorylation and degradation of I{kappa}B{alpha} (18) (Fig. 9GoB). We show here that suppression of NF-{kappa}B activation by TPCK can result in the induction of DNA breaks (TUNEL assay) (Fig. 9GoC) within 24 h, suggesting that inhibition of {kappa}B binding activity through distinct mechanisms can induce the death pathway in naive T cells. Similar results were also obtained with a new BAY 11–7082 inhibitor that selectively blocks I{kappa}B{alpha} degradation (59).



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  		FIGURE 9. Suppression of NF-{kappa}B activation by TPCK can induce DNA breaks in naive T cells. A, T cells were incubated in medium or medium supplemented with TPCK (100 µM) for 1 h before stimulation with PMA/ionomycin for the times indicated. Nuclear extracts were prepared and assessed for {kappa}B binding activity by EMSA. B, With the same samples used in A, cytoplasmic extracts were analyzed by immunoblotting for I{kappa}B{alpha} expression. C, The same cells as described in A were examined after 24 h for DNA breaks by TUNEL.

 
Preactivation promotes survival of T lymphocytes treated with SN50 peptide

We also determined whether preactivation of NF-{kappa}B by external stimuli would alter the sensitivity of T cells to SN50-mediated apoptosis. Within 15 min of T cell activation, there is a significant increase in the nuclear localization of RelA, c-Rel, and p50 dimers (55). This translocation of Rel proteins coincides with an increase in {kappa}B-specific DNA binding activity, where the peak activity occurs within 2 h of stimulation (60). Preactivation of T cells with PMA/ionomycin for 2 h completely blocked DNA fragmentation induced by a 24-h exposure to SN50 (Fig. 10Go). In the same cells that were not preactivated with PMA/ionomycin, SN50 induced significant apoptosis (48%). Similar results were observed with IL-2, which is also known to activate NF-{kappa}B. These results show that prior activation and nuclear translocation of NF-{kappa}B can protect cells from apoptosis mediated by SN50. However, the protective effect of preactivation is eventually reversed by continued exposure to SN50 after 3 days through the inhibition of further NF-{kappa}B activation (our unpublished observations).



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

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The involvement of NF-{kappa}B in regulating the apoptotic response has been suggested previously by several groups (1, 14, 15, 16, 17, 18, 19). Inhibition of NF-{kappa}B nuclear translocation increased the susceptibility of cells to undergo apoptosis induced by TNF-{alpha}, ionizing radiation, and cancer chemotherapeutic drugs (17). The suppression of NF-{kappa}B activation by protease inhibitors that block I{kappa}B{alpha} degradation induced apoptosis in murine splenic B cells (18, 19). Similar findings were also reported following the microinjection of either GST-I{kappa}B{alpha} fusion protein or an Ab to c-Rel (18, 19). In contrast, ectopic expression of c-Rel was found to ablate the induction of apoptosis induced in B cells following suppression of NF-{kappa}B activation by inhibitors of I{kappa}B{alpha} degradation (18, 19). These findings point to a critical role of NF-{kappa}B family members in the protection of cells against various forms of apoptotic cell death.

The data presented here shows that blocking nuclear translocation of NF-{kappa}B dimers by the cell-permeable peptide, SN50, induced apoptosis in normal peripheral blood-derived T lymphocytes. In contrast, the inactive control SN50 M mutant peptide had no inhibitory effect on either {kappa}B binding activity or cell viability. Whether suppression of NF-{kappa}B activation is responsible for the initiation of apoptosis in peripheral T cells is supported by the observation that, at the SN50 concentration of 37.5 µg/ml, induction of apoptosis coincided with selective suppression of {kappa}B binding activity as evident by normal nuclear localization of other transcription factors, such as AP-1 and NF-AT. This conclusion is also supported by the fact that suppression of NF-{kappa}B activation by another mechanism (TPCK) also induced DNA breaks.

Given the finding that SN50-mediated apoptosis was observed in resting T cells leaves open the possibility that low levels of constitutive {kappa}B binding activity are required to maintain cellular viability in this lymphoid population. As noted by Western blotting and by gel mobility shifts assays (Figs. 7Go and 8Go) (60), there are low levels of NF-{kappa}B1 and RelA expression in the nucleus without any external stimulation. The fact that SN50 blocked nuclear translocation of p50 and RelA and subsequently induced apoptosis is consistent with the possibility that the constitutive expression of NF-{kappa}B in resting T cells promotes survival.

Susceptibility of T cells to apoptosis appears to be linked to the state of their activation. This conclusion is supported by previously published data that shows mitogen activation of T cells enhances their resistance to {gamma}-irradiation (56). In agreement with this idea, our study shows that preactivation of naive T cells with either PMA/ionomycin or IL-2 completely blocked DNA fragmentation induced by SN50 peptide. Cells needed to be preactivated for at least 2 h before their exposure to SN50 peptide to become resistant to SN50-mediated apoptosis. The kinetics of induction of resistance paralleled the appearance of {kappa}B DNA binding activity (our unpublished observations). It may be that NF-{kappa}B activation results in transcriptional up-regulation of genes encoding proteins involved in protection against apoptosis. The protective product may be distinct from Bcl-2, since SN50 inhibited NF-{kappa}B activation and induced apoptosis even in the presence of significant levels of Bcl-2. Whether overexpression of anti-apoptotic proteins, such as Bcl-xL, can protect T cells from apoptosis induced by blocking NF-{kappa}B nuclear translocation is currently under investigation.

Activation of caspase proteases was previously shown to be required for the induction of apoptosis in different cell types (21, 22, 23, 24, 25, 26, 27, 28, 29, 30). Our findings with the caspase inhibitors Z-VAD-fmk and Ac-DEVD-fmk are consistent with the possibility that SN50-mediated apoptosis in T cells is caspase-dependent. However, there appears to be a difference in either the level or types of caspases induced as a consequence of NF-{kappa}B suppression by SN50 peptide when compared with other inducers of apoptosis, such as TPEN or FasL. In contrast to FasL or TPEN, the SN50 peptide did not induce detectable enzymatic activity of caspase-1-, caspase-3-, or caspase-6-like proteases using fluorogenic peptide substrates, YVAD-AMC, DEVD-AMC, and VEID-AMC, respectively. Although both caspase-1 and caspase-3 were constitutively expressed in precursor forms, we did not detect processed subunits of these proteins in lysates from T cells treated with SN50 peptide, further suggesting they were not activated by SN50. In support of this conclusion is the observation that no proteolytic cleavage of either PARP or DFF-45, well-established substrates for caspase-3, was detected in T cells treated with SN50 peptide. These data suggest that caspase-1, caspase-3, and caspase-6 may not be the primary caspases required for apoptosis induced by inhibition of NF-{kappa}B nuclear translocation in T cells. Additional studies are needed to identify the caspase pathway involved in SN50-mediated apoptosis.

In certain pathological conditions, such as cancer, down-regulation of NF-{kappa}B activity may represent a mechanism for inhibiting the development of T cell immune responses. Impaired activation of NF-{kappa}B has been reported in T cells derived from tumor-bearing mice and cancer patients (60, 61). Furthermore, products present in the tumor environment, such as IL-10, gangliosides, and prostaglandin E2, are known to inhibit NF-{kappa}B activation and down-regulate immune responses (62, 63, 64). The blocking of NF-{kappa}B translocation may make T cells more susceptible to apoptosis. The evidence presented here suggests that the prevention of nuclear expression of NF-{kappa}B dimers can induce apoptosis in T lymphocytes.


   Footnotes
 
1 This work was supported by U.S. Public Health Service Grant CA56937. Back

2 Address correspondence and reprint requests to Dr. Vladimir Kolenko, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195. E-mail address: Back

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

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


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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