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The Journal of Immunology, 2000, 165: 1854-1862.
Copyright © 00 by The American Association of Immunologists

Distinct Molecular Mechanisms of Fas Resistance in Murine B Lymphoma Cells1

Carolyn M. Mueller and David W. Scott2

Department of Immunology, J. H. Holland Laboratory for the Biomedical Sciences, American Red Cross, Rockville, MD 20855


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
A panel of murine B lymphoma cell lines, which express different levels of Fas, was extensively studied for sensitivity to Fas-mediated death signals via an anti-Fas mAb and Fas ligand-bearing cell lines. Expression of the Fas receptor on the B lymphoma cell lines did not correlate with their capacity to undergo Fas-mediated apoptosis. Moreover, Fas-associated death domain protein recruitment to the death-inducing signaling complex (DISC) complex occurred in all cell lines expressing Fas, regardless of whether they were sensitive to Fas-mediated death. Interestingly, the protein synthesis inhibitor, cycloheximide, and protein kinase C inhibitors, such as bisindolylmaleimide, rendered one of the resistant cell lines, CH33, sensitive to signals from the Fas receptor, although the levels of Fas were unchanged. This suggests that constitutive PKC activation plays a role in Fas resistance, perhaps by up-regulating NF-{kappa}B or Bcl-2 family members. Interestingly, CH33 demonstrated caspase 8 activity upon engagement of the Fas receptor in the absence of pharmacological manipulation, suggesting that the block in apoptosis is downstream of the DISC complex. In contrast, the fact that Fas-associated death domain protein was recruited to the DISC complex in other resistant lines, such as WEHI-231, with no caspase 8 activation indicates that these cells may be blocked within the DISC complex. Indeed, Western blot analysis showed that WEHI-231 expressed an isoform of FLICE-like inhibitory protein (cFLIPL), an antiapoptotic protein within the DISC. These studies provide evidence that murine B lymphoma cells utilize different molecular mechanisms along the Fas-signaling cascade to block apoptosis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Members of the TNFR/TNF family of proteins, specifically Fas and FasL, have been implicated, via the induction of apoptosis, in maintaining immune homeostasis (1, 2). Lack of function mutations in the Fas receptor (e.g., in lpr mice or in humans with autoimmune lymphoproliferative syndrome) or FasL (gld mice) can be associated with immune dysfunction. These include autoantibody production, accumulation of lymphoid cells displaying an activated phenotype that leads to lymphadenopathy and splenomegaly, and increased development of B lymphomas (3, 4, 5, 6, 7, 8, 9, 10, 11). Further mutations downstream of the Fas receptor may also contribute directly to lymphomagenesis by blocking the elimination of cells with a transformed phenotype. Thus, despite expressing the Fas receptor, many tumor cells have evolved a variety of pathways to evade destruction by the host immune system.

In this study, we explore the sensitivity of a series of murine B lymphoma cell lines to Fas-mediated apoptosis, and show that approximately half of these are naturally resistant to anti-Fas mAb or FasL-expressing effector cells. Evidence is provided herein suggesting that murine B lymphoma cell lines use multiple and distinct molecular mechanisms to block Fas-mediated apoptosis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines

Murine B lymphoma cell lines (A20.2J (12), CH12 (13), CH27 (14), CH31 (15), CH33 (15), ECH408 (16), NBL.5 (17), WEHI-231 (18), and WEHI-279 (19)) and the cytotoxic T cell hybridoma D11S (20) were maintained at 37°C, 7% CO2 in RPMI 1640 medium (BioWhittaker, Walkersville, MD) supplemented with 10 mM HEPES, 1 mM sodium pyruvate, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 1x MEM nonessential amino acids, 5 x 10-5 M 2-ME (Sigma, St. Louis, MO), and 5% FCS, as described previously (15); all supplements not indicated were obtained from Life Technologies (Grand Island, NY). After 30 passages, new lots of cells were thawed to prevent genetic drift. The Eµmyc B lymphoma cell line was derived by culturing peritoneal wash cells from Eµmyc transgenic mice (21). The M12.4.1 B lymphoma cell line (22), the murine AIDS B6-1710 B lymphoma cell line (23), and the D11S cell line were provided by Drs. Achsah Keegan, Wendy F. Davidson, and Mark Williams (American Red Cross, Rockville, MD), respectively. The K562 human FasL and K562-neo (control) monocytic cell lines were kindly provided by Dr. Richard C. Duke (University of Colorado Health Sciences Center, Denver, CO). The monocytic cell lines were cultured as stated above in standard media containing 0.5 mg/ml G418 (Life Technologies).

Apoptosis assays

Apoptosis was induced in the B lymphoma cell lines by anti-Fas Ab or by culture with effector cell lines bearing FasL. B lymphoma cells were cultured at 2.5 x 105 cells/ml with 100 ng/ml of monoclonal hamster anti-murine Fas (Jo2; PharMingen, San Diego, CA) for 4–12 h. In some experiments, the B lymphoma cells were precultured with a CD40L membrane preparation derived from Sf9 cells (generous gift of Dr. Marilyn Kehry, Boehringer Ingelheim, Ridgefield, CT), cycloheximide (CHX;3 Sigma), or PKC inhibitors (bisindolylmaleimide (BIS), HBDDE, and Rottlerin; Biomol Research Laboratories, Plymouth Meeting, PA) before anti-Fas treatment. After Fas cross-linking, the cells were fixed in cold 70% ethanol overnight, washed, and then incubated at 37°C for 30 min in PBS containing 10 µg/ml RNase (Sigma). Following the addition of propidium iodide (PI; Sigma) to a final concentration of 50 µg/ml, the samples were analyzed for hypodiploid cells by flow cytometry using FACScalibur and CellQuest software (Becton Dickinson, San Jose, CA).

For apoptosis induced by effector cells, B lymphoma cells were cultured at 1 x 105 cells/ml with D11S cells previously stimulated for 3 h with 5 ng/ml PMA (Sigma) and 3 µM ionomycin (Sigma) to up-regulate FasL. Alternatively, target cells were cultured with K562 human FasL effectors or K562-neo cells as control effectors. In all cases, a 1:1 E:T ratio was used in a 12-h assay. To label the targets, cells were stained with 10 µg/ml biotin-conjugated anti-murine CD19 (PharMingen), a 1/200 dilution of streptavidin-PE (PharMingen), and then a 1/10 dilution of FITC-annexin V (R&D Systems, Minneapolis, MN). In flow cytometry, the percentage of apoptotic cells was determined from the fraction of annexin V bright cells within the gated CD19-positive B lymphoma cells.

Fas expression

After Fc receptor blocking with 10 µg/ml 2.4G2, 1 x 106 B lymphoma cells were stained with 10 µg/ml FITC-conjugated hamster anti-murine Fas (Jo2; PharMingen) or a 1/100 dilution of FITC-conjugated 145-2C11 (hamster anti-murine CD3, made in house) as an isotype control. The cells were then analyzed by flow cytometry.

Western blots

Following various stimulation procedures (see specific figures), a total of 1 x 107 cells was washed in cold PBS and then lysed in lysis buffer (50 mM HEPES, pH 7.5, 0.5% Nonidet P-40, 5 mM EDTA, 50 mM NaCl, 10 mM sodium pyrophosphate, 50 mM NaF, 1 mM sodium orthovanadate, and protease inhibitor mixture (Boehringer Mannheim, GmbH, Germany)) on ice for 15 min. Following centrifugation at 13,000 rpm, 4°C for 10 min to pellet the nuclei, the protein concentration of the supernatants was determined using the BCA protein assay kit (Pierce, Rockford, IL). A total of 50 µg of protein per lane was run on SDS-PAGE gels under nonreducing conditions and then transferred onto nitrocellulose for Western blotting. Membranes were blocked overnight at 4°C in a milk-blocking buffer (a 10 mM Tris-HCl, pH 7.5, 100 mM NaCl solution containing 0.1% Tween 20, and 5% nonfat dry milk) or for caspase 8 Western blots only, membranes were blocked in PBS containing 3% BSA and 0.1% Tween 20. The blots were then probed for 1 h with primary Ab diluted in blocking buffer (rabbit anti-mouse Bag-1 (N-20; Santa Cruz Biotechnology, Santa Cruz, CA; diluted to 1 µg/ml), mouse anti-mouse Bcl-2 (Transduction Laboratories, Lexington, KY; diluted 1/250), rabbit anti-mouse Bcl-XL (PharMingen; diluted 1/1,000), or goat anti-mouse Mch5 p20 (T16, anti-caspase 8; Santa Cruz Biotechnology, diluted 1/800)). Following three washes in blocking buffer lacking protein, the blots were incubated for 1 h with HRP-conjugated secondary Ab (anti-rabbit IgG (Boehringer Mannheim; diluted 1/5,000), anti-mouse IgG (Boehringer Mannheim; diluted 1/5,000), or anti-goat IgG (Santa Cruz Biotechnology; diluted 1/20,000)). After additional washing, the membranes were developed by enhanced chemiluminescence using a kit from Boehringer Mannheim.

Immunoprecipitation of Fas, FADD, and FLIPs

A total of 3 x 107 cells was stimulated with 5 µg of anti-murine Fas for 5 min at room temperature. Cells were subjected to a freeze/thaw cycle using an ethanol bath, washed in cold PBS, and then lysed in 1 ml of immunoprecipitation lysis buffer (30 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 10% glycerol, and protease inhibitor mixture) on ice for 15 min. Following a centrifugation to pellet the nuclei, the lysates were mixed with 100 µl protein G-Sepharose (Pharmacia Biotech AB, Uppsala, Sweden) at 4°C overnight. After three washes of the protein G-Sepharose with lysis buffer, the samples were run on 12% SDS-PAGE gels under nonreducing conditions and transferred to nitrocellulose for Western blotting. Membranes were blocked overnight at 4°C in milk-blocking buffer and then probed for 1 h at room temperature with primary Ab diluted in blocking buffer. Primary Ab included a rat anti-mouse Fas Ab (Upstate Biotechnology, Lake Placid, NY; diluted to 1 µg/ml), goat anti-mouse FADD Ab (M-19; Santa Cruz Biotechnology; diluted 1/200), and rabbit anti-human FLIP (anti-usurpin, a generous gift of Dr. Donald W. Nicholson, Merck Frosst, Pointe Claire-Dorval, Quebec, Canada (24); diluted 1/3000). After three washes, the membranes were probed for 1 h with HRP-conjugated secondary Ab (anti-rat Ig (Boehringer Mannheim; diluted 1/500), anti-goat IgG (1/1000), or anti-rabbit IgG (1/5000)). After a final set of washes, the membranes were developed by enhanced chemiluminescence.

EMSA

The experimental procedure was modified from Lin et al. (25). CH33 cells left untreated, treated with 50 µM N-tosyl-L-phenylalanine-chloromethyl ketone (TPCK; Sigma) for 2 h, or treated with 10 µM BIS for 12–18 h were washed with cold PBS and lysed in 100 µl cellular lysis buffer (10 mM HEPES, pH 7.9, 1 mM EDTA, 60 mM KCl, 1 mM DTT, 0.5% Nonidet P-40, 0.5 mM sodium orthovanadate, and protease inhibitor mixture) on ice for 5 min. Following centrifugation at 5000 rpm, 4°C, nuclear membranes were washed with cellular lysis buffer and resuspended in 100 µl nuclear lysis buffer (250 mM Tris-HCl, pH 7.8, 60 mM KCl, 1 mM DTT, 0.5 mM sodium orthovanadate, and protease inhibitor mixture). The membranes were then subjected to three freeze/thaw cycles in a dry ice, ethanol bath. The samples were centrifuged at 10,000 rpm, 4°C for 15 min, and the protein concentration of the supernatants was determined. For each sample, 10 µg protein was incubated for 10 min at room temperature with 3 µg poly(dI-dC) (Sigma) and 3 µl 5x gel-shift buffer (20% glycerol, 5 mM MgCl2, 2.5 mM EDTA, 2.5 mM DTT, 250 mM NaCl, 50 mM Tris-HCl, pH 7.5, and 0.25 mg/ml poly(dI-dC)) in a total volume of 15 µl. One microliter of a double-stranded oligonucleotide, end labeled with T4 polynucleotide kinase (Boehringer Mannheim) and [{gamma}-32P]ATP (Amersham Pharmacia Biotech, Piscataway, NJ), was then added and the samples were incubated for 20 min at room temperature. The oligonucleotides included AP1 (5'-CGCTTGATGAGTCAGCCGGAA-3') and OCT-1 (5'-TGTCGAATGCAAATCACTAGAA-3') purchased from Promega (Madison, WI) and an oligonucleotide containing the {kappa}B site from the mouse Ig enhancer (5'-GATCGAGGGGACTTTCCGAGAGATC-3', a gift from Dr. Jose Zamorano, American Red Cross). Following the addition of 2 µl of gel-loading buffer (250 mM Tris-HCl, pH 7.5, 0.2% bromophenol blue, and 40% glycerol), the samples were electrophoresed on a 6% polyacrylamide gel. The gel was dried and subjected to autoradiography.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Fas expression and sensitivity to Fas-mediated apoptosis

A panel of 12 murine B lymphoma cell lines was stained for Fas expression and analyzed by flow cytometry (Fig. 1Go). Within this group of cell lines were represented IgM+IgD-, IgM+IgD+, and IgG+ B cell lymphomas. Additionally, we included cells lines that were either sensitive or insensitive to B cell receptor (BCR)-driven apoptosis. Detectable levels of Fas were seen on the majority (8/12) of cell lines. Although the IgG+ cell lines did express slightly higher levels of Fas, surface expression could not be correlated with any known phenotype of the B lymphoma cells.



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  		FIGURE 1. Fas expression on the surface of murine B lymphoma cell lines. Exponentially growing cells, unstimulated (shaded histogram) or stimulated with 6 µg/ml CD40L for 48 h (solid line), were stained for Fas expression with FITC-conjugated Jo2 mAb and analyzed by flow cytometry. A representative subset of six B lymphoma cell lines (of 12 tested) is displayed. Dashed line represents isotype-matched control staining. Data are from one of three experiments with similar results. Treatment of lymphoma cells for 48 h with CD40L induced a 2- to 4-fold shift in mean fluorescence intensity. The relative degree of sensitivity to Fas-mediated apoptosis is indicated for each cell line (see Fig. 2Go and Tables IGo and IIGo). NS, Not sensitive; MS, moderately sensitive; S, sensitive; and VS, very sensitive.

 
When the Fas receptor was cross-linked on the B lymphoma cells using the mAb Jo2, only a subset of the cell lines was induced to undergo apoptosis as measured by PI analysis (Fig. 2GoA; Table IGo). As expected, no increase in the percentage of hypodiploid cells was seen following anti-Fas treatment of cells having very low to no detectable surface expression of Fas (Eµmyc, NBL.5, CH27, CH31). Among the B lymphoma cell lines expressing moderate to high levels of Fas, five of eight cell lines were sensitive to Fas-mediated apoptosis. The IgG+ B lymphoma cell line A20.2J was the most sensitive, with on average greater than 30% hypodiploid cells after 4 h of anti-Fas cross-linking (Fig. 2GoA). However, the levels of Fas did not correlate with sensitivity to Fas-mediated apoptosis within the IgM+ subset of B lymphoma cell lines. For example, WEHI-231 and CH33 cells expressed as high or higher levels of Fas than the other IgM+ cells, and yet were resistant following up to 12 h of incubation with anti-Fas. Interestingly, ECH408, an IgD-transfected subclone of the resistant CH33 cell line, was sensitive to anti-Fas. This result suggests that clonal variation in Fas sensitivity can be obtained.



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  		FIGURE 2. Sensitivity of B lymphoma cell lines to Fas-mediated apoptosis. Four representative cell lines are displayed (see Table IGo). A, Cell lines were treated with 100 ng/ml of anti-Fas (Jo2) for 4 h (A20.2J) or 12 h (WEHI-231, ECH408, and CH33), then harvested for PI staining and cell cycle analysis. The percentage of cells with hypodiploid DNA is indicated in the upper left portion of each histogram. B, B lymphoma cell lines were cultured for 12 h alone, with neo-transfected K562 monocytic cells, or K562 cells engineered to express human FasL. After staining with anti-CD19, the CD19+ B lymphoma cells were gated and the percentage of annexin V bright cells is indicated for each sample. Data are representative of at least three experiments.

 

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  		Table I. Summary of Fas expression and sensitivity to Fas-mediated apoptosis

 
Additionally, the B lymphoma cells were tested for sensitivity to Fas-driven apoptosis using a more physiologically relevant assay with effector cells expressing surface FasL (Fig. 2GoB and Table IGo). Exactly the same sensitivity pattern was observed using the K562 human FasL or D11S effector cells as was observed with anti-Fas cross-linking. Thus, for example, a low percentage of annexin V bright cells was observed when WEHI-231 or CH33 were cultured with K562 human FasL or D11S, whereas A20.2J was extremely sensitive to culture with a FasL-bearing cell line. Equivalent results were found independent of which FasL+ effector cell line was used (data not shown). These data indicate that lack of sensitivity to anti-Fas is not due to the nature of the Fas signal nor the requirement for Fc receptor expression on target cells for presentation of Jo2.

CD40L treatment increases Fas expression and sensitivity in most B cell lines

From the work of others, it is known that stimulation of splenic B cells as well as B lymphoma cell lines via the CD40 receptor can result in the up-regulation of cell surface Fas (12, 26, 27, 28, 29). This increase in expression of Fas is thought in part to account for the increase in sensitivity to Fas-driven apoptosis of B cells and their neoplastic counterparts. A subset of our panel of murine B lymphoma cells was stimulated with 6 µg/ml of CD40L for 48 h before staining for Fas expression. An increase of ~2- to 4-fold in expression of Fas was observed on nearly all Fas+ cell lines tested (Fig. 1Go). However, except for CH31, Faslow cell lines (Eµmyc, NBL.5, and CH27) were unable to up-regulate Fas expression upon CD40L or IL-6 plus IFN-{gamma} treatment (data not shown).

When the CD40L-stimulated B lymphoma lines were also tested for their sensitivity to Fas-driven apoptosis, almost all of the Fas+ cell lines showed an increase in sensitivity to Fas cross-linking. This was especially true for CH31, which was previously insensitive to Fas-mediated death (Table IIGo). It is unclear whether this is solely due to an increase in Fas expression. However, the CD40L-treated WEHI-231 cells remained resistant to Fas-mediated death even though the cells up-regulated surface expression of the Fas receptor. This suggests that limited membrane expression of Fas is not the only explanation for Fas resistance (see below).


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  		Table II. Fas-induced apoptosis of unstimulated and CD40L-stimulated B lymphoma cells

 
Recruitment of FADD to the DISC complex and the activation of caspase 8

Initial studies on a subset of the resistant B lymphoma cell lines examined whether a functional DISC complex was formed upon ligation of the Fas receptor. After cross-linking of the receptor with Jo2 on both resistant and sensitive cell lines, protein G-Sepharose was used to immunoprecipitate the Ab-bound complexes. The samples were then tested by Western blotting to determine whether the FADD molecule had coimmunoprecipitated with the Fas molecule (Fig. 3Go). For all cell lines tested, FADD was recruited to Fas upon engagement of the receptor. This finding suggested that all cell lines tested possessed a functional Fas receptor capable of recruiting FADD. Since FADD was recruited to the DISC complex, it was possible that caspase 8 was not activated in resistant cells. Cell lysates of untreated and anti-Fas-treated cells, from both resistant and sensitive B lymphoma lines, were probed by Western blotting for the p20 cleavage product of caspase 8, indicating an active cysteine protease. Activation of caspase 8 was found in all sensitive cell lines (Fig. 4Go) and also in CH31 cells stimulated with CD40L, as expected (data not shown). Interestingly, caspase 8 was found to be cleaved in the resistant cell line, CH33 (also measured by cleavage of isoleucine-glutamic acid-threnine-aspartate (Ile-Glu-Thr-Asp)-7-amino-4-trifluoromethyl-coumarin in a fluorometric assay; data not shown). Thus, the block in Fas signaling in CH33 appears to be downstream of the DISC complex. However, caspase 8 activation was not found in WEHI-231, suggesting a block within the DISC complex in this resistant B lymphoma cell line. When the immunoprecipitations of the DISC complexes were screened for the cFLIP molecule, the long isoform of cFLIP was only found to be associated with Fas in WEHI-231 (Fig. 3Go). The short isoform of the FLIP molecule could not be confidently analyzed due to Ig light chain contamination in the immunoprecipitation. Thus, WEHI-231 appears to be blocked at the DISC complex due to expression of cFLIPL, whereas the block in Fas-mediated death in CH33 must be downstream of caspase activation.



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  		FIGURE 3. Composition of the DISC complex. The recruitment of FADD and cFLIP by Fas in resistant and sensitive cell lines was analyzed by cross-linking the Fas receptor with Jo2 and then immunoprecipitating the Ab with protein G-Sepharose. The immunoprecipitated products were tested by Western blotting for levels of Fas, FADD, and cFLIP.

 


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  		FIGURE 4. Caspase 8 activation in sensitive as well as resistant cell lines. After cross-linking the Fas receptor on B lymphoma cell lines for at least 2 h with Jo2, cell lysates were examined by Western blotting for the cleaved active form of caspase 8. Arrows indicate the cleaved fragments of pro-caspase 8. No detectable apoptosis was observed in the CH33 cell line under these conditions. The degree of sensitivity to Fas-mediated apoptosis is indicated for each cell line (see Fig. 2Go and Tables IGo and IIGo). NS, Not sensitive; MS, moderately sensitive; S, sensitive; and VS, very sensitive.

 
De novo protein synthesis is required for the resistance of CH33 B lymphoma cells to Fas

To examine whether protein synthesis was required for resistance to Fas-driven apoptosis, the insensitive cell lines WEHI-231 and CH33 were treated with CHX for 6 h before an additional 6 h of anti-Fas treatment. In analogy to results with TNF-{alpha} killing, the CH33 cell line was induced to undergo apoptosis by the anti-Fas Ab when treated with CHX (Fig. 5Go), but WEHI-231 did not. For CH33 cells, either a newly synthesized protein or a short-lived protein is required for the resistance to anti-Fas treatment. Additionally, treatment of CH33 with the RNA transcription inhibitor, actinomycin D, or the demethylating agent, 5-azacytidine, could render these cells sensitive to anti-Fas mAb (data not shown). For WEHI-231, if cFLIPL is the only mechanism by which apoptosis is blocked, then the inability of CHX to render WEHI-231 sensitive to anti-Fas Ab suggests that the cFLIPL protein has a t1/2 longer than 12 h of the assay.



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  		FIGURE 5. Protein synthesis and Fas resistance. WEHI-231 and CH33 cells were either untreated or treated for 6 h with CHX before the addition of anti-Fas for a 6-h incubation. Cells were analyzed by PI staining and flow cytometry for cells having hypodiploid DNA content as in Fig. 2Go. Data representative of three experiments.

 
Role of PKC in Fas resistance

PKC has been demonstrated to be involved in the signaling cascade from the BCR leading to resistance to Fas-mediated apoptosis in both splenic B cells and also B lymphoma cells (12, 30). To test the hypothesis that chronic BCR stimulation and PKC activation may lead to Fas resistance during lymphomagenesis in vivo, the insensitive cell lines were pretreated with the PKC-specific inhibitor BIS for 6 h before Fas cross-linking with Jo2. The CH33 cell line demonstrated a BIS dose-dependent increase to Fas-driven apoptosis (Fig. 6GoA). However, even the highest nontoxic dose of BIS could not render WEHI-231 sensitive to anti-Fas. Thus, active PKC is involved in the resistance of CH33 to Fas. PKC isoform ({alpha}, ß, {delta}, {epsilon}, {gamma}, {iota}, {lambda}, and {theta}) typing of the B lymphoma cell lines did not indicate a specific isoform as being involved in the resistance of CH33 to Fas signaling, except that CH33 does not express PKC-{gamma} or PKC-{theta} (data not shown). However, the use of PKC-specific inhibitors did delineate which isoforms may be involved. Both HBDDE (inhibitor of PKC-{alpha} and {gamma}) and Rottlerin (inhibitor of PCK-{delta}) rendered CH33 sensitive to anti-Fas Ab (Fig. 6GoB). Since CH33 does not express PKC-{gamma}, PKC-{alpha} and PKC-{delta} appear to be the molecules involved in the resistant phenotype. Signal transduction molecules downstream of PKC have begun to be analyzed in CH33. The transcription factor NF-{kappa}B has been implicated in protection from apoptosis (31). NF-{kappa}B EMSAs were performed on nuclear extracts of CH33 cells treated with BIS and compared with untreated cells. A 20 to 35% reduction in active NF-{kappa}B was observed in BIS-treated cells (Fig. 7Go). Interestingly, the basal level of active NF-{kappa}B is 8-fold higher in CH33 cells than in the Fas-sensitive cell line, A20.2J (data not shown). While BIS partially down-regulated NF-{kappa}B activity, no reduction in active AP1 or OCT-1 was observed in BIS-treated CH33 cells compared with untreated cells. TPCK, nonspecific proteosome inhibitor, completely down-regulated NF-{kappa}B, partially reduced OCT-1 activity, and to a lesser extent reduced AP1. Thus, the decrease in NF-{kappa}B levels in BIS-treated CH33 correlates with an increase in sensitivity to Fas-driven apoptosis.



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  		FIGURE 6. Inhibition of PKC and sensitivity to Fas-driven apoptosis. A, WEHI-231 and CH33 cells were left untreated or treated with the indicated doses of BIS for 6 h. A 12-h Jo2 killing assay was then performed before cell cycle analysis. B, CH33 cells were treated with PKC inhibitors HBDDE (30 µM), Rottlerin (1 µM), or BIS (10 µM) for 1 h to minimize drug toxicity effects, and then treated for 12 h with 100 ng/ml anti-Fas. PI analysis was performed to determine the percentage of hypodiploid cells. Representative of three experiments.

 


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  		FIGURE 7. Analysis of NF-{kappa}B, AP1, and OCT-1 activity in the CH33 B lymphoma cell line. Cells were left untreated or treated with 5 µM BIS for 12–18 h or 50 µM TPCK for 2 h. Nuclear extracts were mixed with 32P-labeled oligonucleotides containing binding sites for NF-{kappa}B, AP1, and OCT-1 and then run on a 6% polyacrylamide gel in an EMSA. In cold probe controls, a 100-fold excess of the corresponding unlabeled oligonucleotides was added, to duplicate samples of nuclear extracts from untreated cells, to competitively inhibit binding to the labeled oligonucleotides. Densitometric values for the active transcription factors are indicated at the bottom of the figures; values were obtained using UN-SCAN-IT software (Silk Scientific, Orem, UT).

 
Additionally, members of the Bcl-2 family of proteins have been found to provide protection from Fas-mediated apoptosis in type II target cells (32). A partial reduction in the levels of the antiapoptotic molecules Bcl-2 and Bcl-XL was observed between BIS-treated CH33 cells and control cells (Fig. 8Go). Two bands were observed in Western blotting for the Bcl-2 family members, presumably reflecting unphosphorylated and phosphorylated forms of the proteins. The greatest decrease in BIS-treated cells was in the lower migrating form of Bcl-2. Anti-Fas alone or in combination with BIS had little to no effect on Bcl-2 or Bcl-XL levels. However, in Western blotting for an additional antiapoptotic family member, both BIS and anti-Fas alone decreased the levels of Bag-1. Immunoprecipitation studies to quantitate the stoichiometries of antiapoptotic and proapoptotic Bcl-family members in treated vs control cells will be required to definitively assess the role of these molecules in resistant phenotype of CH33.



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  		FIGURE 8. Bcl-2 family members in CH33. Cells were left untreated or treated with 10 µM BIS for 1 h. Samples then received no Ab or 100 ng/ml anti-Fas for 4 h. Cell lysates were prepared and 50 µg protein was run per lane for transfer to nitrocellulose and Western blotting. Membranes were blotted for a subset of the antiapoptotic Bcl-2 family members (Bcl-2, Bcl-XL, and Bag-1). Densitometry values are indicated below each Western. Representative of at least two experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The dysregulation of Fas-mediated apoptosis has been implicated in autoimmunity, loss of immune homeostasis leading to the accumulation of lymphocytes, and the development of B cell lymphomas. However, B cell lymphomagenesis is also known to occur in an environment in which Fas and FasL are functional. Thus, for B lymphomas that express Fas receptors, the molecular mechanism(s) of resistance to Fas-driven apoptosis needs clarification. A panel of immature and mature murine B lymphoma cell lines was analyzed for expression of the Fas receptor and sensitivity to signaling via this death receptor. The murine B lymphoma cell lines examined could be subdivided into four categories (see Table IIIGo): 1) Fas+- and anti-Fas-sensitive B lymphoma cells, 2) Fas- cell lines whose death receptor levels could not be up-regulated, 3) B lymphoma cells that could be modulated in their sensitivity to anti-Fas, and finally 4) the Fas+ cells that were anti-Fas resistant.


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  		Table III. Four categories of B lymphoma cell lines

 
The first group of cell lines, which were sensitive to anti-Fas, consisted of 5 of the 12 cell lines, including A20.2J, CH12, and ECH408. Both IgG+ and IgM+ B lymphoma cells were represented within this category, which included cells with both moderate and high levels of Fas expression.

The second category included three cell lines (NBL.5, CH27, and Eµmyc.5), which expressed very little to no Fas receptor. Although both Eµmyc and NBL.5 express Fas mRNA, barely detectable levels of Fas protein were observed only in NBL.5 following immunoprecipitation and Western blotting (data not shown). Furthermore, Fas expression could not be up-regulated on these cell lines by stimulation via CD40L, or by IFN-{gamma} or IL-6, all of which are known to up-regulate Fas levels in other cell types (33, 34). These cells could possess a mutation within fas that abrogates surface expression. Mutation of the Fas receptor has been found to occur in human non-Hodgkin’s lymphomas (35), myelomas (36), and T cell leukemias (37, 38). The observed mutations included point mutations within and outside of the death domain of fas (35, 36, 38) as well as insertions and deletions of the gene (37). Additionally, these cell lines could possess a defect in CD40 signaling, a phenomenon that has been suggested for a panel of human B chronic lymphocytic leukemia cells (39). This defect is rendered unlikely by their unresponsiveness to two other stimuli, IFN-{gamma} and IL-6.

Upon CD40L stimulation, the cell line that showed the greatest increase in Fas expression and sensitivity to Fas-induced apoptosis was CH31 (category III). With this phenotype, CH31 represents a cell line that closely resembles naive B cells (26, 30, 40, 41). For this cell line only, the low level of expression of Fas appeared to be a key factor in the determination of resistance, although it is possible that CD40L also initiated proapoptotic changes in Fas signaling per se. The latter suggestion appears inconsistent with the observation that CD40L:CD40 interactions often can lead to protection from apoptosis via up-regulation of bcl-2 family members (42). However, we suggest that CD40L signals both pro- and antiapoptotic changes, which differ in kinetics of expression. Experiments are underway to test this hypothesis.

Eight of twelve cell lines examined expressed detectable levels of Fas, and of these Fas+ cell lines, three were resistant to Fas-mediated apoptosis. In examining the resistant B lymphoma cell lines, they could be separated into the cell lines that could be modulated in their sensitivity and those that remained resistant regardless of stimulating conditions (categories III and IV, respectively). The differential capacity of cell lines to be modulated in their sensitivity indicated blocks at different points along the Fas signaling pathway.

The most revealing B lymphoma cell line was CH33, which could be modulated in its sensitivity to Fas cross-linking by PKC inhibitors. BIS has also been found to increase Fas-induced apoptosis in T cells, suggesting a critical role for PKC in preventing the induction of apoptosis in lymphocytes (43). The finding that PKC inactivation sensitizes CH33 to Fas signaling also suggests that this B lymphoma cell line has an activated phenotype.

Fas signaling in type II targets is proposed to be controlled in part by the Bcl-2 family of proteins (32). Additionally, this family of proteins has been shown to be downstream of PKC activation and involved in resistance to Fas signaling (12, 27, 44, 45). Indeed, partial modulation of antiapoptotic family members (Bcl-2 and Bcl-XL) was observed in CH33 cells treated with BIS. Future experiments defining the stoichiometries of these proteins complexed to proapoptotic family members, such as Bax, will be required to fully assess the role of these proteins in the resistant phenotype of CH33.

Another likely candidate downstream of PKC is NF-{kappa}B (46, 47, 48 ; I. Stancovski and D. Baltimore, unpublished observations). A number of investigators have implicated NF-{kappa}B activation in protection from Fas- and TNFR-mediated apoptosis (31, 49, 50, 51). Again, we find a partial reduction of 20 to 35% in total NF-{kappa}B, but not AP1 or OCT-1, in BIS-treated cells. However, specific isoforms of NF-{kappa}B may be more significantly affected. To fully discern the role of NF-{kappa}B activation, transient tranfection of an I-{kappa}B (inhibitory protein that dissociates from NF-{kappa}B) superrepressor molecule into CH33 is currently being performed in conjunction with anti-Fas killing assays.

Additionally, pretreatment of CH33 with CHX renders the cells highly sensitive to anti-Fas Ab-induced apoptosis. Further studies are required to determine whether a short-lived protein is involved in resistance or if, upon Fas signaling, a new protein is synthesized that protects against apoptosis. It is known that CHX-treated CH33 cells produce increased levels of reactive oxygen intermediates compared with untreated cells following anti-Fas cross-linking (M. Williams and C. M. Mueller, unpublished). Although reactive oxygen species are thought to be involved in the intracellular signals leading to up-regulation of FasL in T cells (52, 53), the role these intermediates play in promoting B cell apoptosis remains to be determined. Interestingly, Giri and Aggarwal (54) have suggested a link between reactive oxygen intermediates and NF-{kappa}B in determining the resistance of a T lymphoma cell line to TNF-mediated apoptosis. This needs to be tested in CHX-treated CH33 cells.

The block in Fas signaling in WEHI-231 appears to be within the DISC complex. The Fas receptor on WEHI-231 was capable of recruiting FADD, suggesting that the death domain of Fas is not mutated. However, no activation of caspase 8 was observed. Western blotting of cFLIP protein levels in DISC immunoprecipitates of WEHI-231 revealed the presence of FLIPL, which could explain the lack of apoptotic signaling in this cell line. It is also possible that a mutation exists in FADD or caspase 8 that prevents the recruitment and activation of caspase 8. A mutation in caspase 8, which increases the size of the molecule by 88 aa and alters the capacity of the enzyme to induce apoptosis, has been found in a human squamous cell carcinoma cell line (55). An intriguing candidate that could play a potential role in the Fas resistance of B lymphomas in vivo is the expression of FasL. FasL has been found to be expressed in several types of human neoplasms, including lung carcinomas (56), esophageal squamous cell carcinomas (57), large granular lymphocytic leukemias (58, 59), NK cell (59) and non-Hodgkin’s lymphomas (60), and myelomas (61). For human myeloma cells, Villunger et al. (61) found that the expression of FasL does not protect cells from apoptosis induced by anti-Fas mAb, as might be expected, or by other FasL+ cells . An additional molecule that has been found to be up-regulated in malignant tissue (lung and colon cancer) and to block FasL is the decoy receptor, DcR3 (62). Since FasL or DcR3 cannot block the effects of anti-Fas, these molecules could not be providing resistance to Fas-induced apoptosis in our assays. However, these molecules could have played a role in the in vivo establishment of the B lymphomas from which both our sensitive and resistant cell lines were derived.

Although much progress has been made, the precise mechanisms by which human lymphomas and leukemias evade immune surveillance in the form of Fas-driven apoptosis remain to be elucidated. Our data emphasize that murine B lymphoma cell lines appear to utilize multiple and distinct molecular mechanisms along the Fas signal transduction cascade to abrogate the induction of apoptosis, as recently described by Gutierrez et al. (63) for human Burkitt lymphoma lines. If Fas-mediated apoptosis is to be harnessed to treat B lymphomas, then therapeutic agents specific to each unique blockade may need to be considered. The panel of B lymphoma cell lines examined herein, as well as their in vivo carried counterparts, provides a useful model system for evaluating conditions or agents that have potential for modifying resistance to Fas-mediated apoptosis.


   Acknowledgments
 
We thank Dr. Wendy Davidson for critically reading the manuscript and providing helpful suggestions. We also thank Drs. Richard Duke, Marilyn Kehry, Donald Nicholson, Wendy Davidson, Achsah Keegan, Mark Williams, and Jose Zamorano for providing reagents for this study.


   Footnotes
 
1 This work was supported by U.S. Public Health Service National Institutes of Health Training Grant, T32 HL07698, as well as ROI CA55644 and the American Red Cross. Back

2 Address correspondence and reprint requests to Dr. David W. Scott, Department of Immunology, Holland Laboratory, American Red Cross, 15601 Crabbs Branch Way, Rockville, MD 20855. Back

3 Abbreviations used in this paper: CHX, cycloheximide; BCR, B cell receptor; BIS, bisindolylmaleimide; cFLIPL, FLICE-like inhibitory protein (long form); DISC, death-inducing signaling complex; FADD, Fas-associated death domain protein; HBDDE, 2,2',3,3',4,4'-hexahydroxyl-l,1'-biophenyl-6,6'-dimethanol dimethyl ether; PI, propidium iodide; PKC, protein kinase C; TPCK, N-tosyl-L-phenylalanine-chloromethyl ketone. Back

Received for publication December 6, 1999. Accepted for publication May 31, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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