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The Journal of Immunology, 2000, 165: 1743-1754.
Copyright © 00 by The American Association of Immunologists

The NF-{kappa}B Cascade Is Important in Bcl-xL Expression and for the Anti-Apoptotic Effects of the CD28 Receptor in Primary Human CD4+ Lymphocytes1

Ali Khoshnan*, Charles Tindell*, Isett Laux*, David Bae*, Brydon Bennett{dagger} and Andre E. Nel2,*

* Division of Clinical Immunology and Allergy, Department of Medicine, University of California, Los Angeles, CA 90095; and {dagger} Signal Pharmaceuticals, San Diego, CA 92121


    Abstract
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We explored the role of the NF-{kappa}B pathway in the survival of primary human CD4+ T lymphocytes during CD28 costimulation. Transduction of proliferating CD4+ T cells with a tetracycline-regulated retrovirus encoding for a dominant-interfering, degradation-resistant I-{kappa}B{alpha} (inhibitor of {kappa}B{alpha} factor) mutant induced apoptosis. Using DNA arrays, we show that Bcl-xL features as a prominent anti-apoptotic member among a number of early CD28-inducible genes. A 1.2-kb segment of the proximal Bcl-xL promoter, linked to a luciferase reporter, responded to CD3/CD28 stimulation in Jurkat cells. Mutation of an NF-{kappa}B site around -840 decreased, while ectopic expression of I-{kappa}B kinase-ß (IKKß) enhanced reporter gene activity. Na+-salicylate and cyclopentenone PGs, direct inhibitors of IKKß, interfered in the activation of the Bcl-xL promoter and induced apoptosis in CD28-costimulated CD4+ T cells. Moreover, salicylate blocked nuclear localization of NF-{kappa}B factors that bind to the NF-{kappa}B binding site in the Bcl-xL promoter, as well as the expression of Bcl-xL protein. HuT-78, a lymphoblastoid T cell line with constitutive NF-{kappa}B activity, contained elevated levels of Bcl-xL protein and, similar to proliferating CD4+ T cells, was resistant to apoptotic stimuli such as anti-Fas and TNF-{alpha}. In contrast, the same stimuli readily induced apoptosis in a Jurkat T cell clone with no detectable Bcl-xL expression. Jurkat BMS2 cells also differed from HuT-78 in collapse of mitochondrial membrane potential and superoxide generation in the mitochondrium. Taken together, these data demonstrate that CD3/CD28-induced activation of IKKß and expression of Bcl-xL promote the survival of primary human CD4+ T lymphocytes.


    Introduction
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Homeostasis in the peripheral T cell compartment is dependent on a complex network of cell surface receptors, cytoplasmic signaling components, and transcription factors that regulate growth, differentiation, and apoptosis of these cells (1, 2, 3). We are particularly interested in the role of the NF-{kappa}B cascade on genes that regulate above biological responses in mature T cells. In this regard, we have recently shown in primary human CD4+ lymphocytes that the TCR is critically dependent on the CD28 accessory receptor for the activation of the multisubunit inhibitor of {kappa}B (I-{kappa}B)3 kinase (IKK) complex, which induces I-{kappa}B{alpha} and I-{kappa}Bß phosphorylation (4, 5). Phosphorylation of these inhibitory proteins leads to degradation and the release of Rel/NF-{kappa}B proteins from the cytosol to the nucleus (6, 7). IL-2 is among the genes that are induced by NF-{kappa}B transcription factors and contains key NF-{kappa}B regulatory sites in its promoter (4, 5, 8). While IL-2 plays a role in the effect of the CD28 receptor on T cell growth and prevention of anergy, CD28 also assists in the expression of genes that regulate cell viability (9, 10, 11, 12, 13). In this communication, we will focus on the role of the CD28 receptor and the NF-{kappa}B cascade in preventing activation-induced cell death in human T lymphocytes.

The Rel/NF-{kappa}B family of proteins has diverse and opposing effects on apoptosis regulation in different tissues (14, 15, 16, 17, 18, 19). While, for instance, Rel/NF-{kappa}B factors suppress apoptosis in immature B lymphocytes and developing thymocytes, c-Rel is highly expressed in cells undergoing apoptosis in developing embryos (20, 21). However, analysis of c-Rel-deficient mice does not support an obligatory role for c-Rel in apoptosis (22). While there are no definitive studies looking at the role of NF-{kappa}B pathway in human peripheral blood T lymphocytes, murine studies indicate that the NF-{kappa}B pathway has anti-apoptotic effects in mature T lymphocytes (1, 23). Included among the anti-apoptotic genes that may be NF-{kappa}B regulated and that have demonstrated links to the CD28 receptor is Bcl-xL (24, 25). Both the human and mouse Bcl-xL promoters contain NF-{kappa}B binding sites that have been shown to be activated in B cell lines by CD40 or in a T cell line by Tax overexpression (26, 27).

We wished to determine which apoptosis-related genes are induced by CD28 costimulation in primary human CD4+ lymphocytes, and whether activation of the NF-{kappa}B pathway plays a role in the expression of anti-apoptotic genes. We show through the use of cDNA expression arrays that while CD28 costimulation is associated with early expression of several pro-apoptotic genes, there is prominent expression of only one anti-apoptotic gene, namely Bcl-xL. Interference in the NF-{kappa}B cascade in CD4+ lymphocytes by a retrovirally expressed, degradation-resistant I-{kappa}B{alpha} mutant or inhibitory drugs induced apoptosis in parallel with decreased Bcl-xL expression. Furthermore, NF-{kappa}B-mediated activation of Bcl-xL in CD4+ T cells is influenced by IKKß function. CD3/CD28 stimulation as well as ectopic expression of IKKß regulated Bcl-xL promoter activity. In addition, Bcl-xL expression correlated with resistance of proliferating CD4+ T cells to a variety of apoptotic stimuli. This indicates that CD3/CD28-mediated activation of IKKß and subsequent expression of Bcl-xL may coordinate the life span of CD4+ T cells.


    Materials and Methods
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Reagents

OKT3 (anti-CD3) was obtained from Ortho Pharmaceuticals (Raritan, NJ), and the 9.3 mAb (anti-CD28) was generously provided by Bristol-Meyer Squibb (Princetown, NJ). For the purpose of cellular activation, these primary stimulating Abs were cross-linked with mAb 187.1 (Bristol-Meyer Squibb). Anti-Bcl-xL Abs and human TNF-{alpha} were purchased from PharMingen (San Diego, CA), while anti-cIAP2 goat Abs were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-Flag (M2) Abs were obtained from Sigma (St. Louis, MO). HRP conjugated to protein A was from Amersham (Arlington Heights, IL). Tosyl-activated magnetic beads and M-450 anti-CD4 beads were purchased from Dynal (Great Neck, NY). PMA (P), ionomycin (I), pyrrolidine-dithiocarbamate, and sodium salicylate (Na+) were from Sigma. Anti-Fas mAb (CH11) was obtained from MBL (Nagoya, Japan). The annexin V/propidium iodide (PI) staining kit system was purchased from Trevigen (Gaithersburg, MD). Retronectin was from Takara Biomedicals (Otsu, Shiga, Japan), and 3,3' dihexyloxacarbocyanine iodide (DiOC6) and hydroethidine (HE) were obtained from Molecular Probes (Eugene, OR). Staurosporine and cycloheximide were purchased from ICN (Costa Mesa, CA). Prostaglandins PGA1, PGB2, and 15-deoxy-{Delta}12–14-PGJ2 (15dPGJ2) were purchased from Cayman Chemical (Ann Arbor, MI).

Cell culture and preparation of primary CD4+ T cells

Jurkat (clone BMS2) and HuT-78 T cell lines were grown in RPMI medium, supplemented with 10 mM HEPES, 10% FCS, 2 mM glutamine, 100 U penicillin, and 100 µg streptomycin/ml. The 293-T epithelial cells were cultivated in DMEM containing 10% FCS, 2 mM glutamine, 100 U penicillin, and 100 µg streptomycin/ml. For preparation of CD4+ T cells, mononuclear cells were isolated from human peripheral blood by density centrifugation and depletion of adherent cells on plastic culture dishes. CD4+ T cells were positively selected with anti-CD4 Dynabeads according to manufacturer’s instructions (Dynal). This yielded a T cell subset that was >98% positive for the CD4 marker, as determined by dual color CD4/CD8 flow cytometry. Isolated CD4+ T cells were stimulated with anti-CD3 and anti-CD28 mAb coupled to tosyl-activated magnetic beads, as previously described (5, 28). Cells were replenished with 40 U/ml rIL-2 (Chiron, Emeryville, CA) on day 3, and allowed to complete their growth cycle over the course of 12–13 days. At this point, IL-2 and beads were removed, and cells allowed to return to their resting state over a 48-h time period. Resting vs activated state was assessed by cell size analysis in a Coulter counter (Coulter Pharmaceutical, Palo Alto, CA). Cells were subjected to flow cytometry for a second time to confirm that >95% of the cells remained CD4+.

Construction and preparation of a tetracycline (tet)-regulated DN/I-{kappa}B{alpha}-expressing retroviral vector

The mutant I-{kappa}B{alpha} construct, with mutational alteration of Ser32 and Ser36 to Ala residues, has been previously described (7). The Hermes HRIgfp tet-regulated retroviral vector was kindly provided by Dr. Helen Blau (Stanford University, Palo Alto, CA) (29). The backbone, in addition to long terminal repeats and packaging signal, contains six copies of a tet operon (Tet O) linked to a minimal CMV promoter (PhCMV-1) (see Fig. 1GoA). Green fluorescent protein (GFP) is expressed from an internal ribosome entry site inserted downstream of the multiple cloning site (29). N-terminal Flag-tagged DN/I-{kappa}B{alpha} generously provided by Dr. G. Cheng (University of California at Los Angeles), was directionally cloned into the HpaI-BamHI site of the Hermes vector. In-frame insertion was confirmed by DNA sequencing (see Fig. 1GoA). The second retroviral vector (pREV Tet-off) encoding for the tet-repressible transactivating (tTA) protein was obtained from Clontech (Palo Alto, CA). Individual retroviruses were produced in the 293-Ampho (American Type Culture Collection, Manassas, VA) packaging cell line. Briefly, packaging cell lines were plated in 10-cm dishes at a confluency of ~70% and transfected with 20 µg of the Hermes or pREV Tet-off vector by calcium-phosphate precipitation procedures in the presence of 25 µM chloroquine. After overnight incubation at 37°C, the culture media were replaced with 6 ml of T cell growth medium and transferred to 32°C. Viral supernatants were harvested 48 h posttransfection, cleared by centrifugation at 2500 rpm for 5 min, and either stored in aliquots at -80°C or used to infect cells. The pREV Tet-off viral titer was determined by selecting neomycin-resistant colonies in NIH 3T3 cells and adjusted to a final concentration of 5–8 x 106 infectious particles/ml. Although a quantitatively exact titer could not be obtained for the DN/I-{kappa}B{alpha} viral stocks, we used supernatants from 293-Ampho cultures that were >70% positive for GFP expression.



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FIGURE 1. Tet-regulated expression of DN/I-{kappa}B{alpha} in 293-T and Jurkat T cells transduced with recombinant retroviruses. A, Physical map of the tTA and DN/I-{kappa}B{alpha} retroviral vectors. B, Western blot analysis of lysates from 293-T and Jurkat cells transduced with the tTA and DN/I-{kappa}B{alpha} retroviruses in the presence and absence of tet. The 293-T and Jurkat cells were sequentially infected with both retroviruses, as described in Materials and Methods. A total of 50 µg of each lysate was examined for DN/I-{kappa}B{alpha} expression by Western blot analysis using anti-Flag Ab (1:1000), followed by sheep anti-mouse (1:2000) conjugated to HRP. Reactive proteins were detected by enhanced chemiluminescence (ECL). The arrow points to the 44-kDa DN/I-{kappa}B{alpha} protein. Longer exposure did show a faint band of similar size in samples incubated with tet (not shown).

 
Assessment of the functionality of the retroviral supernatants

tTA and DN/I-{kappa}B{alpha} encoding viral supernatants were tested in 293-T cells and Jurkat lymphoblastoid cells. The 293-T epithelial cells were seeded in six-well plates to ~70% confluency and sequentially infected with the pREV and Hermes viral supernatants. Briefly, 1 ml of each supernatant was diluted 1/1 with complete DMEM containing 4 µg/ml of polybrene and added to the cells. The virus was centrifuged onto the cells at 2200 rpm for 90 min at 37°C. Cells were rested for 6 h between infections. Infected cells were grown in the absence or presence of 5 µg/ml tet. The infection efficiency of 293-T cells was about 60%, as determined by GFP expression. Jurkat cell infection followed the same guidelines, with slight modification. Briefly, 2 x 105 Jurkat cells were mixed with 1 ml of each viral supernatant, diluted 1/1 in RPMI containing 4 µg/ml of polybrene, transferred to 24-well plates, and centrifuged at 2200 rpm for 90 min. The infection efficiency of Jurkat cells was about 40%, as assessed by flow cytometry. An emission setting of 488 nm was used to detect GFP.

Transduction of primary CD4+ T cells

Primary CD4+ T cells were amplified by CD3/CD28 costimulation, as described above. Three days postamplification, magnetic beads were removed and cells were transferred to 24-well plates coated with Retronectin (Takara Biomedicals) and incubated for 2 h. A total of 1 ml of viral supernatant, diluted 1/1 in T cell-conditioned medium, was added to cells and further centrifuged at 2200 rpm for 90 min. Cells were sequentially treated with the viral supernatants, using three rounds of infection over a 72-h time period in the presence of 5 µg/ml tet and 200 IU/ml of IL-2. On the fourth day postinfection, cells were washed three times in T cell culture medium, and incubated in the presence or absence of 5 µg/ml tet. The percentage of infected T cells was analyzed by flow cytometry, as described above.

Gene expression array

Total cytoplasmic RNA was isolated as described (30). The RNA pellet was resuspended in water and quantitated by absorbance at 260 nm. Approximately 200 µg of cytoplasmic RNA was used to isolate poly(A) RNA using Oligotex resin, as per the manufacturer’s instructions (Qiagen, Valencia, CA). Poly(A) RNA was eluted with 2 x 40 µl elution buffer, and the entire solution was quantitated at 260 nm. Typical recovery was 1.5–3 µg. The poly(A) RNA was precipitated with 95% ethanol in the presence of 0.3 M sodium acetate, pH 5.2, and 4 µg of glycogen. The pellet was washed with 75% ethanol, dried, and resuspended in water to a concentration of 0.5 µg/ml poly(A) RNA. The poly(A) RNA (1.5 µg) was used to produce a complex cDNA probe for screening of the Atlas Human cDNA Expression Array (Clontech), as described in the manufacturer’s protocol. The gene filters were sealed in plastic and the image data collected with a PhosphorImager and ImageQuant software (Molecular Dynamics, Sunnyvale, CA). Spot labeling, hit picking, and densitometric analysis were performed using a series of Excel-based macros developed at Signal Pharmaceuticals (San Diego, CA).

Apoptosis assays

Apoptosis in primary CD4+, HuT-78, and Jurkat cells was assessed by dual color annexin V/PI staining. Apoptosis was assessed in primary T cells 48 h after removal of tet from retrovirus-infected cells or after incubation for 16 h in the presence of sodium salicylate, prostaglandin PGA1, PGB2, and 15dPGJ2 at the indicated concentrations. Apoptosis was also assessed in all three cell types 16 h after the introduction of 100 ng/ml anti-Fas mAb, 50 ng/ml TNF-{alpha} (in the presence of 10 µg/ml cycloheximide), or 1 µM staurosporine. Cells were incubated with 1 µl annexin V plus 10 µl PI in 500 µl binding buffer for 15 min, as recommended by the manufacturer. Flow cytometry was performed using a FACScan (Becton Dickinson, Mountain View, CA), equipped with a single 488-nm Argon laser. Annexin V-FITC and PI were analyzed at emission settings of 535 and 575 nm, respectively. Dual color staining for mitochondrial membrane potential ({Delta}{varphi}m) and superoxide production was performed by DiOC6(3) and HE staining (31). Briefly, 1 x 106 cells were incubated with 20 nM DiOC6 and 2 µM HE for 30 min at 37°C in the dark. Cells were subjected to flow cytometry using emission settings of 501 nm for DiOC6(3) and 605 nM for HE.

EMSA

Gel-shift analysis of nuclear extracts from primary CD4+ T cells and T cell lines was performed as previously described (5). A total of 6 x 107 resting primary CD4+ T cells or Jurkat was left untreated or incubated in the presence of 20 mM sodium salicylate for 2 h before stimulation for 30 min with anti-CD3 mAb or a combination of anti-CD3 plus anti-CD28 mAbs, secondarily cross-linked with 187.1 mAb. Cells were immediately washed and processed for extraction of nuclear extracts. A double-stranded oligonucleotide containing the putative NF-{kappa}B binding site at positions -840 to -848 of the murine Bcl-xL promoter (26) (5'-cgatAAAGGGACTTCCAAGat-3' and 3'-taTTTCCCTGAAGGTTCtagc-5') was end labeled with the Klenow polymerase in the presence of [32P]GTP and [32P]dCTP. DNA binding was performed at room temperature for 20 min in a final volume of 20 µl, which contained 5 µg of nuclear extract, 5 x 104 cpm labeled oligonucleotide, and binding buffer containing 100 µg/ml of poly(dI-dC) as nonspecific competitor. Shift complexes were resolved on nondenaturing 5% polyacrylamide gel in 0.5x TBE. Specific competition was performed with a 50-fold excess of the unlabeled probe as well as Abs to p50 and p65, as previously described (26).

Immune complex kinase assays

A total of 4 x 107 resting CD4+ T cells was treated with 20 mM sodium salicylate for 2 h before stimulation for 30 min with 2 µg/ml anti-CD3 or a combination of 2 µg/ml anti-CD3 + 2 µg/ml anti-CD28 mAb, secondarily cross-linked with 10 µg/ml mAb 187.1. Stimulation with PMA (100 nM) plus ionomycin (1 µg/ml) was used as a positive control. After cell lysates were precleared with protein A-Sepharose beads, 200 µg lysate was treated with 2 µg anti-IKKß absorbed onto protein A-Sepharose beads for 2 h. Immune complexes were washed and equilibrated in kinase buffer, as described (32). Kinase reactions were initiated by the addition of 10 µCi [{gamma}-32P]ATP and 3 µg GST-I-{kappa}B{alpha} (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54) substrate. The reaction was conducted for 30 min at 30°C. Products were analyzed on SDS-PAGE and detected by autoradiography.

Luciferase assays

A 1.2-kb Bcl-xL promoter element, cloned upstream of a luciferase gene (pGL2 vector), was kindly provided by Dr. G. Nunez (University of Michigan, Ann Arbor, MI) (33), while wild-type (pGL848) and NF-{kappa}B mutant (pGL2{kappa}BM) versions of a 848-bp luciferase reporter were a generous gift from Dr. Mari Kannagai (Tokyo Medical and Dental University, Japan) (26). A total of 12 µg of the indicated reporter gene constructs was transiently transfected into 107 Jurkat cells in the presence or absence of indicated amounts of IKKß (34). The cells were rested for 24 h and then stimulated for 6 h with a combination of 2 µg/ml anti-CD3 + 2 µg/ml anti-CD28 mAb, secondarily cross-linked with 187.1 mAb. Where indicated, transfectants were incubated in the presence of 20 mM sodium salicylate or 20 µM 15dPGJ2 prior to stimulation. Control stimulation was with 100 nM PMA + 1 µg/ml ionomycin. The cells were lysed in luciferase buffer (Analytical Luminescence, Ann Arbor, MI), and luciferase activity was measured in 50 µg of lysate in a Monolight 2010 Luminometer (Analytical Luminescence). Transfection efficiency was monitored by cotransfection of a ß-galactosidase plasmid (CMV-ß-gal); ß-galactosidase activity was used for adjusting luciferase values between different transfected cell populations.

Western blot analysis

A total of 50 µg of cellular lysate was separated by SDS-PAGE and transferred to nitrocellulose membranes, as previously described (5). Membranes were sequentially overlaid with specific primary and secondary Abs conjugated to HRP, as indicated in the figure legends. Products were detected by enhanced chemiluminescence (ECL).


    Results
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Tet-regulated expression of a dominant-interfering I-{kappa}B{alpha} mutant leads to apoptosis in primary human CD4+ T cells

Coculture of primary human CD4+ lymphocytes with anti-CD3 plus anti-CD28 mAb coimmobilized on beads induces a prolonged (12–16 days) proliferative cycle during which the majority of CD4+ T cells are protected from activation-induced cell death by a signal imparted via the CD28 receptor (25, 28). This signal may involve the NF-{kappa}B cascade, which is activated in a synergistic fashion by TCR and CD28 costimulation (5). We asked whether the introduction of a degradation-resistant I-{kappa}B{alpha} mutant will affect the survival of CD3/CD28-stimulated human CD4+ lymphocytes. That goal was accomplished by using DN/I-{kappa}B{alpha} expression by a tet-regulated system that uses a combination of the pREV Tet-off and Hermes HRIgfp retroviruses. The former virus encodes for a tTA protein, while the latter expresses a copy of I-{kappa}B{alpha}(32A/36A) downstream a heptamerized tet operator (Fig. 1GoA) (29). The Hermes virus also coexpresses GFP, which allows the tracking of infected cells by fluorescence microscopy or flow cytometry (Fig. 1GoA). The efficiency of these retroviruses was demonstrated in 293 and Jurkat cells, in which tet removal from the culture medium could be seen to induce Flag-tagged DN/I-{kappa}B{alpha} (Fig. 1GoB) and GFP (not shown) expression. These infection conditions were used to develop optimal conditions for infection of primary lymphocytes.

In experiments in which these retroviruses were used to infect CD3/CD28-stimulated CD4+ blasts, we observed GFP expression in >40% of the tet(-) cells (Fig. 2GoA). In contrast, only 7% of tet(+) cells expressed GFP (Fig. 2GoA). When the same populations were used to assess cell viability by flow cytometry, we could demonstrate an increase from 9 to 63% in the percentage of annexin V+/PI+ cells in the tet(-) as compared with tet(+) population (Fig. 2GoB). DN/I-{kappa}B{alpha} expression in tet(-) cells was confirmed by anti-Flag immunoblotting (Fig. 2GoC, top panel). Examination of similar lysates with an antiserum that recognizes I-{kappa}B{alpha} protein revealed that DN/I-{kappa}B{alpha} was overexpressed compared with endogenous I-{kappa}B{alpha} levels (Fig. 2GoC, bottom panel). Taken together, these data indicate that interference in the NF-{kappa}B pathway by a degradation-resistant I-{kappa}B{alpha} mutant negates the anti-apoptotic effect of the CD28 receptor in primary human CD4+ lymphocytes.



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FIGURE 2. Primary human CD4+ T cells transduced by the DN/I-{kappa}B{alpha} retrovirus undergo apoptosis. A, Proliferating CD4+ blasts were transduced with tTA and DN/I-{kappa}B{alpha} retroviruses in the presence or absence of 5 µg/ml tet, as described in Materials and Methods. Two days postinfection, cells were harvested, washed, and examined for GFP expression by flow cytometry. B, Dual color annexin V/PI staining to show apoptotic cells in the same populations as determined by flow cytometry (31 ). C, Western blot analysis showing DN/I-{kappa}B{alpha} expression in the same populations as in A. The top panel shows blotting with an anti-Flag Ab, as described in Fig. 1GoB. The bottom panel shows blotting of similar lysates probed with anti-I-{kappa}B{alpha} Abs to compare the levels of recombinant and endogenous I-{kappa}B{alpha}. These experiments were reproduced twice.

 
NF-{kappa}B activity is linked to Bcl-xL expression and cellular survival in primary CD4+ T lymphocytes

To determine which genes function as early response elements during CD3/CD28 costimulation, we used the human Atlas (Clontech) cDNA expression array, which includes an apoptosis-related panel. Reverse-transcribed RNA from CD4+ lymphocytes, treated for 1 h with the anti-CD3 + anti-CD28 mAb combination or anti-CD3 mAb alone, was used for this analysis. While there was no early message expression for anti-apoptotic proteins such as cIAP1, cIAP2, cIAPx, Bfl-1, and Bcl-2, CD3 + CD28 stimulation induced Bcl-xL expression (Table IGo). In contrast, CD3 ligation alone had a negligible effect on Bcl-xL expression (Table IGo). All of the aforementioned genes have been linked to NF-{kappa}B activation (26, 27, 35, 36, 37, 38). Interestingly, CD3 + CD28 costimulation also up-regulated expression of a number of pro-apoptotic genes, including TNF-{alpha} and Fas ligand (Table IGo). CD28 costimulation also induced nonapoptosis-related genes, belonging to the transcription factor, enzyme, cytokine/chemokine, receptor, and stress response categories (Table IIGo). The expression of some of these genes may involve activation of their promoter by AP-1 and NF-{kappa}B response elements, which are CD28 inducible (Table IIGo).


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Table I. Apoptosis-associated CD3/CD28-inducible early response genes in CD4+ T cells1

 

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Table II. Nonapoptosis-related CD3/CD28-inducible early response genes in human CD4+ lymphocytes

 
The Bcl-xL gene product has a clear link to the anti-apoptotic effects of the CD28 receptor (24, 39, 40). Moreover, a number of investigators have recently identified functional NF-{kappa}B response elements in the human and murine Bcl-xL promoters, which may play a role in expression of the protein in T lymphocytes (26, 27). We confirmed at protein level that Bcl-xL expression in primary human CD4+ lymphocytes is linked to CD28 costimulation (Fig. 3GoA). While CD3 + CD28 costimulation induced Bcl-xL expression in CD4+ T cells, anti-CD3 mAb alone had little effect (Fig. 3GoA). In contrast, cIAP2 was constitutively expressed in these cells, and its relative abundance was not further increased during cellular stimulation (Fig. 3GoB). The same was true for cIAP2 expression in the Jurkat T cell line (Fig. 3GoB).



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FIGURE 3. Sodium salicylate interferes with IKK activity and Bcl-xL expression in CD4+ T cells. A, Western blot analysis of Bcl-xL expression in Na+-salicylate-treated CD4+ cells. A total of 107 resting cells was either left untreated or stimulated with anti-CD3 mAb or a combination of anti-CD3 + anti-CD28 mAb bound to magnetic beads in the absence or presence of 20 mM sodium salicylate for 16 h. A total of 50 µg lysate was examined for Bcl-xL expression using anti-Bcl-xL mAb (1:1000), followed by sheep anti-mouse HRP (1:2000). B, Western blot analysis of CD4+ and Jurkat T cell lysate for cIAP2 expression, using a goat anti-cIAP2 Ab (1:1000), followed by rabbit anti-goat HRP (1:2000). Jurkat and primary CD4+ T cells were stimulated as in A. C, Sodium salicylate blocks IKK activity. Resting primary human CD4+ T cells were either left untreated or exposed to 20 mM sodium salicylate for 2 h before stimulation for 30 min with anti-CD3 or anti-CD3 plus anti-CD28 mAb (2 µg/ml each), secondarily cross-linked with 10 µg/ml 187.1 mAb. Cells were lysed and IKK complexes were immunoprecipitated with 2 µg of an anti-IKKß antiserum bound to protein A-Sepharose beads. Kinase assays were performed using the substrate GST/I-{kappa}B{alpha}(1–54), as described in Materials and Methods. Products were analyzed by SDS-PAGE and autoradiography. The arrow points to phosphorylated GST/I-{kappa}B{alpha}. D, Western blot analysis for Bcl-xL expression using CD3 + CD28 ligation in the absence or presence of 10 µM SB203580 and/or 20 mM sodium salicylate.

 
To explore the link between Bcl-xL expression and NF-{kappa}B activation, we made use of the inhibitory effects of Na+-salicylate on IKKß activation (41). This drug effectively suppressed IKK activation in CD4+ lymphocytes during CD3 + CD28 costimulation (Fig. 3GoC), and also had an inhibitory effect on Bcl-xL expression during immunoblotting (Fig. 3GoA, lane 6). Although it has been suggested that salicylate can induce apoptosis through the activation of the p38MAPK cascade (42), we could not demonstrate that a specific inhibitor of that cascade (SB203580) exerted any effect on Bcl-xL expression or that it could negate the inhibitory effects of Na+-salicylate (Fig. 3GoD). A second chemical inhibitor of the NF-{kappa}B cascade in T lymphocytes, pyrrolidine-dithiocarbamate (43), also interfered in Bcl-xL expression in primary human CD4+ T cells (not shown).

In the next of experiments, we asked whether Na+-salicylate affected CD4+ cell viability, and demonstrated a dose-dependent increase in apoptosis in CD3 + CD28-treated cells (Fig. 4GoA). To confirm the specificity of IKKß in this cellular effect, we also examined the effect of the cyclopentenone PGs, 15dPGJ2 and PGA1, which have recently been shown to directly inhibit the IKKß subunit of the IKK complex in Jurkat cells (44). As shown in Fig. 4GoB, treatment of proliferating CD4+ T cells with these cyclopentenone PGs induced apoptosis in a dose-dependent fashion. In contrast, PGB2, which does not inhibit IKKß, had no effect on CD4+ T cell survival (Fig. 4GoB). Collectively, these data indicate that IKKß activity is essential for the survival of primary human CD4+ T cells.



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FIGURE 4. Dual color annexin V/PI staining to assess induction of apoptosis in CD4+ T cells treated with pharmacological inhibitors of IKKß. A, Primary CD4+ T cells were stimulated with anti-CD3 + anti-CD28 Abs coupled to magnetic beads. On day 3, proliferating T cells were treated for 16 h with indicated concentrations of Na+-salicylate (A) or prostaglandins PGB2, 15dPGJ2, and PGA1 (B). After staining with annexin V and PI, cells were analyzed by flow cytometry. Similar results with Na+-salicylate were obtained with CD4+ T cells from two additional donors.

 
Activation of the Bcl-xL promoter via a NF-{kappa}B response element in T lymphocytes

To show more directly activation of the Bcl-xL gene during CD28 costimulation, we used a 1.2-kb fragment of the murine promoter linked to a luciferase reporter (Bcl-xL-Luc) for transfection into the Jurkat T cell line (33). Although Jurkat cells failed to show endogenous Bcl-xL expression during CD28 costimulation (see Fig. 5GoA), CD3 + CD28 ligation had a stimulatory effect on reporter gene activity (Fig. 5GoA). This response was enhanced by the addition of PMA or cotransfection of IKKß cDNA (Fig. 5GoA). Please notice that exogenously expressed IKKß (Fig. 5GoB) is constitutively active in Jurkat cells (4, 5), which may explain the 2.1-fold increase in basal luciferase activity in these transfected cells (Fig. 5GoA). Moreover, the costimulatory effects of CD3 + CD28 on the Bcl-xL promoter were inhibited by treating the cells with the pharmacological inhibitors, Na+-salicylate and PG 15dPGJ2, confirming that IKKß activity plays a role in Bcl-xL promoter activity (Fig. 5GoC).



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FIGURE 5. CD3 + CD28 ligation activates the murine Bcl-xL promoter in Jurkat cells. A, Jurkat T cells were transfected with 12 µg of the 1.2-kb Bcl-xL-Luc reporter in the absence or presence of 2.5 or 5 µg IKKß. After resting for 24 h, cells were stimulated with anti-CD3 plus anti-CD28 mAb, secondarily cross-linked with 187.1, or PMA (100 nM) + ionomycin (1 µg/ml) for 6 h. Cells were lysed and examined for luciferase activity, as described in Materials and Methods. B, Anti-Flag immunoblotting to show IKKß expression in A. C, Jurkat T cells were transfected with 12 µg of the 1.2-kb Bcl-xL-Luc reporter. After resting for 24 h, cells were either left untreated or treated with 20 mM of sodium salicylate or 20 µM PG 15dPGJ2 for 1 h before stimulation as in Fig. 4GoA. D, Similar to Fig. 4GoA, except a Bcl-xL promoter with mutated NF-{kappa}B binding site was used as a reporter.

 
An NF-{kappa}B-binding motif (GGGACTTCC), similar to the NF-{kappa}B element in the immunoglobin promoter, has been identified in the murine Bcl-xL promoter (26). Mutation of this site, located at bp -848 to -840 from the start site in an 848-bp promoter, significantly decreased the promoter response to NF-{kappa}B activation in a murine CTLL cell line (26). When used in Jurkat cells, the mutant promoter could not be induced by CD3 + CD28 costimulation, and did not respond to ectopic expression of IKKß either (Fig. 5GoD). Moreover, we demonstrated that nuclear extracts from Jurkat cells contain CD3 + CD28-inducible factor(s) that binds to a labeled oligonucleotide probe corresponding to that NF-{kappa}B response element (Fig. 6GoA). Complex formation was competed by a 50-fold excess of the cold probe (Fig. 6GoA, lane 4), and the complex was partially supershifted with Abs to p50 and p65, as previously described (26). It is interesting that P+I treatment, which leads to IKK activation, induced the same shift complex (Fig. 6GoB, lane 5). Similar effects were seen in CD4+ T cells during treatment with anti-CD3 + anti-CD28 or P+I (Fig. 6GoB, lanes 3 and 4). Na+-salicylate interfered in the generation of these shift complexes in Jurkat as well as CD4+ T cells (Fig. 6Go, A and B). Similarly, the cyclopentenone PGA1 inhibited PMA-induced NF-{kappa}B shift complexes and IKK activation in Jurkat cells (44).



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FIGURE 6. EMSA analysis of Jurkat and primary CD4+ T cell nuclear proteins. A, Nuclear extracts were prepared from resting or stimulated cells, which were incubated in the presence or absence of 20 mM Na+-salicylate, as indicated. Cells were stimulated with anti-CD3 + anti-CD28 mAb or P+I for 30 min. These extracts were incubated with 1 x 105 cpm of 32P-labeled oligonucleotide corresponding to the NF-{kappa}B binding site at position -840 to -848 of the murine promoter. Cold competition was performed with a 50-fold excess of unlabeled oligonucleotide. B, Similar EMSA as described in A, except primary CD4+ T cell nuclear extracts were used. Results were reproduced twice.

 
Bcl-xL expression in CD4+ blasts and HuT-78 cells is associated with different apoptosis profiles than in non-Bcl-xL-expressing Jurkat cells

While the anti-apoptotic effects of the NF-{kappa}B cascade cover a wide range of stimuli, the role of Bcl-xL to diverse stimuli has not been studied in any depth in primary human T cells. To perform those studies, we used Bcl-xL-expressing CD4+ blasts to look at the effect of Fas, TNF-{alpha}, and staurosporine. For comparison, we included a Jurkat cell clone, which does not express detectable levels of endogenous Bcl-xL (Fig. 7GoA), as well as HuT-78, which shows constitutive Bcl-xL expression (Fig. 7GoA). Interestingly, this constitutive Bcl-xL expression in HuT-78 cells occurs in parallel with constitutive NF-{kappa}B activation (45). That fact is demonstrated by the ability of nuclear extracts from unstimulated HuT-78 cells to generate a shift complex with a probe representative of the NF-{kappa}B site in the Bcl-xL promoter (Fig. 7GoB). Cellular stimulation with CD3 + CD28 mAb or P+I did not increase the relative abundance of this shift complex (not shown).



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FIGURE 7. Bcl-xL expression in HuT-78 and Jurkat T cells correlates with NF-{kappa}B activation. A, Western blot analysis of HuT-78 and Jurkat T cells for Bcl-xL expression. Cellular stimulation and performance of immunoblotting were as described in the legend of Fig. 3GoA. B, EMSA on Jurkat and HuT-78 nuclear extracts. Samples were processed as described in the legend of Fig. 4Go. Similar data were obtained in duplicate experiment.

 
In additional experiments, proliferating CD4+ blasts, HuT-78, and Jurkat cells were exposed to anti-Fas mAb, TNF-{alpha}, and staurosporine for 16 h, and subjected to two-color annexin V/PI staining. While these stimuli readily induced apoptosis in Jurkat cells, there was a comparatively minor effect in CD4+ blasts and HuT-78 cells (Fig. 8GoA). This is compatible with the wide range of anti-apoptotic effects of Bcl-xL in T cells. Included among those effects is regulation of the mitochondrial permeability transition pore complex, which has an effect on {Delta}{varphi}m as well as release of apoptogenic factors from the mitochondrium (46, 47). The drop in {Delta}{varphi}m during mitochondrial perturbation can be assessed in a flow cytometer with the fluorochrome, DiOC6(3). We therefore looked at the decrease in DiOC6(3) fluorescence in HuT-78 vs Jurkat cells, and combined that with measurement of superoxide (O2-) generation, as determined by HE staining (31). O2- generation is a reflection of damage to the mitochondrial inner membrane, which disrupts electron transfer between complexes I and III (31). This experiment showed that while staurosporine, anti-Fas, and TNF-{alpha} had a considerable effect on {Delta}{varphi}m as well as O2 generation in Jurkat cells, the same stimuli had only a minor effect on HuT-78 cells (Fig. 8GoB). This suggests that the constitutively active NF-{kappa}B cascade and accompanying high levels of Bcl-xL expression in HuT-78 cells exert important anti-apoptotic effects at mitochondrial level.



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FIGURE 8. Contrasting apoptosis profiles in proliferating CD4+ T cells and HuT-78 compared with Jurkat. A, Dual color annexin V/PI flow cytometry to compare apoptosis rates in day 3 CD4+ blasts, HuT-78, and Jurkat cells. A total of 1 x 106 cells was treated with 50 ng/ml TNF-{alpha} in the presence of cycloheximide, 100 ng/ml anti-Fas mAb, or 1 µM staurosporine for 16 h. Annexin V/PI staining and flow cytometry were performed as described in Fig. 3GoD. B, Dual color DiOC6(3)/HE staining in Jurkat and HuT-78 T cells to assess the effect of above stimuli on mitochondrial responses. Cells were incubated with 20 µM DiOC6(3) and 2 µM HE for 15 min in the dark before assessment in a flow cytometer. Results were reproduced three times.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We show that CD3/CD28 costimulation in primary human CD4+ T cells induces Bcl-xL expression in parallel with activation of a NF-{kappa}B response site in the promoter of that gene. Retroviral-mediated expression of a DN/I-{kappa}B{alpha} mutant as well as treatment with direct inhibitors of IKKß activity, namely Na+-salicylate and cyclopentenone PGs, induced apoptosis in primary CD4+ T cells. Since this suggests that the CD3/CD28-induced NF-{kappa}B cascade exerts anti-apoptotic effects in primary T cells via Bcl-xL expression, we showed that the activation of the promoter of that gene required NF-{kappa}B activation and was induced by IKKß expression in Jurkat T cells. Moreover, HuT-78, a T cell line with constitutive activation of the NF-{kappa}B cascade, exhibited high basal levels of Bcl-xL expression. HuT-78 cells, like proliferating primary CD4+ T cells, have a relatively high resistance to Fas-, TNF-{alpha}-, and staurosporine-induced apoptosis compared with a Jurkat cell clone in which Bcl-xL was not detectable. Therefore, among a range of apoptosis-related proteins induced by CD28 costimulation, Bcl-xL plays a major role in apoptosis prevention.

Stimulus-induced degradation of inhibitory I-{kappa}B{alpha} and I-{kappa}Bß proteins activates NF-{kappa}B transcription factors (6, 7). We have been using Ab-coated beads as surrogate APCs to amplify primary CD4+ T cells in studying the role of NF-{kappa}B pathway in proliferation, cell death, and anergy (5, 28). Using a retrovirus-mediated gene transfer approach, we found that interruption of NF-{kappa}B pathway in CD4+ T cells with superinhibitory DN/I-{kappa}B{alpha} inhibited cell proliferation and induced apoptosis (Fig. 2GoB). The prosurvival effect of the NF-{kappa}B pathway has been demonstrated in many systems, including T cells (18, 26, 48). For example, Kolenko et al. (48) have demonstrated that pharmacological inhibition of NF-{kappa}B pathway in PBLs induces caspase-dependent apoptosis. Moreover, CTLA-4 ligation, which has been shown to inhibit the proliferation and survival of T cells, suppresses CD28-mediated activation of NF-{kappa}B by blocking I-{kappa}B{alpha} phosphorylation (49, 50). Therefore, CD28-mediated NF-{kappa}B activation is a prerequisite for the proliferation and survival of mature T cells during ligation of the TCR.

The anti-apoptotic effects of NF-{kappa}B have been attributed to induction of pro-survival gene products, including Bcl-2, Bcl-xL, cIAPs, Al/Bfl-1, IEX-1L, TNFR-associated factor 1 and 2, and superoxide dismutase (26, 27, 35, 36, 37, 38). Examination of CD4+ T cells revealed that among the anti-apoptotic proteins regulated by NF-{kappa}B, CD3/CD28 stimulation resulted in the induction of Bcl-xL (Table IGo). Consistent with previous reports, CD3 + CD28 costimulation also induced expression of the pro-apoptotic Fas ligand (Table IGo). It is interesting, therefore, that Bcl-xL has been associated with inhibition of Fas-induced apoptosis in murine T cells and exogenously transfected Jurkat cells (39, 51). Bcl-xL also has the ability to rescue B cells from Fas-induced apoptosis (27, 39). While interference in Fas-induced cell death may explain the prosurvival effect of Bcl-xL in CD28-costimulated primary CD4+ T cells (Fig. 8GoA), it is equally possible that this may also represent an interference in TNF-{alpha}-induced apoptosis. In this regard, our cDNA arrays showed that CD3/CD28 costimulation also induced TNF-{alpha} message expression (Table IGo). While our studies were not designed to address Bcl-xL interference in a particular cell death pathway, it is noteworthy that the NF-{kappa}B pathway has a known negative regulatory effect on TNF-{alpha}-induced apoptosis (1). Whatever the primary stimulus for cell death in CD4+ lymphocytes during inhibition of the NF-{kappa}B pathway, CD28-costimulated cells were resistant to TNF-{alpha}- and Fas-induced apoptosis (Fig. 8GoA).

Several reports, including ours, have suggested that IKKß activity is responsible for I-{kappa}B{alpha} phosphorylation in response to the majority of NF-{kappa}B-inducing signals (5, 52). Seminal studies in mice in which the IKKß locus has been deleted support that notion (53, 54, 55). Embryonic cells from IKKß knockout mice do not activate the NF-{kappa}B pathway in response to TNF-{alpha} or IL-1 stimulation (53), and die at midgestation due to severe liver apoptosis (55). In this study, we show that treatment of primary CD4+ T cells with Na+-salicylate, an inhibitor of IKKß (41), blocks Bcl-xL expression (Fig. 3GoD) as well as inducing apoptosis (Fig. 3GoE). This notion was further substantiated by the pro-apoptotic effects of recently discovered cyclopentenone IKKß inhibitors, namely prostaglandins PGA1 and 15dPGJ2 (Fig. 4Go) (44). The induction of apoptosis by Na+-salicylate in B cells and myeloid leukemia cells has been associated with activation of caspases (56, 57). It is also interesting that Bcl-xL has been shown to interact with caspases and interferes with the execution of the death phase (58, 59, 60). Therefore, it is plausible that inhibition of IKKß activity by cyclopentenone PGs and Na+-salicylate is responsible for induction of apoptosis in CD28-costimulated T cells.

Proliferating primary CD4+ and HuT-78 lymphoblastoid T cells resisted death in response to various apoptotic stimuli, including TNF-{alpha}, anti-Fas mAb, and staurosporine (Figs. 7Go and 8Go). This resistance to apoptosis was also reflected in the resistance of mitochondrial membrane perturbation in HuT-78 cells (Fig. 8GoB). This is compatible with the regulatory effects of Bcl-xL on the mitochondrial permeability transition pore (47), but does not rule out other anti-apoptotic effects of this regulatory protein. For instance, Bcl-xL has recently been demonstrated to interact with Apaf-1 and several caspases (58, 59, 60). Thus, heterodimerization with pro-apoptotic proteins and competition with Apaf-1 for binding to caspases may represent additional Bcl-xL activities in primary CD4+ T cells.

The 3.2-kb stretch of DNA upstream of the Bcl-xL initiation site contains a variety of different transcription factor binding sites (33). While the contribution of most of these elements in regulating the expression of Bcl-xL remains unclear, some progress has been made. For instance, signaling through IL-6 pathway, which leads to activation of Stat3 and Janus kinases, has been associated with induction of Bcl-xL expression (61). Indeed, constitutive activation of Stat3 has been associated with the pathogenesis of multiple myeloma through inhibition of apoptosis (61). Two reports have also identified independent NF-{kappa}B binding sites in the Bcl-xL promoter (26, 27). In one of those studies on the human promoter, two tandem NF-{kappa}B binding sites were identified in the human promoter approximately 200 bp upstream of the initiation site (27). In the second study, a NF-{kappa}B binding site was identified at ~840 bp upstream of the Bcl-xL initiation site in the murine promoter (26). The activation of the latter binding element by Tax protein is putatively involved in the ability of CTLL-2 cells to survive in the absence of IL-2 (26). DN/I-{kappa}B{alpha} as well as mutation of this putative NF-{kappa}B site abolished Tax-mediated Bcl-xL promoter activity and expression (26). While a 600-bp promoter element did not respond to CD3/CD28-mediated costimulation or IKKß overexpression in our studies (not shown), 1.2-kb and 848-bp Bcl-xL promoter elements were responsive to these stimuli (Fig. 5GoA). The CD3/CD28-induced activation of the 1.2-kb Bcl-xL-Luc promoter element was blocked by pharmacological inhibtion of IKKß (Fig. 5GoC). In addition, mutation of the NF-{kappa}B site at position -840 to -848 in the 848-bp reporter rendered it nonresponsive to CD3 + CD28 ligation (Fig. 5GoD). Moreover, CD3 + CD28 stimulation resulted in nuclear localization of NF-{kappa}B transcription factors that bound to an oligonucleotide probe representative of that NF-{kappa}B binding site (Fig. 6Go) (26). Nuclear localization and binding of these factors were inhibited in primary CD4+ and Jurkat T cells treated with Na+-salicylate (Fig. 6Go). Furthermore, constitutive activation of the NF-{kappa}B pathway in untreated HuT-78 T cells was associated with constitutive Bcl-xL expression and generation of an identical NF-{kappa}B shift complex (Fig. 7Go). These studies indicate that CD3 + CD28-induced activation of NF-{kappa}B pathway influences Bcl-xL expression through this putative binding site. Similar to CD3/CD28 stimulation, Tax-mediated NF-{kappa}B activation, which also leads to Bcl-xL expression, proceeds through IKKß activation (26, 62).

The data presented indicate that regulation of IKKß activity in CD4+ T cells and expression of the anti-apoptotic protein Bcl-xL may coordinate the life span and expansion rate of specific T cell clones when they encounter particular Ags. These findings have important implications in the regulation of the mature T cell compartment and disease induction that may result from dysregulated apoptosis in these cells.


    Acknowledgments
 
We thank Ivonne Castaneda and Peggy Shih for secretarial assistance, and Martin Kasubowski for technical support.


    Footnotes
 
1 This study was supported by the U.S. Public Health Service (AG14992). A.K. was supported by National Institute of Allergy and Infectious Diseases-funded University of California, Los Angeles, Immunology Training Grant. Back

2 Address correspondence and reprint requests to Dr. Andre Nel, Division of Clinical Immunology and Allergy, Department of Medicine, University of California, 10833 Le Conte Avenue 52-175 CHS, Los Angeles, CA 90095. Back

3 Abbreviations used in this paper: I-{kappa}B, inhibitor of {kappa}B factor; cIAP, cellular inhibitor of apoptosis protein; 15dPGJ2, 15-deoxy-{Delta}12–14-PGJ2; DiOC6, dihexyloxacarbocyanine iodide; DN, dominant-negative; GFP, green fluorescent protein; HE, hydroethidine; IKK, I-{kappa}B kinase; P+I, PMA plus ionomycin; PI, propidium iodide; tet, tetracycline; tTA, tet-repressible transactivating; {Delta}{psi}m, mitochondrial membrane potential. Back

Received for publication February 4, 2000. Accepted for publication May 23, 2000.


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