The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Snow, A. L.
Right arrow Articles by Martinez, O. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Snow, A. L.
Right arrow Articles by Martinez, O. M.
The Journal of Immunology, 2001, 167: 5404-5411.
Copyright © 2001 by The American Association of Immunologists

Resistance to Fas-Mediated Apoptosis in EBV-Infected B Cell Lymphomas Is Due to Defects in the Proximal Fas Signaling Pathway1

Andrew L. Snow2,*, Linda J. Chen2, Ronald R. Nepomuceno, Sheri M. Krams*, Carlos O. Esquivel and Olivia M. Martinez3,*

* Program in Immunology and {dagger} Department of Surgery, Stanford University School of Medicine, Stanford, CA 94305


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Post-transplant lymphoproliferative disorder is characterized by the outgrowth of EBV-infected B cell lymphomas in immunosuppressed transplant recipients. Using a panel of EBV-infected spontaneous lymphoblastoid cell lines (SLCL) derived from post-transplant lymphoproliferative disorder patients, we assessed the sensitivity of such lymphomas to Fas-mediated cell death. Treatment with either an agonist anti-Fas mAb or Fas ligand-expressing cells identifies two subsets of SLCL based on their sensitivity or resistance to Fas-driven apoptosis. Fas resistance in these cells cannot be attributed to reduced Fas expression or to mutations in the Fas molecule itself. In addition, all SLCL are sensitive to staurosporine-induced cell death, indicating that there is no global defect in apoptosis. Although all SLCL express comparable levels of Fas signaling molecules including Fas-associated death domain protein, caspase 8, and caspase 3, Fas-resistant SLCL exhibit a block in Fas-signaling before caspase 3 activation. In two SLCL, this block results in impaired assembly of the death-inducing signaling complex, resulting in reduced caspase 8 activation. In a third Fas-resistant SLCL, caspase 3 activation is hindered despite intact death-inducing signaling complex formation and caspase 8 activation. Whereas multiple mechanisms exist by which tumor cells can evade Fas-mediated apoptosis, these studies suggest that the proximal Fas-signaling pathway is impeded in Fas-resistant post-transplant lymphoproliferative disorder-associated EBV+ B cell lymphomas.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Programmed cell death mediated by CD95/Fas, a well-characterized member of the TNF receptor superfamily, is critical for immune homeostasis, including the down-regulation of immune responses and the elimination of autoreactive T cells. Fas-mediated apoptosis is also an important mechanism by which CTLs and NK cells that express Fas ligand (FasL)4 eliminate transformed and virally infected cells (1, 2). FasL binds to and induces the trimerization of Fas on target cells, initiating signal transduction with the formation of the death-inducing signaling complex (DISC). DISC assembly involves the recruitment of the adapter molecule Fas-associated death domain protein (FADD)/Mort1 to the cytoplasmic tail of the Fas receptor through homophilic death domain interactions (3). FADD, in turn, recruits the zymogen form of caspase 8 (also known as FADD-like IL-1{beta}-converting enzyme (FLICE)) through homologous interactions between death effector domains on both molecules (4, 5), resulting in the autocatalytic cleavage of procaspase 8. The active caspase 8 heterotetramer is released from DISC and initiates the caspase cascade by cleaving the effector caspase 3. Effector caspase activation leads to DNA fragmentation and degradation of key cellular proteins, completing the cell death program.

Tumor cells have evolved multiple mechanisms to evade Fas-mediated apoptosis (6). Elevated levels of Bcl-2, a well-characterized anti-apoptotic molecule first discovered in follicular B cell lymphomas, are found in many different human cancers (7, 8). Expression of functional FasL by the tumor cell itself may also confer protection by deleting anti-tumor lymphocytes, a phenomenon known as tumor counterattack (2). Additionally, alternative splicing of Fas transcripts can give rise to soluble forms of Fas, which have been proposed to contribute to apoptosis resistance by competing with membrane-bound Fas for available FasL molecules (9). Furthermore, the loss of function lpr and gld mutations in Fas and FasL, respectively, associated with autoimmune lymphoproliferative disease in mice in which activated lymphocytes are not eliminated by apoptosis also serve to demonstrate the potential for tumor development through Fas dysregulation (10, 11). More recently, viruses such as Kaposi’s sarcoma-associated human herpesvirus and human molluscipoxvius were found to encode catalytically inactive homologues of caspase 8 known as FLICE inhibitory proteins that are suggested to act as dominant negative proteins that interfere with Fas-induced DISC formation (12, 13, 14). Indeed, malignancies linked to oncogenic viruses often demonstrate resistance to apoptosis, although the specific mechanisms through which viruses directly or indirectly prevent cell death programs within tumor cells remain elusive.

Post-transplant lymphoproliferative disorder (PTLD) is a significant complication in immunosuppressed patients following organ transplantation (15, 16). PTLD is a phenotypically complex disorder ranging from benign hyperplasia to malignant lymphoma and is most commonly characterized by the development of EBV-associated B cell lymphomas (16, 17). EBV-specific CTL normally control the population of EBV-infected B cells in immunocompetent individuals, precluding tumorigenicity. However, immunosuppressive drugs used to prevent graft rejection debilitate EBV-specific T cells and permit the hyperproliferation and lymphomagenesis of B cell hosts by EBV, a potent transforming virus in this context. EBV infection is of particular concern in pediatric transplant recipients, since primary infection of seronegative transplant patients is more likely to lead to PTLD (15). Currently, treatment of PTLD primarily involves the reduction of immunosuppressive therapy to restore the T cell compartment, with the unfortunate consequence of increasing the risk of graft rejection. Conversely, potent immunosuppression may select for highly proliferative B cell lymphomas that cannot be brought to regression later by competent EBV-specific CTL.

To study these B cell lymphomas in vitro, we previously generated EBV-infected spontaneous lymphoblastoid cell lines (SLCL) derived from transplant recipients diagnosed with PTLD. We demonstrated that IL-10 is an important autocrine growth factor for these lymphomas (18). To date, mechanisms of apoptosis in PTLD-derived B cell lymphomas have not been examined in detail. In this study we investigate programmed cell death triggered by Fas in these cells and demonstrate that two subsets of SLCL can be identified based on their sensitivity or resistance to Fas-mediated apoptosis. We show that resistance to Fas-driven apoptosis in SLCL is not due to alterations in the Fas molecule itself, but rather occurs via a block in Fas signal transduction that precludes caspase 3 activation. Thus, defects in the proximal Fas signaling pathway contribute to Fas resistance in PTLD-associated EBV+ B cell lymphomas.


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

SLCL are EBV-transformed B cell lines derived from the peripheral blood (MF4, VB5, JB7, JC62) or lymph node (AB5) of patients diagnosed with PTLD. As previously described, the SLCL are phenotypically, morphologically, and functionally similar to EBV B cell lymphomas that arise in vivo during PTLD (18). The AB5 cell line was generated from a lymph node tumor biopsy taken from a pediatric kidney recipient diagnosed with PTLD. The tumor mass was disrupted into a single-cell suspension, established and grown in complete medium (see below) without exogenous growth factors or cyclosporine A. AB5 was classified as an SLCL according to EBV positivity as determined by PCR detection of the viral genome, the expression of B cell activation markers, and cytokine production as described for other SLCL (18). The Jurkat T cell line (TIB-152), the SKW 6.4 EBV+ B cell line (TIB-215), and the Daudi EBV+ Burkitt’s lymphoma cell line (CCL-213) were acquired from American Type Culture Collection (Manassas, VA). Human cell lines were maintained in RPMI 1640 (Life Technologies, Gaithersburg, MD) supplemented with 10% heat-inactivated FBS (Cellgro, Herndon, VA), 50 U/ml penicillin, and 50 µg/ml streptomycin (Life Technologies) at 37°C with 5% CO2. Mouse L fibroblasts transfected with murine FasL (FasL+ cells) and control L cells transfected with a plasmid containing the murine FasL in the reverse orientation were provided by Drs. T. Ferguson and J. Russell (Washington University, St. Louis, MO). L cells were maintained in DMEM with 10% heat-inactivated FBS, 50 U/ml penicillin, and 50 µg/ml streptomycin and cultured at 37°C with 5% CO2. Unless otherwise specified, all other reagents were obtained from Sigma (St. Louis, MO).

Induction and detection of apoptosis

Cells (1 x 106 cells/ml) were cultured for 24 h at 37°C in the presence of mouse anti-human Fas (IgM clone CH-11; Coulter-Immunotech, Marseilles, France; 200 ng/ml) or an isotype-matched IgM control Ab. In some experiments the protein kinase inhibitor staurosporine was used at 2 µM. Alternatively, SLCL and Jurkat cells (1 x 106 cells) were cocultured with FasL+ cells or control L cells or were treated with anti-Fas mAb CH-11 (200 ng/ml) for 24–48 h in 24-well plates. The ratio of L cells to SLCL ranged from 1:2 to 1:32. For both methods of Fas ligation, the percentage of apoptotic cells was determined by cell cycle analysis using propidium iodide (PI) staining and flow cytometry as previously described (19). Briefly, cells were washed twice in HBSS containing 0.2% BSA and 0.02% sodium azide and incubated on ice with 50 µg/ml PI, 0.1% Triton X-100, 1 mg/ml sodium citrate, and 1 mg/ml RNase A for 30 min. Twenty thousand cells were acquired and analyzed on a FACScan flow cytometer using CellQuest software (BD Biosciences, Mountain View, CA). Gates were established on viable cells using forward and side scatter parameters, and apoptotic cells were quantified as the percentage of cells with hypodiploid DNA content. Additionally, apoptosis was quantified using an annexin V-enhanced green fluorescent protein (EGFP) Apoptosis Detection kit (BioVision Research Products, Palo Alto, CA) according to the manufacturer’s protocol. Briefly, cells were pelleted and resuspended in 0.5 ml of the provided 1x binding buffer. Five microliters of annexin V-EGFP and 5 µl PI were added to each sample and incubated at room temperature for 5 min in the dark. Cells were immediately analyzed by flow cytometry as described above. Apoptotic cells were quantified as the percentage of treated cells with increased EGFP fluorescence compared with untreated cells (+IgM control Ab).

Fas cell surface expression

To determine Fas (CD95) expression, 1 x 106 cells were washed twice in cold HBBS containing 0.2% BSA and 0.02% sodium azide (FACS buffer) and incubated for 30 min on ice with 10 µg/ml PE-conjugated mAb to human Fas (BD PharMingen, San Diego, CA) or an isotype-matched control Ab in 100 µl FACS buffer. After washing twice in FACS buffer, 20,000 cells were analyzed by flow cytometry. Only viable cells established using forward and side scatter parameters were used for analysis.

Amplification and cloning of Fas gene transcripts

Total RNA was isolated from the SLCL and Jurkat cells using TRIzol reagent (Life Technologies). Three micrograms of total RNA was used in cDNA synthesis reactions with oligo(dT) primers and AMV reverse transcriptase (Roche, Indianapolis, IN). Fas transcripts were amplified using the oligonucleotide primers 5'-CACTTCGGAGGATTGCTCAACA-3' (sense) and 5'-TATGTTGGCTCTTCAGCGCTA-3' (antisense; Life Technologies), which flank the full-length 1167-bp coding region of the Fas gene. PCR was performed using Gene Amp PCR core reagents (PerkinElmer, Branchburg, NJ). After an initial 5-min denaturation at 95°C, PCR amplification was conducted for 35 cycles of denaturation for 70 s at 94°C, annealing for 30 s at 60°C, and extension for 60 s at 72°C. PCR products were cloned into the pT-Adv vector (Clontech, Palo Alto, CA), and individual clones were sequenced by the Stanford University Protein and Nucleic Acid facility. Sequence analysis was performed using the DNAstar Lasergene for Windows software package (DNAstar, Madison, WI).

Western blots

For analysis of endogenous protein levels, 1 x 107 cells were washed in cold PBS and lysed in 100 µl 50 mM Tris (pH 7.4), 1% Nonidet P-40, 0.5% deoxycholate, 150 mM NaCl, 0.5 mM EDTA, 2 mM PMSF, 5 µg/ml each of aprotinin and leupeptin, and 0.5 µg/ml pepstatin A on ice for 30 min. Lysates were centrifuged at 13,000 rpm for 5 min at 4°C to remove insoluble material, and the protein concentration of the lysates was determined using the DC protein assay (Bio-Rad, Hercules, CA). Thirty micrograms of each lysate was separated by 12% SDS-PAGE under reducing conditions and transferred to nitrocellulose. Following blocking with 5% BSA in TBST, blots were probed for 2 h with primary Abs diluted in 3% BSA in TBST. The following primary Abs were used: rabbit anti-human caspase 8 (BD PharMingen; diluted 1/1,500); mouse anti-human FADD (BD PharMingen; diluted 1/1,000); rabbit anti-human caspase 3 (BD PharMingen; diluted 1/1,000), and mouse anti-human {beta}-actin (AC-15; diluted 1/8,000). Following three 5-min washes in TBST, bound Abs were detected using appropriate secondary Abs (peroxidase-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA; diluted 1/50,000) or peroxidase-conjugated sheep anti-mouse Ig (Amersham Pharmacia, Piscataway, NJ; diluted 1/2,000)) for a 1-h incubation at room temperature. Blots were washed as described above and developed using an ECL kit (Amersham Pharmacia).

To assess caspase activation following Fas ligation, 5 x 106 cells (1 x 106 cells/ml) were treated for 0, 2, or 4 h at 37°C with 200 ng/ml mouse anti-Fas (CH-11), and lysates were prepared as described above. Western blotting was performed on 30 µg of each lysate as described above using rabbit anti-human caspase 8 or rabbit anti-human caspase 3 capable of detecting both pro and cleaved forms of caspase 8 and caspase 3, respectively.

Immunoprecipitation of Fas-DISC complexes

Immunoprecipitation of the Fas-DISC was performed as described previously (20). A total of 1 x 107 cells in 1 ml were cultured in the presence of 3 µg anti-Fas mAb APO1–3 IgG3 {kappa} at 37°C for 2 h. Cells were washed once in cold PBS and lysed in 1 ml lysis buffer (30 mM Tris (pH 7.4), 1% Triton X-100, 10% glycerol, 150 mM NaCl, 1 mM PMSF, 5 µg/ml each of aprotinin and leupeptin, and 0.5 µg/ml pepstatin A) for 15 min on ice. Following removal of insoluble material by centrifugation, Fas-DISC complexes were precipitated using 10 µl GammaBind Sepharose beads (Amersham Pharmacia) for 4 h at 4°C. Precipitates were washed four times in 1 ml cold lysis buffer and resuspended in 20 µl 2x reducing Laemmli SDS-sample buffer. Samples were boiled for 3 min, separated by 12% SDS-PAGE, and transferred to nitrocellulose. Membranes were immunoblotted with anti-FADD and anti-caspase 8 Abs as described above or with anti-Fas (C20, Santa Cruz Biotechnology, Santa Cruz, CA; diluted 1/1000) Abs and detected by ECL.

Caspase colorimetric assays

Caspase 8 and caspase 3 activities were examined using commercially available colorimetric kits (BioVision, Palo Alto, CA). Briefly, 5 x 106 cells (1 x 106 cells/ml) were cultured for 24 h with anti-Fas mAb (CH-11) or an IgM isotype control Ab as described above. Cells were recovered, washed twice using PBS/10% FBS, and lysed with the provided buffer for 10 min on ice. Lysates were centrifuged at 13,000 rpm for 5 min at 4°C to remove insoluble material, and the protein concentration of the lysates was determined using the DC protein assay (Bio-Rad). The substrates Asp-Glu-Val-Asp-p-nitroaniline (pNA) or Ile-Glu-Thr-Asp-pNA (200 µM, final concentration) were incubated with 80 µg total protein, and the OD at 405 nm was determined at 1 and 120 min for both caspase 3 or caspase 8, respectively. The data are recorded as the OD405 at 120 min - the OD405 at 1 min.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Sensitivity of SLCL to Fas-mediated apoptosis

To study EBV+ B cell lymphomas associated with PTLD, we generated several SLCL from the peripheral blood of pediatric transplant patients diagnosed with PTLD following liver transplantation. These B cell lines were transformed by EBV in vivo and were propagated from primary culture of PBMC in the absence of exogenous virus or growth factors. In one case (AB5) the SLCL was derived from a lymph node tumor biopsy originating from a kidney transplant patient with PTLD. We have previously shown that these cell lines possess an activated B cell phenotype and depend on IL-10 as an autocrine growth factor (18).

A panel of five SLCL was tested for sensitivity to Fas-induced apoptosis using the Fas cross-linking mAb CH-11. Following 24-h treatment with CH-11, cell cycle analysis with PI was used to distinguish apoptotic cells with sub-G1 hypodiploid DNA content. Annexin V staining was also used to identify cells in the early stages of apoptosis. Both PI analysis and annexin V staining identified two subsets of SLCL based on relative sensitivity to Fas-driven apoptosis, and the histograms of representative cell lines are shown in Fig. 1Go, A and B. Two SLCL, VB5 and MF4, showed similar Fas-dependent increases in apoptosis compared with the control Jurkat T cell line as demonstrated by both PI analysis and annexin V staining (Fig. 1Go, C and D). In contrast, the SLCL JC62, JB7, and AB5 all exhibited marked resistance to Fas-mediated apoptosis (Fig. 1Go, C and D). Daudi cells, an EBV+ Burkitt’s lymphoma line, served as an additional control for Fas resistance. A higher percentage of apoptotic cells was identified by annexin V staining compared with PI analysis, reflecting cells in the early stages of apoptosis before DNA fragmentation (Fig. 1Go, compare D with C). Nevertheless, the same pattern of relative sensitivity to Fas-induced apoptosis emerged when measured by either assay. To verify that the Fas-resistant SLCL were not simply refractory to CH-11 stimulation, SLCL were cultured with mouse L cell fibroblasts expressing recombinant FasL. The same pattern of Fas-induced apoptotic sensitivity and resistance by SLCL was seen (data not shown), demonstrating that the response by SLCL to Fas ligation is physiologically relevant.



View larger version (18K):
[in this window]
[in a new window]
  		FIGURE 1. Sensitivity of SLCL to Fas-mediated apoptosis. A, Cells (1 x 106 cells/ml) were cultured in 1 ml 200 ng/ml anti-Fas mAb CH-11 or an IgM isotype-matched control mAb. After 24 h cells were stained with PI and analyzed by flow cytometry. The percentage of viable cells with hypodiploid DNA content is indicated on the left of each histogram. Data from four representative cell lines are shown. B, Cells were cultured as described above, stained with annexin V-EGFP, and analyzed by flow cytometry. Histogram data from four representative cell lines are shown. C, Cells were cultured and analyzed as described above, and the percentage of apoptotic cells is shown for each cell line. Data are the mean ± SEM of three separate experiments. D, Cells were cultured as described above, stained with annexin V-EGFP, and analyzed by flow cytometry. The percentage of annexin V-positive apoptotic cells is shown for each cell line. Data are the mean ± SEM of three separate experiments.

 
Fas cell surface expression

To begin to address the differential sensitivity to Fas-mediated cell death in our cell lines, SLCL were stained for surface expression of Fas and analyzed by flow cytometry. All five SLCL express high levels of Fas compared with the Fas+ Jurkat T cells, whereas Daudi cells express low levels of Fas (Fig. 2Go). Although it should be noted that VB5 and MF4 cells have slightly higher levels of Fas expression than the other SLCL, resistance to Fas-induced apoptosis in JC62, JB7, and AB5 cells cannot be explained by a marked difference in the extent of Fas expression. Indeed, Jurkat T cells demonstrate high levels of Fas-mediated apoptosis (~70%; Fig. 1GoC) despite expressing less Fas than the Fas-resistant SLCL. Furthermore, cloning and sequencing of the Fas molecule from each SLCL revealed no sequence mutations or deletions (data not shown), implying the Fas receptor itself is not defective in Fas-resistant cell lines.



View larger version (34K):
[in this window]
[in a new window]
  		FIGURE 2. Cell surface expression of Fas on SLCL. Cells (1 x 106) were stained for Fas expression using a PE-conjugated anti-CD95/Fas mAb ({blacksquare}) or a PE-conjugated isotype-matched control mAb ({square}) and analyzed by flow cytometry. Mean fluorescence intensities of each cell line: JC62, 376.51; JB7, 277.13; AB5, 436.49; VB5, 447.58; MF4, 543.47; Jurkat, 126.66; and Daudi, 11.01. Data are representative of at least two separate experiments.

 
Staurosporine-induced apoptosis in SLCL

We next tested whether SLCL were sensitive to a non-Fas-dependent apoptotic stimulus using the protein kinase inhibitor staurosporine. All SLCL underwent increased apoptotic cell death following 24-h treatment with staurosporine as measured by cell cycle analysis (Fig. 3Go). The SLCL AB5 showed less cell death in response to staurosporine, which may reflect broader resistance to apoptotic stimuli in the cells that were derived from a more advanced, solid tumor. In addition, Daudi cells consistently demonstrated resistance to staurosporine-induced apoptosis, suggesting that Daudi cells have additional mechanisms to resist apoptotic stimuli. Nevertheless, these results indicate that none of the SLCL possesses a significant global defect in programmed cell death.



View larger version (19K):
[in this window]
[in a new window]
  		FIGURE 3. Sensitivity of SLCL to staurosporine-induced apoptosis. Cells (1 x 106) were cultured for 24 h in the absence or the presence of the protein kinase inhibitor staurosporine (2 µM), then stained with PI and analyzed by flow cytometry. The percentage of apoptotic cells is shown for each cell line. Data are the mean ± SEM of three separate experiments.

 
Expression of Fas signaling molecules in SLCL

Following our observations of differential sensitivity to Fas-induced apoptosis in the SLCL, we examined components of the Fas signaling pathway in these cell lines to explore the possible mechanism of Fas resistance. First, basal levels of important Fas signaling proteins in each cell line were determined by Western blotting of lysates prepared from untreated cells. The expression of FADD, caspase 8, and caspase 3 in all five SLCL examined was comparable to that in Fas-sensitive Jurkat control cells (Fig. 4Go), implying that each SLCL contains the critical proteins necessary for propagating signals for cell death following ligation of the Fas receptor.



View larger version (46K):
[in this window]
[in a new window]
  		FIGURE 4. Expression of FADD, caspase 8, and caspase 3 in SLCL. Thirty micrograms of protein from whole cell lysates of each cell line was separated by 12% SDS-PAGE under reducing conditions. After transfer to nitrocellulose, expression levels of FADD, caspase 8, and caspase 3 were determined by Western blotting using Abs against the proteins indicated on the left. {beta}-Actin served as a loading control. The blot shown is representative of at least three separate experiments.

 
DISC formation in SLCL

To determine whether there are defects in DISC formation in Fas-resistant cell lines, experiments were conducted to compare the recruitment of FADD and caspase 8 to the Fas-induced DISC in each SLCL. Following cross-linking of the Fas receptor with the agonist anti-Fas mAb APO1–3, immunoprecipitated protein complexes were examined by Western blotting to compare levels of coimmunoprecipitated FADD and caspase 8 in each cell line. Treatment with 1 µg/ml APO1–3 mAb induced a similar pattern of relative sensitivity to Fas-mediated apoptosis when assayed by cell cycle analysis (data not shown). As expected, both FADD and caspase 8 coimmunoprecipitated with the Fas receptor in the sensitive VB5 and MF4 cells following anti-Fas treatment, suggesting that DISC formation is intact in these cell lines (Fig. 5Go, lanes 14 and 16). Furthermore, bands corresponding to activated forms of caspase 8 (p43/41, p23) were clearly detected in VB5 and MF4 cells following 2 h of anti-Fas treatment, implying that the Fas-induced DISC in these cells is also functional for signaling. However, significantly reduced amounts of FADD and caspase 8 were found in the Fas-DISC complexes precipitated from JB7 and AB5 cells, consistent with their resistance to Fas-driven apoptosis (Fig. 5Go, lanes 10 and 12). This result suggests that a block or defect in Fas signaling exists at the level of DISC formation in these Fas-resistant cell lines. Interestingly, the small amounts of caspase 8 that are detected in JB7 and AB5 lysates also display some degree of activation, as demonstrated by the presence of p43/41 and p23 cleavage products.



View larger version (43K):
[in this window]
[in a new window]
  		FIGURE 5. DISC formation in SLCL following Fas ligation. The recruitment of FADD and caspase 8 to the Fas receptor was assessed in each cell line by cross-linking Fas with the anti-Fas mAb APO1–3 for 2 h (+) and immunoprecipitating Ab-bound complexes using protein G-Sepharose beads. As a control, APO1–3 was added to untreated cells after lysis (-). Coprecipitating proteins were detected by Western blotting using the Abs indicated on the left. The positions of the detected proteins are indicated on the right. IgH, H chain of the immunoprecipitating anti-Fas mAb. The blots shown are representative of at least three separate experiments.

 
Surprisingly, JC62 cells that are resistant to Fas-mediated apoptosis have higher levels of FADD and caspase 8 in the Fas-induced DISC than the resistant JB7 and AB5 cell lines (Fig. 5Go, compare lane 8 to lanes 10 and 12). This implies that a defect in Fas signaling may occur farther downstream in the JC62 cell line. Conversely, the p23 subunit of activated caspase 8 is much less prevalent in the JC62 Fas-DISC consistent with the lower levels detected in the other Fas-resistant cells. This reduction could contribute to the hampered Fas signaling in the JC62 cells despite proper DISC formation.

As an additional positive control for DISC formation, the EBV-transformed B cell line SKW 6.4 was used. Previous reports have established SKW 6.4 cells as type I cells, for which most procaspase 8 molecules are activated at the DISC following Fas ligation (21). Consistent with these findings, SKW 6.4 cells demonstrated significant DISC formation following anti-Fas treatment, with large amounts of caspase 8 and FADD coprecipitating with the Fas receptor (Fig. 5Go, lane 6), similar to the VB5 and MF4 SLCL.

Jurkat T cells demonstrate no appreciable DISC formation upon Fas ligation, as evidenced by the lack of detectable FADD or caspase 8 recruitment to the Fas receptor (Fig. 5Go, lane 2). This observation is consistent with previous studies establishing Jurkat cells as type II Fas-sensitive cells, in which almost all caspase 8 activation occurs without DISC formation (21) (Fig. 6Go). Daudi cells also show no detectable FADD or caspase 8 recruitment following Fas cross-linking, which may be partially explained by the lower levels of cell surface Fas expression observed by both flow cytometry (Fig. 2Go) and Western blotting (Fig. 5Go, lanes 3 and 4). It should be noted that less Fas receptor was detected in Jurkat and Daudi cells treated with anti-Fas to induce DISC formation before lysis (Fig. 5Go, lanes 2 and 4, +), as opposed to control samples in which the anti-Fas mAb was added after lysis (Fig. 5Go, lanes 1 and 3, -). It is likely that some anti-Fas mAb was lost while washing the cells that were treated with Ab before lysis, explaining why less Fas receptor was immunoprecipitated in those samples. Indeed, a faint band corresponding to the APO1–3 Ab H chain can be seen in all samples treated with Ab after lysis due to cross-reactivity with the peroxidase-conjugated secondary Abs, implying that more immunoprecipitating Ab is present in those precipitates (Fig. 5Go, - lanes). When supplementary Ab is added to lysates made from cells treated with APO1–3 before lysis, comparable amounts of Fas was immunoprecipitated (data not shown).



View larger version (38K):
[in this window]
[in a new window]
  		FIGURE 6. Caspase 8 and caspase 3 activation in SLCL. Cells (1 x 106/ml) were stimulated for 0, 2, or 4 h in 5 ml 200 ng/ml anti-Fas mAb CH-11. After lysis 30 µg protein from each sample was separated by 12% SDS-PAGE (reduced) and transferred to nitrocellulose. Blots were probed with Abs capable of recognizing both the zymogen and cleaved subunits of caspase 8 and caspase 3, indicated on the right. The blots shown are representative of at least three separate experiments.

 
Caspase 8 and caspase 3 activation in SLCL

Results derived from examining DISC formation in each SLCL led us to investigate the kinetics of caspase 8 and caspase 3 activation following anti-Fas treatment. Cells were stimulated with the anti-Fas mAb CH-11 for 0, 2, or 4 h, and lysates made from these cells were immunoblotted with Abs capable of recognizing both the zymogen and cleaved forms of each caspase. In Jurkat T cells, both caspase 8 and caspase 3 activation peaked at 2 h, as evidenced from the presence of distinct bands corresponding to the p43/41 and p17 cleavage products of caspase 8 and caspase 3, respectively (Fig. 6Go, lane 2). By 4 h the levels of these activated subunits only decreased slightly (Fig. 6Go, lane 3). These results agree with previous reports on caspase 8 and 3 activation in Jurkat cells (21).

In Fas-sensitive VB5 and MF4 SLCL, both caspase 8 and caspase 3 are activated with similar kinetics compared with Jurkat T cells (Fig. 6Go, lanes 16–21), even though most caspase 8 activation is presumably occurring within the Fas-induced DISC in these cell lines as opposed to type II Jurkat cells. Interestingly, the presence of activated forms of both caspases decreases by 4 h of treatment in conjunction with an apparent increase in the amounts of zymogen forms (Fig. 6Go, lanes 18 and 21). This result is even more pronounced at 8 h (data not shown), suggesting that both caspase 8 and caspase 3 activations are transient in these cells despite efficient Fas-induced apoptosis by 24 h of treatment.

In Fas-resistant JB7 and AB5 cells, little or no caspase 8 and caspase 3 activation is detected by Western blot (Fig. 6Go, lanes 10–15), consistent with the levels of caspase activation in the DISC immunoprecipitation experiments (Fig. 5Go). The absence of cleaved subunits of either caspase persisted even after 8 h of Fas ligation (data not shown), suggesting the activation of caspase 8, and subsequently caspase 3, in these cell lines is not delayed, but rather is substantially blocked. In contrast, the activated p43/41 forms of caspase 8 are readily detectable in Fas-resistant JC62 cells (Fig. 6Go, upper panel, lanes 8 and 9), also consistent with the presence of these activated subunits in the DISC. However, the activated p17 form of caspase 3 is absent in JC62 cells following Fas cross-linking (Fig. 6Go, lower panel, lanes 8 and 9), suggesting a potential Fas signaling defect in this cell line that can be localized between caspase 8 and caspase 3 activation. Daudi cells again served as a negative control, showing no detectable caspase activation (Fig. 6Go, lanes 5 and 6) even after 8 h of anti-Fas treatment (data not shown). Caspase activation was also confirmed using colorimetric assays, in which specific pNA-labeled peptide substrates for caspase 8 and caspase 3 are incubated with cell lysates treated for 24 h with anti-Fas mAb or an IgM control. Substrate cleavage by activated caspases releases free pNA, which can be quantified at 405 nm on a spectrophotometer. Data generated from colorimetric assays confirmed the same pattern of caspase 8 and caspase 3 activation noted by Western blot for all cell lines (data not shown). Thus, the block in Fas-mediated apoptosis in resistant SLCL appears to occur before caspase 3 activation, probably resulting from inhibition of caspase 8 activation in the DISC or inhibition of caspase 3 cleavage by activated caspase 8.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study we investigated Fas-mediated apoptosis in a panel of B cell lymphomas (SLCL) derived from transplant patients with PTLD, a severe complication arising from the uncontrolled outgrowth of EBV-infected B cells in the context of immunosuppression. We show that a subset of these SLCL is resistant to Fas-induced apoptosis when triggered by either an agonist anti-Fas mAb or FasL-expressing cells. This resistance is not due to reduced Fas expression at the cell surface or to mutations in the full-length Fas receptor that might render it incapable of oligomerization or signaling. Instead, we show that proximal Fas signaling is interrupted in resistant SLCL before appreciable caspase 3 activation. Defects or blocks in the Fas signaling pathway contribute to the impairment of proper DISC formation following Fas stimulation in two cell lines (JB7 and AB5), whereas in a third Fas-resistant cell line (JC62), caspase 3 activation is blocked despite intact DISC formation and caspase 8 cleavage. In contrast, the Fas signaling pathway is functional in Fas-sensitive SLCL (VB5 and MF4), driving the activation of the effector caspase 3 and leading to increased apoptosis.

Cells can be classified into two types according to the Fas signaling pathway used (21). In type I cells caspase 8 is primarily activated at the DISC, as seen in the SKW 6.4 B cell line and our Fas-sensitive SLCL. Caspase 8 activation is evident in the DISC as early as 10 min following Fas engagement (data not shown). In contrast, type II cells such as Jurkat T cells demonstrate reduced DISC assembly, with caspase 8 activation following loss of mitochondrial membrane potential. Although we have yet to extensively study the role of mitochondria in SLCL following Fas stimulation, our data suggest that Fas-resistant JB7 and AB5 cells are not type II cells. Despite impaired DISC formation, the low amounts of activated caspase 8 detected in our blotting experiments appear to be cleaved at the DISC level. Furthermore, we do not detect large amounts of activated caspase 8 following 2 h of anti-Fas treatment in these Fas-resistant SLCL, as would be expected for type II cells.

Fas signaling in JC62 cells appears to be obstructed before caspase 3 activation despite substantial caspase 8 activation at the DISC and the presence of undiminished levels of procaspase 3. It is difficult to discern whether this defect may be due to the specific targeting of caspase 3 or suboptimal generation of activated caspase 8 subunits (i.e., p23 subunit) for signal propagation. One novel explanation is that activated caspase 8 is not released from the DISC to cleave downstream substrates, although this is unprecedented in other Fas-resistant cell types. Another explanation might involve the inhibitor of apoptosis (IAP) family of proteins, which can specifically bind to and inhibit caspase 3 (22, 23). In fact, the human IAP survivin is often expressed in human neoplasms at higher levels than in normal adult tissues (24). IAPs could potentially inhibit caspase 3 activity and subsequent apoptosis in JC62 cells following Fas stimulation, while allowing staurosporine-induced apoptosis to occur through the release of Smac/DIABLO, an IAP inhibitor, from the mitochondria (25, 26).

In the Fas-resistant JB7 and AB5 cell lines, a defect in Fas signaling was detected at the level of DISC formation, resulting in suboptimal recruitment of both FADD and caspase 8 molecules. Although DISC formation appears to be impaired in these resistant cells, the caspase 8 molecules that are recruited to the DISC are capable of autocatalytic cleavage. However, the activated caspase 8 generated appears to be insufficient to induce the downstream apoptotic caspase cascade. Hence, the completion of the Fas-driven apoptotic program may require a higher threshold of caspase 8 activation than generated in JB7 and AB5 cells. Alternatively, the kinetics of DISC formation and/or caspase 8 activation may be delayed in these Fas-resistant cell lines, which may also be attributed to a specific block in proximal Fas signaling. The latter possibility is unlikely, however, since procaspase 8 remains uncleaved in JB7 and AB5 cells up to 8 h following Fas ligation (data not shown).

Another attractive possibility for explaining poor caspase 8 recruitment and activation at the Fas-induced DISC involves cellular FLICE inhibitory protein (cFLIP, also referred to as I-FLICE/FLAME-1/CASH/CLARP/Casper/MRIT/Usurpin) (27, 28, 29, 30, 31, 32, 33, 34). cFLIPexists as two isoforms, cFLIPL and cFLIPS, that structurally resemble caspase 8 and FLICE inhibitory protein, respectively (27, 32, 34). cFLIPL contains two death effector domains and a catalytically inactive caspase domain and has been proposed to have either an anti-apoptotic or a proapoptotic function in regulating Fas-mediated apoptosis depending on cellular context. For instance, Scaffidi and colleagues (35) demonstrated that cFLIPL is cleaved at the DISC upon Fas ligation in stably transfected BJAB cells and remains bound to prevent further recruitment of procaspase 8 into the DISC. Because this effect was only demonstrated when cFLIPL is overexpressed, endogenous levels of cFLIPL may not play a significant role in blocking Fas signaling in SLCL. Furthermore, small amounts of procaspase 8 can be detected at the DISC following 2-h anti-Fas stimulation, suggesting that cFLIP is not blocking the continuous recruitment of procaspase 8. On the other hand, other groups have suggested the C-terminal caspase-like domain of cFLIPL is critical for inducing apoptosis in mammalian cells in overexpression studies (30, 31, 32, 33). cFLIP is expressed in all SLCL at comparable levels (A. L. Snow, unpublished observations), and studies are ongoing to determine the role of cFLIP in Fas signaling in SLCL.

Research conducted on other types of B cell lymphomas to date reveals a myriad of mechanisms by which Fas-mediated apoptosis may be disrupted. For example, a recent report by Mueller and colleagues (36) described decreased Fas expression, activation of protein kinase C and NF-{kappa}B, and increased cFLIP expression all as distinct ways in which Fas-driven apoptosis is dysregulated in murine B cell lymphomas. Other groups have identified a soluble decoy receptor for Fas (DcR3) that is expressed in several types of tumors, including EBV-infected human B cell lymphomas (37, 38). Although this form of immune evasion could prove important for tumor protection against CTL and NK cells expressing FasL, it does not explain resistance to Fas-driven apoptosis in SLCL stimulated with an agonist anti-Fas Ab. More important, a recent report by Tepper and colleagues (39) uncovered defects in DISC formation and reduced caspase 8 activation in EBV-infected Burkitt’s lymphoma cells. They demonstrated that higher ratios of cFLIPL to caspase 8 transcripts in EBV+ Burkitt’s lymphoma cells correlate to increased resistance to Fas-mediated apoptosis, suggesting the modulation of this ratio may be a novel mechanism by which EBV imparts Fas resistance on Burkitt’s lymphoma hosts. Similar EBV-influenced mechanisms may also be at work in SLCL, which display a LCL phenotype as evidenced by constitutive expression of latent membrane protein-1 (LMP-1; A. L. Snow, unpublished observations).

LMP-1 itself has been implicated in host cell resistance to apoptosis through NF-{kappa}B-mediated up-regulation of Bcl-2 and related anti-apoptotic molecules (40, 41, 42, 43, 44). Latency in EBV-infected lymphoblastoid B cells is marked by high expression of LMP-1, which can engage TRAF molecules and force the constitutive activation of NF-{kappa}B in B cell hosts, including SLCL (A. L. Snow, unpublished observations). Both antisense targeting of LMP-1 and direct NF-{kappa}B inhibition can suppress growth and promote apoptosis in otherwise resistant EBV+ LCLs (45, 46). Although the up-regulation of Bcl-2 family members by LMP-1 is probably unrelated to direct type I Fas signaling, viral interference with death receptor-mediated apoptosis remains an intriguing possibility that has yet to be demonstrated in SLCL and related EBV+ cells.

Another study examining susceptibility to Fas-driven apoptosis in PTLD-derived cells showed that EBV+ LCL established from heart or bone marrow allograft recipients are extremely sensitive to apoptosis triggered by high dose agonistic anti-Fas Ab (47). Tumors arising from LCL inoculation in SCID mice could be forced into transient remission when treated with the anti-Fas Ab. In contrast, our study demonstrates that other PTLD-derived EBV+ B cell lymphomas can exhibit strong resistance to Fas-mediated apoptosis. Understanding this resistance on a molecular level, particularly in relation to viral modulation, may aid in the development of more effective therapeutic options involving Fas or other apoptotic stimuli for the treatment of PTLD-associated lymphomas.


   Acknowledgments
 
We thank Dr. Maria Millan (Stanford University School of Medicine) for acquiring the AB5 lymph node biopsy and for helpful discussions.


   Footnotes
 
1 This work was supported by National Institutes of Health Grant R01AI41769 (to O.M.M.) and the Lucile Salter Packard Foundation. A.L.S. was supported by a Howard Hughes predoctoral fellowship in Immunology and a Stanford Graduate Fellowship. L.J.C. was supported by a fellowship from the Northern California Kidney Foundation. Back

2 A.L.S. and L.J.C. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Olivia M. Martinez, Stanford University School of Medicine, 1201 Welch Road, MSLS P312, Stanford, CA 94305-5492. E-mail address: omm{at}stanford.edu Back

4 Abbreviations used in this paper: FasL, Fas ligand; DISC, death-inducing signaling complex; EGFP, enhanced green fluorescent protein; FADD, Fas-associated death domain protein; FLICE, FADD-like IL-1{beta}-converting enzyme; FLIP, FLICE inhibitory protein; cFLIP, cellular FLIP; IAP, inhibitor of apoptosis; LCL, lymphoblastoid cell line; LMP-1, latent membrane protein-1; PI, propidium iodide; pNA, p-nitroaniline; PTLD, post-transplant lymphoproliferative disorder; SLCL, spontaneous lymphoblastoid cell lines. Back

Received for publication May 14, 2001. Accepted for publication August 27, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Krammer, P. H.. 1999. CD95(APO-1/Fas)-mediated apoptosis: live and let die. Adv. Immunol. 71:163.[Medline]
  2. Krammer, P. H.. 2000. CD95’s deadly mission in the immune system. Nature 407:789.[Medline]
  3. Kischkel, F. C., S. Hellbardt, I. Behrmann, M. Germer, M. Pawlita, P. H. Krammer, M. E. Peter. 1995. Cytotoxicity-dependent APO-1 (Fas/CD95)-associated proteins form a death-inducing signaling complex (DISC) with the receptor. EMBO J. 14:5579.[Medline]
  4. Chinnaiyan, A. M., K. O’Rourke, M. Tewari, V. M. Dixit. 1995. FADD, a novel death domain-containing protein, interacts with the death domain of Fas and initiates apoptosis. Cell 81:505.[Medline]
  5. Muzio, M., A. M. Chinnaiyan, F. C. Kischkel, K. O’Rourke, A. Shevchenko, J. Ni, C. Scaffidi, J. D. Bretz, M. Zhang, R. Gentz, et al 1996. FLICE, a novel FADD-homologous ICE/CED-3-like protease, is recruited to the CD95 (Fas/APO-1) death-inducing signaling complex. Cell 85:817.[Medline]
  6. Schmitz, I., S. Kirchhoff, P. H. Krammer. 2000. Regulation of death receptor-mediated apoptosis pathways. Int. J. Biochem. Cell Biol. 32:1123.[Medline]
  7. Chao, D. T., S. J. Korsmeyer. 1998. BCL-2 family: regulators of cell death. Annu. Rev. Immunol. 16:395.[Medline]
  8. Cory, S.. 1995. Regulation of lymphocyte survival by the bcl-2 gene family. Annu. Rev. Immunol. 13:513.[Medline]
  9. Tanaka, M., T. Suda, T. Takahashi, S. Nagata. 1995. Expression of the functional soluble form of human fas ligand in activated lymphocytes. EMBO J. 14:1129.[Medline]
  10. Fisher, G. H., F. J. Rosenberg, S. E. Straus, J. K. Dale, L. A. Middleton, A. Y. Lin, W. Strober, M. J. Lenardo, J. M. Puck. 1995. Dominant interfering Fas gene mutations impair apoptosis in a human autoimmune lymphoproliferative syndrome. Cell 81:935.[Medline]
  11. Rieux-Laucat, F., F. Le Deist, C. Hivroz, I. A. Roberts, K. M. Debatin, A. Fischer, J. P. de Villartay. 1995. Mutations in Fas associated with human lymphoproliferative syndrome and autoimmunity. Science 268:1347.[Abstract/Free Full Text]
  12. Thome, M., P. Schneider, K. Hofmann, H. Fickenscher, E. Meinl, F. Neipel, C. Mattmann, K. Burns, J. L. Bodmer, M. Schröter, et al 1997. Viral FLICE-inhibitory proteins (FLIPs) prevent apoptosis induced by death receptors. Nature 386:517.[Medline]
  13. Hu, S., C. Vincenz, M. Buller, V. M. Dixit. 1997. A novel family of viral death effector domain-containing molecules that inhibit both CD95- and tumor necrosis factor receptor-1-induced apoptosis. J. Biol. Chem. 272:9621.[Abstract/Free Full Text]
  14. Bertin, J., R. C. Armstrong, S. Ottilie, D. A. Martin, Y. Wang, S. Banks, G. H. Wang, T. G. Senkevich, E. S. Alnemri, B. Moss, M. J. Lenardo, K. J. Tomaselli, J. I. Cohen. 1997. Death effector domain-containing herpesvirus and poxvirus proteins inhibit both Fas- and TNFR1-induced apoptosis. Proc. Natl. Acad. Sci. USA 94:1172.[Abstract/Free Full Text]
  15. Penn, I.. 1998. De novo malignances in pediatric organ transplant recipients. Pediatr. Transplant. 2:56.[Medline]
  16. Klein, G.. 1998. Post-transplantation lymphoproliferative disease: introduction. Springer Semin. Immunopathol. 20:319.[Medline]
  17. Nalesnik, M. A.. 1998. Clinical and pathological features of post-transplant lymphoproliferative disorders (PTLD). Springer Semin. Immunopathol. 20:325.[Medline]
  18. Beatty, P. R., S. M. Krams, O. M. Martinez. 1997. Involvement of IL-10 in the autonomous growth of EBV-transformed B cell lines. J. Immunol. 158:4045.[Abstract]
  19. Nicoletti, I., G. Migliorati, M. C. Pagliacci, F. Grignani, C. Riccardi. 1991. A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J. Immunol. Methods 139:271.[Medline]
  20. Hennino, A., M. Berard, M. Casamayor-Pallejà, P. H. Krammer, T. Defrance. 2000. Regulation of the Fas death pathway by FLICE-inhibitory protein in primary human B cells. J. Immunol. 165:3023.[Abstract/Free Full Text]
  21. Scaffidi, C., S. Fulda, A. Srinivasan, C. Friesen, F. Li, K. J. Tomaselli, K. M. Debatin, P. H. Krammer, M. E. Peter. 1998. Two CD95 (APO-1/Fas) signaling pathways. EMBO J. 17:1675.[Medline]
  22. Deveraux, Q. L., J. C. Reed. 1999. IAP family proteins: suppressors of apoptosis. Genes Dev. 13:239.[Free Full Text]
  23. Deveraux, Q. L., H. R. Stennicke, G. S. Salvesen, J. C. Reed. 1999. Endogenous inhibitors of caspases. J. Clin. Immunol. 19:388.[Medline]
  24. Ambrosini, G., C. Adida, D. C. Altieri. 1997. A novel anti-apoptosis gene, survivin, expressed in cancer and lymphoma. Nat. Med. 3:917.[Medline]
  25. Du, C., M. Fang, Y. Li, L. Li, X. Wang. 2000. Smac, a mitochondrial protein that promotes cytochrome c-dependent caspase activation by eliminating IAP inhibition. Cell 102:33.[Medline]
  26. Verhagen, A. M., P. G. Ekert, M. Pakusch, J. Silke, L. M. Connolly, G. E. Reid, R. L. Moritz, R. J. Simpson, D. L. Vaux. 2000. Identification of DIABLO, a mammalian protein that promotes apoptosis by binding to and antagonizing IAP proteins. Cell 102:43.[Medline]
  27. Irmler, M., M. Thome, M. Hahne, P. Schneider, K. Hofmann, V. Steiner, J. L. Bodmer, M. Schröter, K. Burns, C. Mattmann, et al 1997. Inhibition of death receptor signals by cellular FLIP. Nature 388:190.[Medline]
  28. Hu, S., C. Vincenz, J. Ni, R. Gentz, V. M. Dixit. 1997. I-FLICE, a novel inhibitor of tumor necrosis factor receptor-1- and CD-95-induced apoptosis. J. Biol. Chem. 272:17255.[Abstract/Free Full Text]
  29. Srinivasula, S. M., M. Ahmad, S. Ottilie, F. Bullrich, S. Banks, Y. Wang, T. Fernandes-Alnemri, C. M. Croce, G. Litwack, K. J. Tomaselli, et al 1997. FLAME-1, a novel FADD-like anti-apoptotic molecule that regulates Fas/TNFR1-induced apoptosis. J. Biol. Chem. 272:18542.[Abstract/Free Full Text]
  30. Goltsev, Y. V., A. V. Kovalenko, E. Arnold, E. E. Varfolomeev, V. M. Brodianskii, D. Wallach. 1997. CASH, a novel caspase homologue with death effector domains. J. Biol. Chem. 272:19641.[Abstract/Free Full Text]
  31. Inohara, N., T. Koseki, Y. Hu, S. Chen, G. Núñez. 1997. CLARP, a death effector domain-containing protein interacts with caspase-8 and regulates apoptosis. Proc. Natl. Acad. Sci. USA 94:10717.[Abstract/Free Full Text]
  32. Shu, H. B., D. R. Halpin, D. V. Goeddel. 1997. Casper is a FADD- and caspase-related inducer of apoptosis. Immunity 6:751.[Medline]
  33. Han, D. K., P. M. Chaudhary, M. E. Wright, C. Friedman, B. J. Trask, R. T. Riedel, D. G. Baskin, S. M. Schwartz, L. Hood. 1997. MRIT, a novel death-effector domain-containing protein, interacts with caspases and Bcl-XL and initiates cell death. Proc. Natl. Acad. Sci. USA 94:11333.[Abstract/Free Full Text]
  34. Rasper, D. M., J. P. Vaillancourt, S. Hadano, V. M. Houtzager, I. Seiden, S. L. Keen, P. Tawa, S. Xanthoudakis, J. Nasir, D. Martindale, et al 1998. Cell death attenuation by ‘usurpin,’ a mammalian DED-caspase homologue that precludes caspase-8 recruitment and activation by the CD-95 (Fas, APO-1) receptor complex. Cell Death Differ. 5:271.[Medline]
  35. Scaffidi, C., I. Schmitz, P. H. Krammer, M. E. Peter. 1999. The role of c-FLIP in modulation of CD95-induced apoptosis. J. Biol. Chem. 274:1541.[Abstract/Free Full Text]
  36. Mueller, C. M., D. W. Scott. 2000. Distinct molecular mechanisms of Fas resistance in murine B lymphoma cells. J. Immunol. 165:1854.[Abstract/Free Full Text]
  37. Pitti, R. M., S. A. Marsters, D. A. Lawrence, M. Roy, F. C. Kischkel, P. Dowd, A. Huang, C. J. Donahue, S. W. Sherwood, D. T. Baldwin, et al 1998. Genomic amplification of a decoy receptor for Fas ligand in lung and colon cancer. Nature 396:699.[Medline]
  38. Ohshima, K., S. Haraoka, M. Sugihara, J. Suzumiya, C. Kawasaki, M. Kanda, M. Kikuchi. 2000. Amplification and expression of a decoy receptor for fas ligand (DcR3) in virus (EBV or HTLV-I) associated lymphomas. Cancer Lett. 160:89.[Medline]
  39. Tepper, C. G., M. F. Seldin. 1999. Modulation of caspase-8 and FLICE-inhibitory protein expression as a potential mechanism of Epstein-Barr virus tumorigenesis in Burkitt’s lymphoma. Blood 94:1727.[Abstract/Free Full Text]
  40. Henderson, S., M. Rowe, C. Gregory, D. Croom-Carter, F. Wang, R. Longnecker, E. Kieff, A. Rickinson. 1991. Induction of bcl-2 expression by Epstein-Barr virus latent membrane protein 1 protects infected B cells from programmed cell death. Cell 65:1107.[Medline]
  41. Laherty, C. D., H. M. Hu, A. W. Opipari, F. Wang, V. M. Dixit. 1992. The Epstein-Barr virus LMP1 gene product induces A20 zinc finger protein expression by activating nuclear factor {kappa}B. J. Biol. Chem. 267:24157.[Abstract/Free Full Text]
  42. Fries, K. L., W. E. Miller, N. Raab-Traub. 1996. Epstein-Barr virus latent membrane protein 1 blocks p53-mediated apoptosis through the induction of the A20 gene. J. Virol. 70:8653.[Abstract]
  43. Wang, S., M. Rowe, E. Lundgren. 1996. Expression of the Epstein Barr virus transforming protein LMP1 causes a rapid and transient stimulation of the Bcl-2 homologue Mcl-1 levels in B-cell lines. Cancer Res. 56:4610.[Abstract/Free Full Text]
  44. D’Souza, B., M. Rowe, D. Walls. 2000. The bfl-1 gene is transcriptionally upregulated by the Epstein-Barr virus LMP1, and its expression promotes the survival of a Burkitt’s lymphoma cell line. J. Virol. 74:6652.[Abstract/Free Full Text]
  45. Kenney, J. L., M. E. Guinness, T. Curiel, J. Lacy. 1998. Antisense to the Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP-1) suppresses LMP-1 and bcl-2 expression and promotes apoptosis in EBV-immortalized B cells. Blood 92:1721.[Abstract/Free Full Text]
  46. Cahir-McFarland, E. D., D. M. Davidson, S. L. Schauer, J. Duong, E. Kieff. 2000. NF-{kappa}B inhibition causes spontaneous apoptosis in Epstein-Barr virus-transformed lymphoblastoid cells. Proc. Natl. Acad. Sci. USA 97:6055.[Abstract/Free Full Text]
  47. Durandy, A., F. Le Deist, J. F. Emile, K. Debatin, A. Fischer. 1997. Sensitivity of Epstein-Barr virus-induced B cell tumor to apoptosis mediated by anti-CD95/Apo-1/fas antibody. Eur. J. Immunol. 27:538.[Medline]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
A. L. Snow, S. L. Lambert, Y. Natkunam, C. O. Esquivel, S. M. Krams, and O. M. Martinez
EBV Can Protect Latently Infected B Cell Lymphomas from Death Receptor-Induced Apoptosis.
J. Immunol., September 1, 2006; 177(5): 3283 - 3293.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
C. Le Clorennec, I. Youlyouz-Marfak, E. Adriaenssens, J. Coll, G. W. Bornkamm, and J. Feuillard
EBV latency III immortalization program sensitizes B cells to induction of CD95-mediated apoptosis via LMP1: role of NF-{kappa}B, STAT1, and p53
Blood, March 1, 2006; 107(5): 2070 - 2078.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
I. Schmitz, H. Weyd, A. Krueger, S. Baumann, S. C. Fas, P. H. Krammer, and S. Kirchhoff
Resistance of Short Term Activated T Cells to CD95-Mediated Apoptosis Correlates with De Novo Protein Synthesis of c-FLIPshort
J. Immunol., February 15, 2004; 172(4): 2194 - 2200.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
Y.-J. Kim, R. R. Brutkiewicz, and H. E. Broxmeyer
Role of 4-1BB (CD137) in the functional activation of cord blood CD28-CD8+ T cells
Blood, October 16, 2002; 100(9): 3253 - 3260.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
A. Spencer, S.-L. Yeh, K. Koutrevelis, C. Baulch-Brown ;, N. Mitsiades, C. Mitsiades, K. C. Anderson, and S. P. Treon
TRAIL-induced apoptosis of authentic myeloma cells does not correlate with the procaspase-8/cFLIP ratio
Blood, September 26, 2002; 100(8): 3049 - 3050.
[Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Snow, A. L.
Right arrow Articles by Martinez, O. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Snow, A. L.
Right arrow Articles by Martinez, O. M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS