From the Tumor Immunology Program, German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany
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ABSTRACT |
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The Bcl-2 family member Bcl-xL has often been correlated with apoptosis resistance. We have shown recently that in peripheral human T cells resistance to CD95-mediated apoptosis is characterized by a lack of caspase-8 recruitment to the CD95 death-inducing signaling complex (DISC) and by increased expression of Bcl-xL (Peter, M. E., Kischkel, F. C., Scheuerpflug, C. G., Medema, J. P., Debatin, K.-M., and Krammer, P. H. (1997) Eur. J. Immunol. 27, 1207-1212). This raises the possibility that Bcl-xL directly prevents caspase-8 activation by the DISC. To test this hypothesis a cell line in which CD95 signaling was inhibited by overexpression of Bcl-xL was used. In these MCF7-Fas-bcl-xL cells Bcl-xL had no effect on the recruitment of caspase-8 to the DISC. It did not affect the activity of the DISC nor the generation of the caspase-8 active subunits p18 and p10. In contrast, cleavage of a typical substrate for caspase-3-like proteases, poly(ADP-ribose) polymerase, was inhibited in comparison with the control-transfected CD95-sensitive MCF7-Fas cells. To test whether Bcl-xL would inhibit active caspase-8 subunits in the cytoplasm, a number of immunoprecipitation experiments were performed. Using monoclonal antibodies directed against different domains of caspase-8, anti-Bcl-xL antibodies, or fusion proteins of glutathione S-transferase with different domains of caspase-8, no evidence for a direct or indirect physical interaction between caspase-8 and Bcl-xL was found. Moreover, overexpression of Bcl-xL did not inhibit the activity of the caspase-8 active subunits p18/p10. Therefore, in this cell line that has become resistant to CD95-induced apoptosis due to overexpression of Bcl-xL, Bcl-xL acts independently and downstream of caspase-8.
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INTRODUCTION |
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Apoptosis is essential for development and tissue homeostasis (1).
In the nematode Caenorhabditis elegans genetic analysis identified three genes, ced-3, ced-4, and ced-9,
that are essential to apoptosis during worm development (2). Genetical
ordering of the three genes revealed that ced-9 negatively
regulates the induction of apoptosis by ced-3 (3) and that
this regulation requires the presence of ced-4 (4). This
suggests that ced-9 inhibits ced-3 through ced-4. Mammalian homologues
of the C. elegans genes ced-3 and
ced-9 have been identified. ced-3 shares high homology with the family of interleukin 1-converting enzyme-like proteases (caspases) (5), essential mediators of many forms of
apoptosis in mammalian cells (reviewed in Ref. 6), while ced-9 has high homology to bcl-2
(bcl-xL) (3).
Cross-linking of CD95 (APO-1/Fas) results in the recruitment of a set of proteins that include FADD/MORT1 (7, 8) and caspase-8 (FLICE/MACH/Mch5; Refs. 9-11) to the receptor forming the death-inducing signaling complex (DISC)1 (12, 9). Recruitment of caspase-8, which results in its activation (12), requires the presence of FADD and is mediated by an interaction of the death effector domain (DED) of FADD and the first DED of caspase-8 (13, 14). Interestingly, this DED was a region found to share weak homology with two areas in CED-4 (15, 16). In addition, a recent report demonstrated coimmunoprecipitation of Bcl-xL with caspase-8 and vice versa (17). These data suggest a regulatory mechanism in mammalian cells in which the CED-9 homolog Bcl-xL influences the activity of the CED-3 homolog caspase-8 possibly involving the CED-4 homology region of caspase-8.
We have recently reported that in a CD95-resistant peripheral T cell population, FADD was recruited to the activated CD95 receptor, but recruitment and activation of procaspase-8 was not detected. In addition, this apoptosis resistance correlated with the expression of the anti-apoptotic molecule Bcl-xL (18), suggesting that it might directly render cells CD95 apoptosis resistant by interfering with the DISC. Recently, it was shown that overexpression of Bcl-xL inhibited CD95-mediated apoptosis without blocking initial caspase activation (19). We therefore tested whether Bcl-xL might interfere with recruitment of caspase-8 to the DISC or with the activity of caspase-8 using a breast carcinoma cell line MCF7 expressing high levels of CD95 (MCF7-Fas) (20). Overexpression of Bcl-xL in these cells renders them resistant to CD95-induced apoptosis (20). In addition, no DNA fragmentation, morphological changes, or cleavage of poly(ADP-ribose) polymerase (PARP) could be detected in these cells. However, cleavage of caspase-8 was not affected by overexpression of Bcl-xL. No direct or indirect association between Bcl-xL and caspase-8 could be detected, indicating that in these cells Bcl-xL acted independently and downstream of caspase-8.
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EXPERIMENTAL PROCEDURES |
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Cell Lines-- The breast carcinoma cell line MCF7 transfected with CD95 and a control plasmid (MCF7-Fas-vector) or CD95 and Bcl-xL (MCF7-Fas-bcl-xL) were a kind gift from M. Jäättelä (Kopenhagen, Denmark). They were cultured as described elsewhere (20). The SKW6.4 cells were cultured in RPMI + 10% fetal calf serum and 0.05 mg/ml gentamycin.
Antibodies and Reagents--
An affinity-purified rabbit
antipeptide antibody against the C terminus of caspase-8 was generated
as described previously (13). The C5 mAb (IgG2a) recognizes the p10
subunit, the C1 mAb (IgG1) and C15 mAb (IgG2b) recognize the p18
subunit of caspase-8, and the N2 mAb (IgG1) recognizes the caspase-8
prodomain (21). The mouse mAb anti-APO-1 (IgG3, ) recognizes an
epitope on the extracellular part of APO-1 (22). The anti-PARP antibody
(C-II-10) was a kind gift of Dr. A. Bürkle (Heidelberg, Germany).
The rabbit polyclonal serum against Bcl-xL and the
anti-FADD mAb (IgG1) were purchased from Transduction Laboratories
(Lexington, Kentucky). The horseradish peroxidase-conjugated antibodies
goat anti-rabbit IgG, goat anti-mouse IgG1, IgG2a, and IgG2b were
purchased from Dianova (Hamburg, Germany). All chemicals used were of
analytical grade and purchased from Merck (Darmstadt, Germany) or
Sigma.
Cytotoxicity Assay-- 106 cells were incubated in 24-well plates (Costar, Cambridge, MA) with 1 µg/ml anti-APO-1 in medium (5 × 105 cells/ml) for 16 h at 37 °C. After incubation cells were photographed at a 400-fold magnification. Quantification of DNA fragmentation as a specific measure of apoptosis was carried out essentially as described elsewhere (23, 24).
GST Fusion Proteins and Immunoprecipitation-- Using standard polymerase chain reaction and cloning techniques the following GST fusion proteins were generated: GST-FADD, GST-C-FADD (amino acids 89-207), GST-caspase-8, GST-N-caspase-8 (amino acids 1-180), and GST-C-caspase-8 (amino acids 181-478). GST fusion proteins were purified as described previously (10), and 20 µg of each GST fusion protein precoupled to glutathione beads was used per precipitation. For immunoprecipitation 10 µg mAbs (25 µg in the case of C5) were coupled to either anti-IgG1 Agarose beads (C1, N2) (Sigma) or to protein A/G-plus agarose (C5) (Santa Cruz Biotechnology), and postnuclear supernatants of 107 cells were added and incubated for more than 1 h at 4 °C. Beads were then washed three times with lysis buffer (30 mM Tris/HCl, pH 7.5, 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride and small peptide inhibitors (12), 1% Triton X-100 (Serva) and 10% glycerol). The amount of DISC-associated FADD was determined as follows: 107 MCF7 cells were either first treated with 2 µg/ml anti-APO-1 for 5 min at 37 °C and then lysed (stimulated condition) or first lysed and then supplemented with 2 µg/ml anti-APO-1 (unstimulated condition) as described previously (12). Triton X-100 solubilization of cellular proteins using the above lysis buffer and immunoprecipitation of CD95 was done as described previously (12). All immunoprecipitates were separated on 12% SDS-PAGE.
Western Blotting-- For Western blot detection of cytosolic proteins, cellular lysates equivalent to 5 × 105 cells were prepared as described previously (12) and separated by 12.5% SDS-PAGE. After electrophoresis all samples were transferred to Hybond nitrocellulose membrane (Amersham Corp.), blocked with 2% bovine serum albumin in PBS/Tween (phosphate-buffered saline with 0.05% (v/v) Tween 20 (Serva)) for at least 1 h, washed with PBS/Tween, and incubated with the following antibodies: anti-Bcl-xL serum (1:1000), anti-caspase-8 mAbs (1 µg/ml), or the mouse anti-FADD mAb (1 µg/ml). All antibodies were diluted in PBS/Tween. The blots were washed with PBS/Tween and developed with either goat anti-rabbit IgG (1:20,000) or goat anti-mouse IgG1, IgG2a, or IgG2b (1:20,000). After washing with PBS/Tween the blots were developed with the chemiluminescence method (ECL) following the manufacturer's protocol (Amersham).
In Vitro Caspase-8 Activation Assay-- CD95 DISC was immunoprecipitated from 5 × 107 activated or nonactivated cells as described above, and immunoprecipitates were incubated with in vitro translated 35S-labeled caspase-8 in caspase-8 cleavage buffer (50 mM Hepes, pH 7.4, 100 mM NaCl, 0.1% 3-(cyclohexylamino)-1-propanesulfonic acid, 10 mM dithiothreitol, and 10% sucrose) for 24 h at 4 °C. The cleavage reactions were stopped by addition of standard reducing sample buffer. After boiling for 3 min samples were subjected to 15% SDS-PAGE, amplified (Amplify, Amersham), dried, and subjected to autoradiography. For in vitro PARP cleavage of active caspase-8 subunits, 5 × 107 MCF7-Fas cells were left untreated or stimulated with 2 µg/ml anti-APO-1. After 2 h cells were lysed, and lysates were made as described above. Lysates were precleared with mouse IgG, and the active caspase-8 subunits were subsequently precipitated with the C5 mAb as described above. After intensive washing precipitates were incubated in caspase-8 cleavage buffer together with 300 ng of recombinant PARP for 4 h at 37 °C. Reactions were stopped by the addition of standard reducing sample buffer, separated on 10% SDS-PAGE, and immunoblotted with C-II-10.
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RESULTS |
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MCF7-Fas-bcl-xL Cells Are Resistant to CD95-mediated Apoptosis-- Since the CD95 DISC seemed to determine apoptosis sensitivity in peripheral T cells (18), we investigated whether expression of Bcl-xL would directly affect the composition of the DISC. However, normal T cells can hardly be transfected and overexpression of Bcl-2 or Bcl-xL in lymphoid cells does not necessarily block the CD95 pathway (25-29). Hence, we tested the effect of overexpression of Bcl-xL in a breast carcinoma cell MCF7-Fas, one of the few cell lines that have been reported to become resistant to CD95-mediated apoptosis upon overexpression of Bcl-xL (20). Indeed overexpression of Bcl-xL blocked the morphological changes (i.e. rounding up and detaching) induced by cross-linking of CD95 (Fig. 1A). In addition, fragmentation of chromosomal DNA as compared with control-transfected MCF7-Fas cells was almost completely inhibited in the Bcl-xL transfectants (Fig. 1B). This difference in sensitivity toward cross-linking of the CD95 receptor was not due to a difference in expression of the receptor, since both transfectants express similar amounts (data not shown), but due to the expression of Bcl-xL (Fig. 1B).
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Bcl-xL Does Not Associate with Caspase-8 or Block Its Activity-- Recently, Chinnaiyan et al. (17) demonstrated in transient cotransfection experiments that Bcl-xL could be coimmunoprecipitated together with caspase-8 and vice versa. This association was indirect as it required an additional factor believed to be a mammalian CED-4 homolog (17). Most importantly the data suggested that the CED-9 homolog Bcl-xL might act upstream of the CED-3 homolog caspase-8. Since in the MCF7-Fas cells apoptosis through CD95 involved activation of caspase-8 and CD95-mediated apoptosis in these cells was blocked by overexpression of Bcl-xL, we could now test this hypothesis in vivo in stably transfected cells. To this end we generated a number of reagents to use in immunoprecipitation experiments. All following experiments were done both with untreated and anti-APO-1-treated MCF7-Fas and MCF7-Fas-bcl-xL cells. First we tested whether Bcl-xL could be immunoprecipitated from MCF7 cells using fusion proteins of GST with different functional domains of caspase-8 or FADD (Fig. 2). Using GST-caspase-8 and GST-N-caspase-8 (comprising the first 188 amino acids of caspase-8 containing both DED), we could precipitate FADD from lysates of both transfectants (Fig. 2, B and D, lanes 7-10). Therefore, both DED containing GST-caspase-8 constructs were functionally active in binding the physiologically important signaling molecule FADD through their N termini. FADD was not precipitated when GST-C-caspase-8 was used (lacking the two N-terminal DED) (Fig. 2, B and D, lanes 11 and 12). We then tested whether cytoplasmic Bcl-xL from anti-APO-1-treated and untreated cells would coprecipitate with GST-caspase-8 or GST-C-caspase-8. As a control for specificity, we used GST, GST-FADD, or GST-C-FADD (lacking the DED). Bcl-xL was clearly detectable by Western blot analysis in Triton X-100 lysates from 5 × 105 MCF7-Fas-bcl-xL cells (Fig. 2C, lanes 13 and 14). However, no associated Bcl-xL could be detected with any of the GST fusion proteins tested, although 20-fold more lysate (107 cells) was used for precipitation (Fig. 2C, lanes 1-12).
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Formation of the DISC and Activation of Caspase-8 Are Unaffected in MCF7-Fas-bcl-xL Cells-- The above data indicate that Bcl-xL likely does not exert its inhibitory activity by physically interacting with procaspase-8 or active caspase-8 subunits in the cytoplasm, regardless whether anti-APO-1-treated or untreated cells were tested. We have shown recently that in vivo caspase-8 is activated by recruitment to the CD95 DISC (13). If Bcl-xL functioned upstream of caspase-8, it should interfere with caspase-8 cleavage by the DISC. The first step in DISC formation is recruitment of FADD (12). This can be visualized by immunoprecipitation of CD95 and subsequent immunoblotting with a FADD-specific mAb. Treatment of SKW6.4 B cell lymphomas with anti-APO-1 clearly resulted in an association of FADD with CD95 (Fig. 5A, lanes 7 and 8). Similarly, both MCF7-Fas-vector and MCF7-Fas-bcl-xL cells, which express comparable amounts of FADD (Fig. 5A, lanes 1 and 2), efficiently recruited FADD to CD95 after treatment with anti-APO-1 (Fig. 5A, lanes 3-6). We next tested whether the activity of the DISC to process caspase-8 was affected by Bcl-xL overexpression. We have recently shown that this activity can be determined in vitro by incubation of immunoprecipitated DISC with in vitro translated 35S-labeled caspase-8 (13). Testing the MCF7-Fas transfectants in this assay demonstrated that Bcl-xL overexpression did also not alter the activity of the DISC to activate caspase-8 in vitro (Fig. 5B).
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In Vivo Bcl-xL Acts Downstream of Caspase-8 and Upstream of a PARP Cleaving Caspase-- To test whether Bcl-xL would inhibit cleavage of caspase-8 in vivo, the C15 mAb recognizing the p18 subunit of caspase-8 was used. This antibody allowed to follow caspase-8 activation in vivo by detecting both the full-length caspase-8 isoforms as well as the active caspase-8 subunit p18 (Fig. 6A). Importantly, in both control transfected and Bcl-xL overexpressing MCF7-Fas cells the p18 subunit was formed to a similar extent and with identical kinetics (Fig. 6A, lower panel). Furthermore, as has been shown previously for the SKW6.4 cell line (21), all full-length caspase-8 was processed after cross-linking of CD95 in both cell lines (Fig. 6A, upper panel). In contrast to caspase-8 cleavage, cleavage of PARP, a typical substrate of caspase-3-like proteases (9, 30, 31), was blocked in the Bcl-xL-overexpressing MCF7-Fas cells, while PARP was cleaved in the control-transfected MCF7-Fas cells upon anti-APO-1 treatment (Fig. 6B). Taken together our data suggest that Bcl-xL inhibits the CD95 signaling pathway downstream of caspase-8 cleavage and upstream of the activity of caspase-3-like proteases.
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DISCUSSION |
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Regulation of apoptosis in the nematode C. elegans serves as a paradigm for apoptosis regulation in mammalian cells. In the worm three genes dictate apoptosis during development (2). Mutations in ced-3 or ced-4 abolish apoptosis, while mutation of ced-9 results in aberrant cell death. CED-9 is homologous to the mammalian Bcl-2 family and inhibits apoptosis induced by CED-3, which is homolog to the mammalian family of caspases and therefore responsible for execution of apoptosis. CED-4, another apoptosis-promoting gene product, physically interacts with CED-9 (17, 32, 33) and is required for CED-9 to inhibit the function of CED-3. No mammalian homolog for CED-4 has yet been discovered. CED-3 is most homologous to caspase-3-like proteases. It has been shown that Bcl-2 functions upstream of these caspases (27, 34-38). Since caspase-8 has a caspase-3-like activity, the question arose whether Bcl-2 family members could act upstream of caspase-8. Recently, it was suggested that Bcl-xL inhibits caspase-8 activity by physically interacting with caspase-8 (17). This association required CED-4 and was postulated to occur at the level of the mitochondria (39). We have demonstrated previously that during CD95-mediated apoptosis caspase-8 is activated by recruitment to the CD95 DISC. After completion of this process all cytosolic caspase-8 can be converted to active caspase-8 subunits detectable in the cytoplasm (13). If Bcl-xL acts upstream of caspase-8, it would have to bind to either procaspase-8 in the DISC or to procaspase-8 or the active caspase-8 subunits in the cytoplasm. We now show that CD95-induced caspase-8 activation or activity is not affected by overexpression of Bcl-xL in the CD95-resistant cell line MCF7-Fas-bcl-xL. Bcl-xL did not affect the activity of the DISC. In addition, Bcl-xL did not interfere with PARP cleavage activity of the caspase-8 active subunits p10/p18 when immunoprecipitated using mAbs against the caspase-8 active subunits p10 (Fig. 6E) or p18 (data not shown).
Recently, the existence of a caspase activity upstream of Bcl-xL was suggested studying Bcl-xL-transfected Jurkat cells using PARP cleavage as a read-out (19). In this study Bcl-xL-transfected Jurkat cells were resistant to CD95-mediated apoptosis, but PARP was still cleaved. It was suggested that caspase-8 was responsible for the observed PARP cleavage in these cells. However, the identity of the PARP cleaving caspase was not determined. We now show that in MCF7-Fas-bcl-xL cells PARP cleavage was completely blocked by overexpression of Bcl-xL, whereas caspase-8 activity was unchanged. The discrepancy between the two reports is likely due to different cell lines used. Our data demonstrate that first, in vivo PARP was not directly cleaved by caspase-8, and second, another caspase-3-like protease that cleaves PARP was blocked by Bcl-xL.
Our data suggest that Bcl-xL acts downstream of caspase-8. The indirect association between caspase-8 and Bcl-xL that has been reported previously was based on experiments that used a transient transfection assay (17). We now have tested this interaction in vivo in stably transfected MCF7 cells by performing immunoprecipitation experiments using highly sensitive anti-Bcl-xL and anti-caspase-8 antibodies. To prevent competition of the anti-caspase-8 mAbs and Bcl-xL for the same binding site, we used mAbs against three different regions of caspase-8 (the prodomain and the p18 and p10 subunits). Consistent with the lack of functional inhibition of caspase-8 in the DISC, no coimmunoprecipitation of Bcl-xL with caspase-8 or vice versa was detectable.
We have recently shown that CD95 resistance in short term activated peripheral T cells is characterized by a lack of caspase-8 recruitment to the DISC despite high expression of caspase-8 in the cytoplasm (18). In addition, we found that the expression of Bcl-xL correlated with CD95 apoptosis sensitivity in these T cells, making it possible that Bcl-xL directly prevented caspase-8 recruitment to the DISC. However, for caspase-8 being the main target for Bcl-xL is in contradiction to the reported effects of Bcl-2 or Bcl-xL on the CD95 signaling pathway in lymphoid cells. Reports range from no inhibition (27-29, 40) to partial inhibition (25) to substantial inhibition of CD95-mediated apoptosis (19, 34, 38). One of the few cell lines that have been reported to be completely resistant to CD95 apoptosis after overexpression of Bcl-xL is the breast carcinoma cell line MCF7-Fas (20). Using these cells we did not find any association of Bcl-xL with procaspase-8 or caspase-8 active subunits or any effect of Bcl-xL on caspase-8 activation. However, our experiments do not exclude that Bcl-xL could influence the action of caspase-8 by preventing a possible translocation of the caspase-8 active subunits within the cell from one location to their physiological target(s) without blocking their enzymatic activity.
The presence of homologous functionally interchangeable proteins in C. elegans and mammals suggests a universal principle in apoptosis regulation conserved throughout evolution. The data presented here demonstrate that the CED-9 homolog Bcl-xL does not act by interfering with the activation of the CED-3 homolog caspase-8. Therefore, apoptosis sensitivity is at least regulated at two separate levels: at the level of the DISC and independently further downstream at the level of Bcl-xL likely regulating the activity of other CED-3 homologs. In contrast to C. elegans mammalian cells seem to have developed at least two levels at which caspases act. First level caspases, such as caspase-8, may couple the intracellular death machinery to death receptors and may not be regulated by CED-9-like molecules. Second level caspases likely representing caspase-3-like proteases located downstream of mitochondria may represent the CED-3 homologs likely to be regulated by the CED-9 homolog Bcl-xL (Bcl-2).
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ACKNOWLEDGEMENTS |
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We thank Uschi Silberzahn for excellent technical assistance and Alexander Stegh for antibody purification. We thank Marja Jäättelä for the MCF7-Fas cells and Andrea Murmann for the use of the photographic equipment.
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Addendum |
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While this manuscript was being reviewed cloning of a human CED-4 homolog Apaf-1 was reported (41). Upon binding of cytochrome c Apaf-1 was demonstrated to activate caspase-3. This finding is in complete agreement with our observation that Bcl-xL acts downstream of caspase-8, since it also places the action of a human CED-4 homolog downstream of caspase-8.
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FOOTNOTES |
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* This work was supported by grants from the Dutch Cancer Society (to J. P. M.), the Deutsche Forschungsgemeinschaft (to M. E. P.), the Bundesministerium für Forschung und Technologie, and the Tumor Center Heidelberg/Mannheim.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Both authors contributed equally.
§ Present address: Dept. of Immunohematology and Bloodbank, University Hospital Leiden, Albinusdreef 2, 2300RC, Leiden, The Netherlands.
¶ To whom correspondence should be addressed. Tel.: 49-6221-423765; Fax: 49-6221-411715; E-mail: m.peter{at}dkfz-heidelberg.de.
1 The abbreviations used are: DISC, death-inducing signaling complex; FADD, Fas-associated death domain protein; DED, death effector domain; PARP, poly(ADP-ribose) polymerase; mAb, monoclonal antibody; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline.
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REFERENCES |
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