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The Journal of Immunology, 1998, 160: 6083-6092.
Copyright © 1998 by The American Association of Immunologists

Dissection of Pathways Leading to Antigen Receptor-Induced and Fas/CD95-Induced Apoptosis in Human B Cells1

Susanne M. A. Lens2,*, Bianca F. A. den Drijver*, Andy J. G. Pötgens{dagger}, Kiki Tesselaar*, Marinus H. J. van Oers{ddagger} and René A. W. van Lier*

Departments of * Clinical Viro-Immunology and {dagger} Experimental Immunohematology, Central Laboratory of The Netherlands Red Cross Blood Transfusion Service, and Laboratory of Experimental and Clinical Immunology, Academic Medical Center, University of Amsterdam, and {ddagger} Department of Hematology, Academic Medical Center, Amsterdam, The Netherlands


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To dissect intracellular pathways involved in B cell Ag receptor (BCR)-mediated and Fas-induced human B cell death, we isolated clones of the Burkitt lymphoma cell line Ramos with different apoptosis sensitivities. Selection for sensitivity to Fas-induced apoptosis also selected for clones with enhanced BCR death sensitivity and vice versa. In contrast, clones resistant to Fas-mediated apoptosis could still undergo BCR-induced cell death. Based on the functional phenotypes of these clones, we hypothesized that both receptor-induced apoptosis pathways are initially distinct but may eventually converge. Indeed, ligation of both Fas and BCR resulted in cleavage of the IL-1ß-converting enzyme/Ced-3-like protease caspase 3 and its substrates Ac-Asp-Glu-Val-Asp-aldehyde and poly(ADP-ribose) polymerase. Markedly, qualitative differences in the caspase 3 cleavage pattern induced by Fas or BCR ligation were observed; whereas Fas ligation generated caspase 3 cleavage products of 19/20 and 17 kDa, only the latter cleavage product was found upon BCR cross-linking. The caspase inhibitor Val-Ala-Asp-fluoromethylketone blocked both Fas- and BCR-mediated apoptosis, but differentially affected caspase 3 cleavage induced by either stimulus. Finally, overexpression of a Fas-associated death domain (FADD) dominant-negative mutant protein was found to inhibit Fas-induced apoptosis but not BCR-induced apoptosis. Together our findings imply that Fas and BCR couple, via FADD-dependent and FADD-independent mechanisms, respectively, to distinct proteases upstream of caspase 3.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
To ensure self tolerance, sequential selection processes take place during lymphocyte differentiation. A prerequisite for either positive or negative selection of B cells is Ag binding to the B cell Ag receptor (BCR).3 The outcome of this interaction (activation or negative selection) is dependent on the differentiation stage of the B cell, the nature of the Ag (soluble vs membrane bound), the avidity of Ag-BCR interaction, and the availability of Th cell signals (1, 2). Ag receptor-triggered deletion of autoreactive B cell clones was initially thought to occur only in immature B cells (2). However, the fact that in germinal centers, potential autoreactive B cells may arise due to somatic hypermutations, stresses the need for BCR-mediated negative selection of mature B cells. Indeed, studies with murine B cells have shown that cross-linking of the BCR by membrane-bound Ag can result in elimination of mature B cells (3, 4, 5, 6), and recent in vivo experiments demonstrated that germinal center B cells can undergo apoptosis in an Ag-specific manner upon injection of soluble Ag (7, 8, 9). In addition, several mature human B lymphoma cell lines undergo apoptosis upon BCR ligation (10, 11, 12). Unlike Ag receptor-induced cell death in T lymphocytes (9, 13), BCR-induced apoptosis does not involve the interaction between the TNF receptor/TNF family members Fas and Fas ligand (12, 14, 15). However, ligation of Fas does play an important role in negative selection of mature B cells, since humans and mice lacking a functional Fas receptor or Fas ligand show signs of lymphoproliferation and autoimmune diseases with circulating antoantibodies (16, 17, 18, 19).

Apoptosis via BCR or Fas is differentially regulated by Th cell signals. Whereas CD40 ligand and membrane-bound TNF-{alpha} can rescue Burkitt lymphoma cells from Ag receptor-induced apoptosis, both molecules enhance Fas expression and Fas-mediated B cell apoptosis of Burkitt lymphoma cells and germinal center B cells (10, 11, 12, 20, 21, 22). The latter idea suggests that within germinal centers, final positive selection of high affinity, Ag-specific B cells may only occur in the presence of signals that counteract the Fas death signal (23, 24, 25).

Recently, a number of intracellular molecules that couple Fas to the apoptotic pathway have been identified. Ligation of Fas results in binding of the adaptor molecule FADD/MORT to the death domain present in the intracellular tail of Fas (26, 27, 28). FADD, in turn, recruits caspase 8 (FLICE, MACH, Mch5) into the death-signaling complex (29, 30, 31). Caspase 8 is therefore thought to be the first component in a cascade of Fas death-associated ICE/Ced-3-like proteases (31, 32). These evolutionarily conserved cysteine proteases (caspases) are effector molecules of the apoptotic pathway that exist in the cell as inactive proenzymes and become activated upon cleavage. Heterotetramers of the cleaved subunits eventually form the active enzyme (33, 34). Substrates of the caspases include other members of the ICE/Ced-3 family of proteases and structural cellular proteins such as lamin, poly(ADP-ribose) polymerase (PARP), and fodrin, which are components of the DNA repair machinery and cytoskeleton (35, 36, 37, 38). Studies in the T cell line Jurkat have shown that cleavage of caspase 3 (CPP32) into a large (17-kDa) and a small (12-kDa) subunit is a key event in Fas-mediated apoptosis (38, 39), implicating that activation of caspase 3 occurs downstream of that of caspase 8. To date, the caspases involved in BCR-mediated apoptosis have not been identified.

Considering the importance of both BCR- and Fas-mediated cell death in B cell selection, we have here dissected the intracellular apoptotic pathways induced by both receptors. To this end, we have selected clones of the Burkitt lymphoma cell line Ramos that differ in susceptibility to Fas- and BCR-mediated apoptosis, and we have analyzed caspase activity in relation to Fas- and BCR-mediated apoptosis in these clones.


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

The following Abs were produced at the Central Laboratory of The Netherlands Red Cross Blood Transfusion Service (CLB, Amsterdam, The Netherlands): CLB/MH15 (anti-human IgM mAb), CLB-CD40/1 mAb, Fas 10 mAb (a gift from Prof. Dr. L. A. Aarden), horseradish peroxidase-labeled goat anti-mouse and horse anti-rabbit antisera, and FITC-conjugated goat anti-mouse Ig. Caspase 3/CPP32 mAb and FADD mAb were purchased from Transduction Laboratories (Lexington, KY). PARP mAb (clone C-2-10), Ac-DEVD-CHO, Ac-DEVD-pNA, and Ac-YVAD-CHO were purchased from Biomol (Plymouth Meeting, PA), and the caspase inhibitor z-VAD-fmk was obatined from Alexis Biochemicals (Lauferlingen, Switzerland). FITC-labeled annexin V (APOPTEST-FITC) was obtained from Nexins Research (Hoeven, The Netherlands).

Cell culture and cell stimulation

The Burkitt lymphoma cell line Ramos and clones derived from this cell line were cultured in IMDM supplemented with 10% FCS and antibiotics. Cells were stimulated in 24-well plates with the indicated mAb (5 µg/ml or ascites; dilution, 1/1000) at a cell concentration of 0.5 x 106 cells/ml when stimulated for 24 h and at a concentration of 0.2 x 106/ml when longer time-course experiments were conducted.

Flow cytometry

Cells (2 x 105) were washed with ice-cold PBS containing 0.5% BSA (v/v; FACS buffer) and incubated with saturating amounts of mAb (unlabeled or FITC-conjugated) or isotype-matched control mAb for 30 min at 4°C. After washing twice with FACS buffer, FITC-conjugated goat anti-mouse Ig was added to cells that had been incubated with unlabeled mAbs. After washing, 104 viable cells were analyzed on a FACScan (Becton Dickinson, San Jose, CA) using the Consort 30 program.

Detection of apoptotic cells using FITC-labeled annexin V

Phosphatidyl serine exposure on apoptotic cells was measured as described previously (40). Cells (2 x 105) were washed once in ice-cold HEPES buffer (10 mM HEPES, 150 mM NaCl, 5 mM KCl, 1 mM MgCl2, and 1.8 mM CaCl2, pH 7.4) supplemented with 1 mg/ml glucose and 0.5% BSA, and FITC-labeled annexin V was added at a final concentration of 2.5 µg/ml. Cells were incubated for 20 min at 4°C and washed twice with HEPES buffer. Just before analysis on a FACScan, using the Consort 30 program, propidium iodide (PI) was added (final concentration, 5 µg/ml) to the samples to discriminate necrotic cells (annexin V-, PI+) from apoptotic cells (annexin V+, PI- and annexin V+, PI+)

Isolation of Fas death-resistant Ramos clones

Ramos clones resistant to anti-Fas-mediated apoptosis were selected via a three-step procedure. First, bulk cultures of Ramos cells were stimulated with a combination of Fas mAb (5 µg/ml) and CD40 mAb (1/1000 dilution) for 3 days. CD40 mAb was included in the cultures to enhance sensitivity to Fas mAb-mediated apoptosis (12). Remaining viable cells were recovered in IMDM containing 10% FCS for 1 wk and subsequently subjected to another two rounds of stimulation with Fas mAb/CD40 mAb and recovery. Second, limiting dilution was conducted with the resultant cells in the presence of Fas mAb (5 µg/ml) in 96-well plates. Clones that emerged were expanded in IMDM plus 10% FCS. Fas and CD40 expression was measured, and clones that expressed normal levels of both molecules were selected and stimulated with Fas mAb (5 µg/ml) and CD40 mAb (1/1000 dilution), and the percentage of apoptotic cells was determined using FITC-labeled annexin V. Two clones, partially resistant to anti-Fas-mediated cell death, were isolated. Finally, limiting dilution of these two clones was conducted in the absence of Fas mAb. After 2 wk of culture, cell suspensions were split and incubated with medium or a combination of Fas and CD40 mAbs for 3 days. [3H]Thymidine was added during the last 18 h of culture. Three of 288 subclones showed similar levels of [3H]thymidine incorporation in the absence and the presence of Fas mAb and CD40 mAb. Clones were expanded and checked for IgM, Fas, and CD40 expression and for Fas death sensitivity. Eventually one clone (Ramos.FR3) that was consistently (>3 mo) resistant to anti-Fas-mediated apoptosis was selected for further studies.

Isolation of Fas death-sensitive and anti-IgM death-sensitive Ramos clones

Limiting dilution was conducted with wild-type Ramos cells (Ramos.WT). After 2 wk of culture, cell suspensions were split and incubated with medium, Fas mAb (5 µg/ml), or anti-IgM mAb (5 µg/ml) for 3 days. [3H]Thymidine was added during the last 18 h of culture. One of one hundred clones obtained from Ramos.WT was very sensitive to Fas mAb-induced cell death, and 2 of 100 clones showed enhanced sensitivity to anti-IgM mAb-induced apoptosis as measured by [3H]thymidine incorporation. All these clones were expanded and checked for membrane IgM and Fas expression and for their sensitivities to Fas- and anti-IgM-mediated apoptosis using FITC-labeled annexin V and FACS analysis. Eventually, one clone was selected that was consistently highly sensitive to Fas mAb-induced apoptosis (Ramos.FSA), and one was selected that was permanently highly sensitive to anti-IgM mAb induced apoptosis (Ramos.M4).

Generation of retrovirally transduced Ramos.FSA cells

A bicistronic retroviral vector encoding a FADD dominant-negative protein and green fluorescent protein (GFP) was constructed as follows. The internal ribosome entry site (IRES) from encephalomyocarditis virus (41) was ligated as an EcoRI-NcoI fragment to an RcaI-BamHI-blunt fragment containing the coding region of GFP (S65T) (42) and cloned into pBluescript/EcoRI/BamHI-blunt. The IRES-GFP cassette was subsequently cut out with EcoRI and NotI and ligated into the retroviral vector LZRS, which was prepared by cutting out the lacZ sequence from LZRS-pBMN-lacZ (43) with EcoRI and NotI, resulting in the vector LZRS-IG.

A dominant-negative mutant of FADD (FADD-DN; deletion of residues 1–80) (27, 44) was prepared by RT-PCR using the following primers: forward, 5'-CCACCATGGTCGACTTCGAGGCG-3'; and reverse, 5'-CAAAGTCCAGGCTGTGTAGATGCC-3'. The PCR product was cloned into the pGEM-T vector (Promega, Madison, WI). One clone with the expected nucleotide sequence and no additional mutations, as determined by sequence analysis (Dye Terminator Cycle Sequencing Kit, Perkin-Elmer, Gouda, The Netherlands), was subcloned (NcoI/SpeI) into the pGEX-2T plasmid. Subsequently, the FADD-DN fragment was cut out with BamHI and EcoRI and ligated between the unique BamHI and EcoRI sites of the LZRS-IG vector, placing it upstream of the IRES-GFP sequence. As a negative control we used the LZRS-IG vector containing a cDNA from soybean that was partially homologous to human gp91-phox, a subunit of phagocyte NADPH oxidase (45) (provided by Dr. R. Tenhaken, Kaiserlautern, Germany). The soybean gp91-phox protein was nontoxic when expressed in human cells and appeared to be nonfunctional in a cell-free NADPH oxidase assay in which analogous constructs containing human gp91-phox cDNA did show activity (A. J. G. Pötgens, unpublished observations).

For production of amphotropic retrovirus, LZRS-IG plasmids containing soybean or human FADD-DN cDNA were transfected into Phoenix-ampho retroviral packaging cells (expressing the Moloney murine leukemia virus gag, pol, and amphotropic env genes) in the presence of 30 µM chloroquine, using a calcium phosphate transfection kit (Life Technologies, Grand Island, NY). Transfected cells were selected and maintained in 1 µg/ml puromycin (Sigma, St. Louis, MO). The percentage of packaging cells expressing GFP, as analyzed by FACS, was around 90% after 2 wk of selection. Cell-free supernatants of these cells together with DOTAP (Boehringer Mannheim, Mannheim, Germany) were used to transduce Ramos.FSA cells. GFP-expressing Ramos.FSA cells were sorted on a FACStar. After three rounds of sorting, we obtained stably transduced Ramos.FSA cells that were >90% GFP+. Expression of mutated human FADD protein was confirmed by Western blot.

Immunoblot analysis

Lysates of stimulated or unstimulated cells (2.5 x 106) were prepared by suspending cells in 50 µl of lysis buffer (1% Nonidet P-40, 0.01 M triethanolamine-HCl (pH 7.8), 0.15 M NaCl, 5 mM EDTA, 1 mM N-{alpha}-p-tosyl-L-lysine chloromethyl ketone, 0.02 mg/ml ovomucoid trypsin inhibitor, 1 mM PMSF, 0.02 mg/ml leupeptin, 0.4 mM sodium vanadate, and 25 µM phenylarsine oxide). Lysates were cleared by centrifugation for 15 min at 13,000 x g. Protein contents in the cell lysates were determined using the bicinchoninic acid protein assay (Pierce, Rockford, IL). Sixty micrograms of protein per lane was subjected to electrophoresis under reducing conditions in a 12.5% (caspase 3 and FADD) or a 7.5% (PARP) SDS-polyacrylamide gel. Proteins were then transferred to nitrocellulose (Hybond-PVDF or Hybond-C membranes, Amersham, Aylesbury, U.K.) employing a semidry electroblotting chamber (Multipore II, Pharmacia, Uppsala, Sweden), and blots were blocked with 3 to 5% nonfat dry milk in TBST (10 mM Tris, 150 mM NaCl, and 0.05% Tween-20, pH 8.0). Blots were probed with the indicated Abs diluted in TBST containing 2.5% nonfat dry milk. Immunoreactive proteins were visualized using BM chemiluminescence blotting substrate (POD, Boehringer Mannheim).

Protease assay

Protease activity was measured using tetrapeptide pNA substrates in a colometric assay as described previously (46). The assays were performed in 1-ml disposable cuvettes (Greiner Labortecknik, Frickenhausen, Germany) by incubating 20 µl of total cell lysates in 1 ml of reaction buffer (100 mM HEPES (pH 7.5), 20% (v/v) glycerol, 5 mM DTT, and 0.5 mM EDTA) containing 100 µmol of peptide substrate (Ac-DEVD-pNA). Absorbance at 405 nm was monitored at 37°C for 30 min using a UV/VIS spectrophotometer (Perkin-Elmer, Gouda, The Netherlands). In parallel, protein contents were determined using the bicinchoninic acid method (Pierce). Enzyme activity is expressed as the change in milli-OD405 per minute per milligram of protein.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Phenotype of Fas death-resistant and Fas and BCR death-sensitive Ramos clones

To dissect the pathways involved in BCR- and Fas-mediated B cell apoptosis, we generated a set of clones from the Burkitt lymphoma cell line Ramos with differential susceptibility to either BCR- or Fas-induced cell death. After limiting dilution of Ramos cells, a clone was selected that was highly susceptible to Fas mAb-induced apoptosis even in the absence of additional CD40 mAb stimulation (Ramos.FSA), and a clone was selected that was extremely sensitive to anti-IgM mAb-induced apoptosis (Ramos.M4; Fig. 1Go). Interestingly, selection for Fas death sensitivity coincided with enhanced sensitivity to anti-IgM-mediated cell death and, vice versa, selection for anti-IgM induced apoptosis was linked to increased sensitivity for anti-Fas-mediated death (Fig. 1Go). Markedly, clones that were resistant to Fas-mediated cell death could still undergo apoptosis after ligation of the BCR (Fig. 1Go, Ramos.FR3).



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  		FIGURE 1. Functional phenotype of Ramos clones. Clones that were derived from the Burkitt lymphoma cell line Ramos, as described in Materials and Methods, were stimulated with anti-IgM mAb (5 µg/ml) and Fas mAb (5 µg/ml) in the presence or the absence of CD40 mAb (ascites dilution, 1/1000). After 1, 2, and 3 days of stimulation, apoptotic cells were detected by FACS analysis using FITC-labeled annexin V and PI. The mean ± SEM of four independent experiments are shown.

 
Determination of the expression levels of Fas and IgM on these clones revealed no difference in IgM expression (Fig. 2Go). Ramos.FR3 expressed slightly lower levels of Fas than Ramos.WT, whereas both Ramos.FSA and Ramos.M4 expressed higher levels of Fas compared with Ramos.WT (Fig. 2Go). However, when the cells were stimulated with CD40 mAb for 24 h, all clones expressed similar levels of Fas protein (Fig. 2Go). Nevertheless, triggering of Fas in the presence of CD40 mAb did not induce apoptosis in Ramos.FR3 (Fig. 1Go). Furthermore, Fas death resistance in Ramos.FR3 was not caused by mutations in the Fas molecule or by decreased protein levels of the adaptor protein FADD (data not shown).



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  		FIGURE 2. Surface IgM and Fas expression on Ramos clones. Cells from the different Ramos clones were stained with FITC-labeled anti-IgM mAb (fat solid line, upper panel), FITC-labeled Fas mAb (hatched thin line, lower panel) or an FITC-labeled isotype-matched negative control mAb (thin solid line in both panels). In the case of Fas expression, cells that had been stimulated with CD40 mAb for 24 h were also analyzed (fat solid line, lower panel).

 
The fact that in Ramos.FR3, Fas death resistance does not influence anti-IgM-mediated cell death implies that both receptors employ distinct apoptosis signal transduction pathways. On the other hand, since selection for either Fas death sensitivity (Ramos.FSA) or BCR death sensitivity (Ramos.M4) resulted in clones with identical functional phenotypes, it was suggested that both receptors eventually use common apoptosis effector pathways. The level at which both pathways might converge was investigated.

Induction of caspase 3-like protease activity by ligation of Fas and BCR in Ramos.FSA

Recently, many groups have shown that caspases act as effector molecules of the apoptotic program switched on by many different apoptotic stimuli (47). In the various cell systems used, caspase 3 (CPP32) especially appeared to be a central apoptosis-related protease (38, 47, 48, 49). These observations prompted us to investigate whether in human B cells both Fas- and BCR-induced apoptosis signals can activate CPP32-like caspase activity. For this purpose, Ramos.FSA was employed, since in this cell line triggering of either Fas or BCR induces strong apoptosis signals.

Ramos.FSA B cells were stimulated with Fas mAb or anti-IgM mAb, and induction of apoptosis was monitored in parallel with cleavage of Ac-DEVD-pNA (tetrapeptide substrate for caspase 3-like proteases). Fas triggering rapidly induced apoptosis in Ramos.FSA (at 4 h, 25–30% annexin V+ cells). Apoptosis was paralleled by the induction of Ac-DEVD-pNA cleavage activity (Fig. 3GoA). In Jurkat cells (positive control) a similar correlation between Fas mAb-induced apoptosis and Ac-DEVD-pNA cleavage was observed (Fig. 3GoC). Kinetics of apoptosis induction via ligation of the BCR were much slower than those of Fas-induced apoptosis (Fig. 3GoB). Induction of apoptosis was preceded by Ac-DEVD-pNA cleavage activity (starting after 8 h of stimulation; Fig. 3GoB), and both events increased over time. Thus in B cells, ligation of both Fas and BCR can induce activation of caspase 3-like proteases.



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  		FIGURE 3. Ac-DEVD-pNA cleavage activity in Jurkat T cells and Ramos.FSA B-cells upon Fas and BCR ligation. Ramos.FSA cells (2.5 x 106; A and B) or Jurkat cells (2.5 x 106; C) were stimulated with Fas mAb (5 µg/ml; A and C) or with anti-IgM mAb (5 µg/ml; B). At the indicated time points the percentage of apoptotic cells was determined using FITC-labeled annexin V and PI (left y-axis, solid dots). In parallel, cell lysates were prepared, protein contents were determined, and protease activity for Ac-DEVD-pNA (caspase 3-like proteases, right y-axis, open dots) was monitored as described in Materials and Methods. The experiment shown is representative of three performed.

 
Differential cleavage of caspase 3 upon ligation of Fas and BCR

Using an mAb directed against the large p17 subunit of caspase 3, we found that induction of apoptosis via Fas in Ramos.FSA (starting at 2 h) coincided with a decrease in p32 caspase 3 proenzyme and the occurrence of cleavage fragments of 19/20 and 17 kDa (Fig. 4Go, A and B). In addition, cleavage of PARP, a substrate for caspase 3, occurred at the point when apoptotic cells could be visualized (Fig. 4GoC). The 19/20- and 17-kDa caspase 3 cleavage products as well as PARP cleavage were also found in cell lysates of Fas-stimulated Jurkat cells (Fig. 4Go). Evaluation of the caspase 3 status after induction of apoptosis via BCR ligation revealed a caspase 3 cleavage product of 17 kDa, but not one of 19/20 kDa (Fig. 5GoB). The proteins of approximately 25 and 29 kDa (indicated with small squares in Fig. 5GoB) appeared to be the light chain and a degradation product of the heavy chain of the stimulating anti-IgM mAb, respectively (Fig. 5GoB). PARP cleavage was observed just after appearance of the p17 cleavage product of caspase 3, and both events corresponded with the emergence of apoptotic cells upon BCR cross-linking (Fig. 5Go).



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  		FIGURE 4. Caspase 3 and PARP cleavage in Ramos.FSA B cells and Jurkat T cells upon Fas ligation. Ramos.FSA and Jurkat cells (2.5 x 106) were stimulated with Fas mAb (5 µg/ml). At the indicated time points the percentage of apoptotic cells was determined using FITC-labeled annexin V and PI (A). In parallel, cell lysates were prepared, and protein contents were determined. Sixty micrograms of protein per lane was analyzed by SDS-PAGE and subsequently immunoblotted with mAb against the p17 subunit of caspase 3 (B) or PARP mAb (C). In the case of Ramos.FSA, different exposures are shown for p32 and p19/20 plus p17 of the same blot.

 


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  		FIGURE 5. Caspase 3 and PARP cleavage in Ramos.FSA cells upon ligation of surface IgM. Ramos.FSA cells (2.5 x 106) were stimulated with anti-IgM mAb (5 µg/ml). At the indicated time points the percentage of apoptotic cells was determined using FITC-labeled annexin V and PI (A). In parallel, cell lysates were prepared, and protein contents were determined. Sixty micrograms of protein per lane was analyzed by SDS-PAGE and subsequently immunoblotted with mAb against the p17 subunit of caspase 3 (B) or PARP mAb (C). To demonstrate the position of the light chain of the stimulating anti-IgM mAb, 30 µg of anti-IgM mAb was analyzed (last lane). Small squares indicate the light chain and a degradation product of the heavy chain of the stimulating anti-IgM mAb.

 
The observation that cross-linking of the BCR resulted in a different caspase 3 cleavage pattern (p17) compared with that of Fas ligation (p19/20 and p17) indicates that signals transduced via the BCR and Fas initially activate distinct proteases, but that both sets of proteases can eventually cleave and activate caspase 3.

Influence of caspase inhibitors on Fas- and BCR-mediated cell death

To further explore the involvement of caspase 3 in both Fas- and BCR-induced death, Ramos.FSA and Jurkat cells were preincubated with reversible tetrapeptide inhibitor for caspase 3-like (Ac-DEVD-CHO) proteases (38), and cells were subsequently triggered with either Fas or BCR mAbs. At time points where each stimulus induced comparable levels of apoptosis, cells were harvested and analyzed for binding of FITC-labeled annexin V. Although Ac-DEVD-CHO, but not Ac-YVAD-CHO (a compound known to inhibit caspase 1-like protease activity (50)), potently inhibited Fas-mediated apoptosis in Jurkat cells, no effect was found on either Fas- or BCR-mediated apoptosis in Ramos.FSA (Fig. 6GoA). An increase in concentration (up to 0.5 mM) or a short hypotonic shock did not improve the antiapoptotic effect of the caspase 3 inhibitor in Ramos.FSA (data not shown). Interestingly, immunoblot analysis of caspase 3 and PARP status revealed a remarkable difference between the two cell types. Inhibition of Fas-induced cell death by Ac-DEVD-CHO in Jurkat T cells was accompanied by inhibition of PARP cleavage and disappearance of the 17-kDa caspase 3 cleavage product, but accumulation of the 19/20-kDa caspase 3 cleavage product. In contrast, Ac-DEVD-CHO did not inhibit PARP cleavage induced by Fas or BCR ligation in Ramos.FSA. Strikingly, in Ramos.FSA cells the 17-kDa caspase 3 cleavage form did not disappear, but was accumulated when Fas or BCR were triggered in the presence of Ac-DEVD-CHO. No effect in either cell line was found for Ac-YVAD-CHO or the carrier DMSO (Fig. 6Go, B and C), and incubation of Ramos.FSA and Jurkat cells with the inhibitors in the absence of apoptosis-inducing mAbs did not induce any caspase 3 cleavage (not shown).



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  		FIGURE 6. Influence of caspase inhibitors on Fas- and BCR-mediated cell death, and caspase 3 and PARP cleavage. Ramos.FSA cells or Jurkat cells (2.5 x 106) were preincubated for 1 h with 250 µM Ac-DEVD-CHO, Ac-YVAD-CHO, the carrier DMSO, z-VAD-fmk, or its carrier methanol. Subsequently, cells were stimulated with Fas mAb (5 µg/ml) or anti-IgM mAb (5 µg/ml). After 5 h (Ramos.FSA and Fas mAb), 8 h (Jurkat and Fas mAb), or 18 h (Ramos.FSA and anti-IgM mAb) of stimulation, the percentage of apoptotic cells was determined using FITC-labeled annexin V and PI. The mean ± SEM of three independent experiments are shown (A and D). In parallel, cell lysates were prepared, and 60 µg protein/lane was analyzed by SDS-PAGE and subsequently immunoblotted with mAb directed against the p17 subunit of caspase 3 (B and E) or PARP mAb (C and F).

 
In contrast, when using z-VAD-fmk, a broad spectrum caspase inhibitor (51), we observed a strong inhibition of both anti-Fas- and anti-IgM-induced apoptosis in Ramos.FSA (Fig. 6GoD). In the case of Fas-mediated apoptosis, the antiapoptotic effect of the inhibitor was accompanied by the complete absence of caspase 3 and PARP cleavage (Fig. 6Go, E and F). Remarkably, the inhibition of BCR-induced cell death by z-VAD-fmk was paralleled by the inhibition of PARP cleavage and the absence of caspase 3 p17, but the occurrence of the p19/20 cleavage product of caspase 3 (Fig. 6Go, E and F).

Effect of FADD-DN protein expression on BCR-induced apoptosis

The above findings strongly implicate divergence of the two receptor-induced apoptosis pathways upstream of caspase 3. A proximal component of many death receptor-activated apoptosis pathways is the adaptor protein FADD/MORT (44, 52, 53). To explore whether FADD is involved in BCR-mediated apoptosis, we transduced Ramos.FSA cells with plasmids containing either a FADD-DN ({Delta}1–80) cDNA or a soybean gp91-phox cDNA as control (mock). Next to that of endogenous FADD (±24 kDa), expression of mutated FADD (±14 kDa) was readily detected in cell lysates of FSA.FADD-DN (FADD{Delta}1–80)-transduced cells (Fig. 7GoA). Compared with FSA.WT and FSA.mock, Fas-induced apoptosis was strongly inhibited in these cells. In marked contrast, BCR-induced apoptosis was completely unaffected by expression of the FADD-DN mutant protein (Fig. 7Go, B and C).



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  		FIGURE 7. Influence of FADD-DN (fadd {Delta}1–80) mutant protein expression on BCR- and Fas-induced apoptosis. Ramos.FSA cells were retrovirally transduced with plasmids containing either soybean gp91-phox cDNA (mock) or mutated FADD (FADD{Delta}1–80) cDNA. Cell lysates were prepared of the stably transduced cells, protein contents were determined, and 60 µg protein/lane was analyzed by SDS-PAGE and subsequently immunoblotted with FADD mAb (A). In parallel, the different cell lines were stimulated with different concentrations of Fas mAb (B) or anti-IgM mAb (C) for 18 h. Apoptosis was analyzed on a FACScan using FITC-labeled annexin V and PI. The experiment shown is representative of three performed.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Isolation of different apoptosis-sensitive and -resistant variants of the Burkitt lymphoma cell line Ramos enabled us to compare the death pathways employed by Fas and the Ag receptor within one cell type. Selection for high sensitivity to either Fas- or BCR-mediated apoptosis yielded clones with identical functional phenotypes. In contrast, in Fas-death resistant cells, anti-IgM-mediated cell death could still occur. The functional phenotypes of these clones suggest 1) that both Fas- and BCR-induced apoptosis pathways are initially distinct but eventually converge; and 2) that BCR-induced cell death is not only Fas ligand independent (12, 14) but also Fas independent, excluding the possibility of alternative Fas ligands.

Involvement of caspase 1/ICE in Fas-induced apoptosis is controversial (48, 49, 54). Consistent with the observations of Schlegel et al. (39) and Darmon et al. (54), we did not observe any effect of the ICE inhibitor Ac-YVAD-CHO on Fas-induced apoptosis in Jurkat T and Ramos B cells or on BCR-induced death in Ramos cells. Part of the controversy may be explained by variations in caspase 1 expression in the different cell lines used. We found no expression of the 45-kDa caspase 1 protein when analyzing lysates of the Ramos and Jurkat cells used in our study (data not shown).

The level at which the Fas-induced and BCR-induced cell death pathways converge is likely to be caspase 3, as ligation of both receptors results in cleavage of Ac-DEVD-pNA and PARP, substrates for caspase 3-like proteases, and is accompanied by cleavage of the caspase 3 itself. However, major differences were observed in the caspase 3 cleavage pattern. Fas-induced apoptosis is paralleled by the appearance of the large subunit of caspase 3, which either includes (19/20 kDa) or excludes (17 kDa) the prodomain. Strikingly, however, during BCR-induced apoptosis only the 17-kDa, but never the 19/20-kDa, cleavage product of CPP32 could be detected. Thus, the biochemical data infer that Fas and BCR activate distinct proteases upstream of caspase 3.

Although the above findings strongly suggested the involvement of caspase 3 in both types of receptor-induced apoptosis in B cells, the reversible tetrapeptide inhibitor of caspase 3-like proteases, Ac-DEVD-CHO (38), could not block Fas- or BCR-induced apoptosis or PARP cleavage in Ramos B cells, whereas it could block Fas-induced apoptosis and PARP cleavage in Jurkat T cells. Biochemical comparison of B and T cells revealed that ligation of Fas on Jurkat T cells in the presence of Ac-DEVD-CHO resulted in the disappearance of the p17 protein and the accumulation of the p19/20 caspase 3 cleavage product, whereas in inhibitor-treated Ramos cells the p17 cleavage product was accumulated regardless of the apoptosis-inducing stimulus. Therefore, differences in the biologic activity of Ac-DEVD-CHO on Jurkat T and Ramos B cells are most likely due to its differential effect on caspase 3 cleavage. It could be that apart from interference with caspase 3 activity, Ac-DEVD-CHO may also act on caspase 3-related proteases that function upstream of caspase 3, which may be different in T and B cells. Additionally, our data infer that when active caspase 3 (i.e., p17) is generated, Ac-DEVD-CHO inefficiently inhibits PARP cleavage and further propagation of the apoptosis process.

Remarkably, a broad spectrum caspase inhibitor, z-VAD-fmk (51), potently inhibited Fas- and BCR-mediated apoptosis and PARP cleavage in Ramos cells. Whereas Fas-mediated caspase 3 cleavage was completely inhibited by z-VAD-fmk, BCR-induced caspase 3 proteolysis was only partially affected; the p17 caspase 3 cleavage product was no longer found, but the p19/20 form was detected. These findings strengthen the idea that Fas and BCR initiate distinct proteolytic enzymes upstream of caspase 3, most likely the proteases responsible for cleavage between p19/20 and p12 that are either sensitive (Fas) or insensitive (BCR) to z-VAD-fmk. Since p19/20 was found in z-VAD-fmk-treated, BCR-ligated Ramos cells, this compound apparently also interferes with prodomain cleavage from the p19/20 caspase 3 protein (Fig. 8Go). The fact that we never observed the p19/20 protein when the BCR was triggered in the absence of z-VAD-fmk implies that the rate at which p19/20 is produced from the caspase 3 p32 proform is equivalent to the rate at which p19/20 is converted to p17. However, upon Fas ligation, the p32 to p19/20 conversion rate is probably higher than the p19/20 to p17 conversion rate, explaining the detection of both the p19/20 and p17 proteins on Western blots (Fig. 8Go). Catalytic activity of the caspase 3 p19/20 protein has been suggested on the basis of its ability to be labeled with a biotinylated irreversible DEVD acyloxymethyl ketone probe (39). However, we found that in both Jurkat T cells and Ramos B cells, the presence of p19/20 in the absence of p17 is correlated with the absence of PARP cleavage (which contains the DEVD cleavage site) (38) and with a marked reduction in apoptosis, strongly suggesting that p17 is the most catalytic active protein.



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  		FIGURE 8. Model explaining the effects of the caspase inhibitors z-VAD-fmk and Ac-DEVD-CHO on Fas- and BCR-induced caspase 3 (CPP32) cleavage in Ramos B cells. See text for a more detailed description. The number of arrows represents the relative rate at which the caspase 3 proenzyme (p32) is cleaved to form p19/20 plus p12 and p17.

 
The molecular ordering of the proteases involved in death receptor-induced apoptosis is becoming more and more clear (31, 32), and a prominent role for the adaptor protein FADD in coupling extracellular signals to the intracellular proteolytic cascades has been shown in many different settings (44, 52, 53, 55). In this respect it is of particular interest that our findings with FADD-DN-transduced Ramos B cells show FADD independence of BCR-induced apoptosis. Moreover, these latter findings support the idea that although ligation of Fas or BCR eventually leads to the activation of caspase 3, the proximal pathways that couple to these receptors are distinct. It is unclear at this point whether BCR directly activates these proteases or whether an autocrine pathway is used that depends on the activation of FADD-independent death receptors.


   Acknowledgments
 
We thank Dr. J. Borst and Prof. Dr. F. Miedema for critical reading of the manuscript and helpful discussions. We are indebted to Dr. A. Tool for technical assistance, and to Drs. C. Reutelingsperger and R. Tenhaken for their generous gift of FITC-labeled annexin V and soybean gp91-phox cDNA, respectively. Drs. A. Smith and R. Y. Tsien are acknowledged for providing the internal ribosome entry site from encephalomyocarditis virus and GFP(S65T) sequences, respectively.


   Footnotes
 
1 This work was supported by Grant 94-704 from the Dutch Cancer Society (to S.M.A.L.). Back

2 Address correspondence and reprint requests to Dr. S. M. A. Lens, Department of Clinical Viro-Immunology, Central Laboratory of The Netherlands Red Cross Blood Transfusion Service, Plesmanlaan 125, 1066 CX Amsterdam, The Netherlands. E-mail address: Back

3 Abbreviations used in this paper: BCR, B cell antigen receptor; PARP, poly(ADP-ribose) polymerase; ICE, interleukin-1ß-converting enzyme; Ac-DEVD-CHO, Ac-Asp-Glu-Val-Asp-aldehyde; pNA, p-nitrilamide; Ac-YVAD-CHO, Ac-Tyr-Val-Ala-Asp-aldehyde; z-VAD-fmk, Val-Ala-Asp-fluoromethylketone; IMDM, Iscove’s modified Dulbecco’s medium; PI, propidium iodide; GFP, green fluorescent protein; IRES, internal ribosome entry site; DN, dominant negative; FADD, Fas-associated death domain. Back

Received for publication September 23, 1997. Accepted for publication February 17, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Goodnow, C. C.. 1996. Balancing immunity and tolerance: deleting and tuning lymphocyte repertoires. Proc. Natl. Acad. Sci. USA 93:2264.[Abstract/Free Full Text]
  2. Klinman, N. R.. 1996. The "clonal selection hypothesis" and current concepts of B cell tolerance. Immunity 5:189.[Medline]
  3. Russell, D. M., Z. Dembic, G. Morahan, J. F. A. P. Miller, K. Bürki, D. Nemazee. 1991. Peripheral deletion of self-reactive B cells. Nature 354:308.[Medline]
  4. Hartley, S., J. Crosbie, R. Brink, A. B. Kantor, A. Basten, C. C. Goodnow. 1991. Elimination from peripheral lymphoid tissues of self-reactive B lymphocytes recognizing membrane-bound antigens. Nature 353:765.[Medline]
  5. Murakami, M., T. Tsubata, M. Okamoto, A. Shimizu, S. Kumagai, H. Imura, T. Honjo. 1992. Antigen-induced apoptotic death of Ly-1 B cells responsible for autoimmune disease in transgenic mice. Nature 357:77.[Medline]
  6. Finkelman, F. D., J. M. Holmes, O. I. Dukhanina, S. C. Morris. 1995. Cross-linking of membrane Immunoglobulin D, in the absence of T cell help, kills mature B cells in vivo. J. Exp. Med. 181:515.[Abstract/Free Full Text]
  7. Shokat, K. M., C. C. Goodnow. 1995. Antigen-induced B-cell death and elimination during germinal centre immune responses. Nature 375:334.[Medline]
  8. Pulendran, B., G. Kannourakis, S. Nouri, K. G. C. Smith, G. J. V. Nossal. 1995. Soluble antigen can cause enhanced apoptosis of germinal-centre B cells. Nature 375:331.[Medline]
  9. Han, S., B. Zheng, J. Dal Porto, G. Kelsoe. 1995. In situ studies of the primary immune response to (4-hydroxy-3-nitrophenyl)acetyl. IV. Affinity-dependent, antigen-driven B cell apoptosis in germinal centers as a mechanism for maintaining self-tolerance. J. Exp. Med. 182:1635.[Abstract/Free Full Text]
  10. Tsubata, T., J. Wu, T. Honjo. 1993. B-cell apoptosis induced by antigen receptor crosslinking is blocked by a T-cell signal through CD40. Nature 364:645.[Medline]
  11. Valentine, M. A., K. A. Licciardi. 1992. Rescue from anti-IgM-induced programmed cell death by the B cell surface proteins CD20 and CD40. Eur. J. Immunol. 22:3141.[Medline]
  12. Lens, S. M. A., K. Tesselaar, B. F. A. den Drijver, M. H. J. van Oers, R. A. W. van Lier. 1996. A dual role for both CD40L and TNF-{alpha} in controlling human B cell death. J. Immunol. 156:507.[Abstract]
  13. Dhein, J., H. Walczak, C. Baumler, K.-M. Debatin, P. H. Krammer. 1995. Autocrine T-cell suicide mediated by APO-1/(Fas/CD95). Nature 373:438.[Medline]
  14. Peter, M. E., J. Dhein, A. Ehret, S. Hellbardt, H. Walczak, G. Moldenhauer, P. H. Krammer. 1995. APO-1(CD95)-dependent and -independent antigen receptor-induced apoptosis in human T and B cell lines. Int. Immunol. 7:1873.[Abstract/Free Full Text]
  15. Scott, D. W., T. Grdina, Y. Shi. 1996. T cells commit suicide, but B cells are murdered. J. Immunol. 156:2352.[Abstract]
  16. Watanabe-Fukunaga, R., C. I. Brannan, N. G. Copeland, N. A. Jenkins, S. Nagata. 1992. Lymphoproliferation disorder in mice explained by defects in Fas antigen that mediates apoptosis. Nature 356:314.[Medline]
  17. Takahashi, T., M. Tanaka, C. I. Brannan, N. A. Jenkins, N. G. Copeland, T. Suda, S. Nagata. 1994. Generalized lymphoproliferative disease in mice, caused by a point mutation in the Fas ligand. Cell 76:969.[Medline]
  18. 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]
  19. Rieux-Laucat, F., F. Le Deist, C. Hivroz, I. A. G. 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]
  20. Schattner, E. J., K. B. Elkon, D.-H. Yoo, J. Tumang, P. H. Krammer, M. K. Crow, S. M. Friedman. 1995. CD40 ligation induces Apo-1/Fas expression on human B lymphocytes and facilitates apoptosis through the Apo-1/Fas pathway. J. Exp. Med. 182:1557.[Abstract/Free Full Text]
  21. Lagresle, C., C. Bella, P. T. Daniel, P. H. Krammer, T. Defrance. 1995. Regulation of germinal center B cell differentiation: role of the human APO-1/Fas (CD95) molecule. J. Immunol. 154:5746.[Abstract]
  22. Garrone, P., E.-M. Neidhardt, E. Garcia, L. Galibert, C. Van Kooten, J. Banchereau. 1995. Fas ligation induces apoptosis of CD40-activated human B lymphocytes. J. Exp. Med. 182:1265.[Abstract/Free Full Text]
  23. Rothstein, T. L., J. K. M. Wang, D. J. Panka, L. C. Foote, A. Wang, B. Stanger, H. Cui, S-T. Ju, A. Marshak-Rothstein. 1995. Protection against Fas-dependent Th1-mediated apoptosis by antigen receptor engagement in B cells. Nature 374:163.[Medline]
  24. Lagresle, C., P. Mondiere, C. Bella, P. H. Krammer, T. Defrance. 1996. Concurrent engagement of CD40 and the antigen receptor protects naive and memory human B cells from APO-1/Fas-mediated apoptosis. J. Exp. Med. 183:1377.[Abstract/Free Full Text]
  25. Foote, L. C., R. G. Howard, A. Marshak-Rothstein, T. L. Rothstein. 1996. IL-4 induces Fas resistance in B cells. J. Immunol. 157:2749.[Abstract]
  26. Boldin, M. P., E. E. Varfolomeev, Z. Pancer, I. L. Mett, J. H. Camonis, D. Wallach. 1995. A novel protein that interacts with the death domain of Fas/APO1 contains a sequence motif related to the death domain. J. Biol. Chem. 270:7795.[Abstract/Free Full Text]
  27. 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]
  28. 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 (CAP) form a death-inducing signalling complex (DISC) with the receptor. EMBO J. 14:5579.[Medline]
  29. Muzio, M., A. M. Chinnaiyan, F. C. Kischkel, K. O’Rourke, A. Shevchenko, J. Ni, C. Scadiffi, J. D. Bretz, M. Zhang, R. Gentz, M. Mann, P. H. Krammer, M. E. Peter, V. M. Dixit. 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]
  30. Boldin, M. P., T. M. Goncharow, Y. V. Goltsev, D. Wallach. 1996. Involvement of MACH, a novel MORT1/FADD-interacting protease, in Fas/Apo-1- and TNF receptor-induced cell death. Cell 85:803.[Medline]
  31. Srinivasula, S. M., M. Ahmad, T. Fernandes-Alnemri, G. Litwack, E. S. Alnemri. 1996. Molecular ordering of the Fas-apoptotic pathway: the Fas/APO-1 protease Mch5 is a CrmA-inhibitable protease that activates multiple Ced-3/ICE-like cysteine proteases. Proc. Natl. Acad. Sci. USA 93:14486.[Abstract/Free Full Text]
  32. Muzio, M., G. S. Salvesen, V. M. Dixit. 1997. FLICE induced apoptosis in a cell-free system: cleavage of caspase zymogens. J. Biol. Chem. 272:2957.[Abstract/Free Full Text]
  33. Chinnaiyan, A. M., V. M. Dixit. 1996. The cell-death machine. Curr. Biol. 6:555.[Medline]
  34. Fraser, A., G. Evan. 1996. A license to kill. Cell 85:781.[Medline]
  35. Orth, K., A. M. Chinnaiyan, M. Garg, C. J. Froelich, V. M. Dixit. 1996. The Ced-3/ICE-like protease Mch2 is activated during apoptosis and cleaves the death substrate lamin A. J. Biol. Chem. 271:16443.[Abstract/Free Full Text]
  36. Tewari, M., L. T. Quan, K. O’Rourke, S. Desnoyers, Z. Zeng, D. R. Beidler, G. G. Poirier, G. S. Salvesen, V. M. Dixit. 1995. Yama/CPP32ß, a mammalian homolog of Ced-3, is a CrmA-inhibitable protease that cleaves the death substrate poly(ADP-ribose) polymerase. Cell 81:801.[Medline]
  37. Martin, S. J., G. A. O’Brien, W. K. Nishioka, A. J. McGahon, A. Mahboubi, T. C. Saido, D. R. Green. 1995. Proteolysis of fodrin (non-erythroid spectrin) during apoptosis. J. Biol. Chem. 270:6425.[Abstract/Free Full Text]
  38. Nicholson, D. W., A. Ali, N. A. Thornberry, J. P. Vaillancourt, C. K. Ding, M. Gallant, Y. Gareau, P. R. Griffin, M. Labelle, Y. A. Lazebnik, N. A. Munday, S. M. Raju, M. E. Smulson, T. T. Yamin, V. L. Yu, D. K. Miller. 1995. Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis. Nature 376:37.[Medline]
  39. Schlegel, J., I. Peters, S. Orrenius, D. K. Miller, N. A. Thornberry, T.-T. Yamin, D. W. Nicholson. 1996. CPP32/apopain is a key interleukin 1ß converting enzyme-like protease involved in Fas-mediated apoptosis. J. Biol. Chem. 271:1841.[Abstract/Free Full Text]
  40. Koopman, G., C. P. M. Reutelingsperger, G. A. M. Kuijten, R. M. J. Keehnen, S. T. Pals, M. H. J. van Oers. 1994. Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis. Blood 84:1415.[Abstract/Free Full Text]
  41. Mountford, P. S., A. G. Smith. 1995. Internal ribosome entry sites and dicistronic RNAs in mammalian transgenesis. Trends Genet. 11:179.[Medline]
  42. Heim, R., A. B. Cubitt, R. Y. Tsien. 1995. Improved green fluorescence. Nature 373:663.[Medline]
  43. Kinsella, T. M., G. P. Nolan. 1996. Episomal vectors rapidly and stably produce high-titer recombinant retrovirus. Hum. Gene Ther. 7:1405.[Medline]
  44. Chinnaiyan, A. M., C. G. Tepper, M. F. Seldin, K. O’Rourke, F. C. Kischkel, S. Hellbardt, P. H. Krammer, M. E. Peter, V. M. Dixit. 1996. FADD/MORT1 is a common mediator of CD95 (Fas/APO-1) and tumor necrosis factor receptor-induced apoptosis. J. Biol. Chem. 271:4961.[Abstract/Free Full Text]
  45. Roos, D., M. De Boer, F. Kuribayashi, C. Meischl, R. S. Weening, A. W. Segal, A. Ahlin, K. Nemet, J. P. Hossle, E. Bernatowska-Matuszkiewicz, H. Middleton-Price. 1996. Mutations in the X-linked and autosomal recessive forms of chronic chranulomatous disease. Blood 87:1663.[Free Full Text]
  46. Datta, R., D. Banach, H. Kojima, R. V. Talanian, E. S. Alnemri, W. W. Wong, D. W. Kufe. 1996. Activation of the CPP32 protease in apoptosis induced by 1ß-D-arabinofuranosylcytosine and other DNA-damaging agents. Blood 88:1936.[Abstract/Free Full Text]
  47. Henkart, P. A.. 1996. ICE family proteases: mediators of all apoptotic cell death?. Immunity 4:195.[Medline]
  48. Enari, M., R. V. Talanian, W. W. Wong, S. Nagata. 1996. Sequential activation of ICE-like and CPP32-like proteases during Fas-mediated apoptosis. Nature 380:723.[Medline]
  49. Los, M., M. Van de Craen, L. C. Penning, H. Schenk, M. Westendorp, P. A. Baeuerle, W. Droge, P. H. Krammer, W. Fiers, K. Schulze-Osthoff. 1995. Requirement of an ICE/CED-3 protease for Fas/APO-1 protease for Fas/APO-1-mediated apoptosis. Nature 375:81.[Medline]
  50. Thornberry, N. A., H. G. Bull, J. R. Calaycay, K. T. Chapman, A. D. Howard, M. J. Kostura, D. K. Miller, S. M. Molineax, J. R. Weidner, J. Aunins, K. O. Elliston, J. M. Ayala, F. J. Casano, J. Chin, G. J. F. Ding, L. A. Egger, E. P. Gaffney, G. Limjuco, O. C. Palyha, S. M. Raju, A. M. Rolando, J. P. Salley, T. T. Yamin, T. D. Lee, J. E. Shively, M. MacCross, R. A. Mumford, J. A. Schmidt, M. J. Tocci. 1992. A novel heterodimeric cysteine protease is required for interleukin-1ß processing in monocytes. Nature 356:768.[Medline]
  51. Zhu, H., H. O. Fearnhead, G. M. Cohen. 1995. An ICE-like protease is a common mediator of apoptosis induced by diverse stimuli in human monocytic THP.1 cells. FEBS Lett. 374:303.[Medline]
  52. Chinnaiyan, A. M., K. O’Rourke, G.-Y. Yu, R. H. Lyons, M. Garg, D. R. Duan, L. Xing, R. Gentz, J. Ni, V. M. Dixit. 1996. Signal transduction by DR3, a death domain-containing receptor related to TNFR-1 and CD95. Science 274:990.[Abstract/Free Full Text]
  53. Bodmer, J.-C., K. Burns, P. Schneider, K. Hofmann, V. Steiner, M. Thome, T. Bornand, M. Hahne, M. Schroter, T. Becker, A. Wilson, L. E. French, J. L. Browning, H. R. Macdonald, J. Tschopp. 1997. TRAMP, a novel apoptosis-mediating receptor with sequence homology to tumor necrosis factor receptor 1 and Fas (Apo-1/CD95). Immunity 6:79.[Medline]
  54. Darmon, A. J., R. C. Bleackley. 1996. An interleukin-1ß converting enzyme-like protease is a key component of Fas-mediated apoptosis. J. Biol. Chem. 271:21699.[Abstract/Free Full Text]
  55. Walczak, H., M. A. Degli-Esposti, R. S. Johnson, P. J. Smolak, J. Y. Waugh, N. Boiani, M. S. Timour, M. J. Gerhart, K. A. Schooley, C. A. Smith, R. G. Goodwin, C. T. Rauch. 1997. TRAIL-R2: a novel apoptosis-mediating receptor for TRAIL. EMBO J. 16:5386.[Medline]



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