Inhibition of Fas-Induced Apoptosis by Bcl-2

Atsuo Kawahara1,2,4, Toshiko Kobayashi3 and Shigekazu Nagata1,2

1Osaka Bioscience Institute, 6-2-4 Furuedai, Suita, Osaka 565-0874; 2Department of Genetics, Osaka University Medical School, B3, 2-2 Yamada-oka, Suita, Osaka 565-0871; 3Ina Laboratories, MBL Co. Ltd, Oohara, 1063-103 Terasawaoka, Ina, Nagano 396-0002, Japan

Jurkat cells express Fas, and rapidly undergo apoptosis receptor by the binding of FasL to Fas, or by cross- in response to Fas ligand or an agonistic anti-Fas linking with an agonistic anti-Fas antibody induces the antibody. This apoptotic pathway is mediated by a formation of a death-inducing signaling complex cascade of caspases. In this report, we show that Fas (DISC) of proteins composed of Fas, an adaptor activation induced the processing of caspase 8 in Jurkat called FADD/MORT1, and the inactive proform of cells with a time frame similar to the activation of caspase 8 (Kischkel et al., 1995). Oligomerization of caspase 3 and the proteolysis of nuclear proteins. Jurkat procaspase 8 in the DISC seems to induce its self- cell transformants that overexpress Bcl-2 were partially processing into the mature, active protease, containing but not completely resistant to the Fas-induced apopto- p20 and p10 subunits. The activated caspase 8 is then sis. Little processing of caspase 8 was observed upon Fas released from the DISC, and activates other down- activation in these transformants. Furthermore, although stream caspases by proteolytic cleavage of their caspase 8 was recruited to Fas upon Fas activation in the zymogen forms (Medema et al., 1997). parental Jurkat cells, the recruitment of caspase 8 to Fas In many cells, overexpression of anti-apoptotic was inhibited in the transformants overexpressing Bcl-2. members of the Bcl-2 gene family, such as Bcl-2 and

These results suggest that Bcl-2 inhibits Fas-induced Bcl-XL, inhibits the apoptosis induced by a variety of apoptosis by preventing the formation of the death- stimuli (Cory, 1995; Korsmeyer, 1995; Reed, 1997). inducing signaling complex that is composed of Fas, Bcl-2 and Bcl-XL localize in cells to the cytoplasmic FADD/MORT1, and caspase 8. membranes of mitochondria, endoplasmic reticulum, and nuclei. Despite extensive studies, the molecular

Keywords: apoptosis; Bcl-2; caspase; Fas mechanism by which Bcl-2 and Bcl-XL inhibits apoptosis is still not clearly understood. Fas-induced

apoptosis is also inhibited by Bcl-2 and Bcl-XL in variety of cells (Boise and Thompson, 1997; Itoh et al., Introduction 1993; JaÈ aÈ ttelaÈ et al., 1995; Rodriguez et al., 1996), although perhaps not in all Fas-expressing cells Apoptosis is an innate cell suicide mechanism that (Memon et al., 1995; Strasser et al., 1995). Since the abates unwanted, harmful, and neoplastic cells signaling pathway for Fas-induced apoptosis is rather (Jacobson et al., 1997). Various stimuli, including well characterized, we investigated the eect of Bcl-2 cytokines, anti-cancer drugs, and UV- or g-irradiation on each step of this process. In this report, we show trigger the apoptotic program (Nagata, 1997), which is that overexpression of Bcl-2 in human Jurkat cells characterized morphologically by condensation and partially inhibits the cell death triggered by Fas fragmentation of cells and nuclei, and biochemically activation. The cleavage of cellular substrates such as by fragmentation of the chromosomal DNA (Wyllie et poly(ADP-ribose) polymerase (PARP) and lamins al., 1980). Regardless of the stimuli, apoptotic signals occurred signi®cantly more slowly in transformants ultimately lead to the same biochemical events down- overexpressing Bcl-2 than in the parental Jurkat cells. stream in the signaling cascade. That is, all apoptotic Similarly, the Fas-induced processing of procaspases 3 stimuli activate members of the caspase family, which and 8 was retarded in Bcl-2-overexpressing cells. cleave various cellular substrates, leading to morpho- Finally, the FasL-induced DISC formation was logical changes in the cells as well as to the inecient in Bcl-2-overexpressing cells. These results fragmentation of chromosomal DNA (Chinnaiyan suggest that the partial inhibition of Fas-induced and Dixit, 1996; Enari et al., 1998; Nicholson and apoptosis by Bcl-2 is due at least in part, to the Thornberry, 1997; Sakahira et al., 1998). inhibition of the recruitment of procaspase 8 to the Fas Fas (also called APO-1 or CD95), a receptor for Fas receptor. ligand (FasL), belongs to the tumor necrosis factor (TNF) receptor family, and transduces the apoptotic signal into cells (Nagata and Golstein, 1995). The Results molecular mechanism for Fas-induced apoptosis is currently being elucidated. Aggregation of the Fas Inhibition of Fas-induced apoptosis by Bcl-2 We have previously shown that overexpression of Bcl- 2 partially inhibits Fas-induced apoptosis in mouse Correspondence: S Nagata WR19L cells in vitro and mouse hepatocytes in vivo 4 Current address: Laboratory of Molecular Genetics, National (Itoh et al., 1993; Rodriguez et al., 1996). To examine Institute of Child Health and Human Development, Building 6B, Room 420, NIH, Bethesda, Maryland 20892, USA whether Bcl-2 inhibits Fas-induced apoptosis in other Received 23 March 1998; revised 8 June 1998; accepted 9 June 1998 cell types, we established Jurkat cell transformants Inhibition of Fas-induced apoptosis by Bcl-2 A Kawahara et al 2550 that overexpress human Bcl-2. As shown in Figure 1a, clones (JB-2 and JB-3) expressed similar high levels the parental Jurkat cells expressed only very low levels of Bcl-2. Jurkat cells endogenously express Fas on of Bcl-2, whereas both independent transformant their surface, and Fas expression was not aected by

Figure 1 Inhibition of Fas-mediated apoptosis by Bcl-2. (a) Expression of Bcl-2 in Jurkat-derived cell lines. Cell lysates (56104 cells/lane) were subjected to SDS ± PAGE, followed by Western blotting using anti-Bcl-2 and anti-a-Tubulin as probes. Arrows indicate the positions of Bcl-2 and a-Tubulin. (b) Expression of Fas in Jurkat-derived cell lines. Cells were incubated with anti-Fas antibody (CH11) and subsequently with FITC-conjugated anti-mouse IgM ( ). The pro®le of cells stained with the secondary 4 antibody alone is also indicated (.....). (c) Eect of Bcl-2 overexpression on Fas-induced apoptosis. Cells (5610 cells/100 ml) were incubated with the indicated concentrations of anti-Fas antibody (CH11; Lot 120) for 12 h. Cell viability was determined by the WST-1 assay as described in Materials and methods. Results are expressed as a percentage of live cells seen in the absence of anti- Fas antibody. The numbers represent the average of three separate assays Inhibition of Fas-induced apoptosis by Bcl-2 A Kawahara et al 2551 the ectopic expression of Bcl-2 (Figure 1b). The the processing of procaspase 8 was similar to that for sensitivity of the parental Jurkat cells and the procaspase 3 as well as for the cleavage of PARP and transformant clones to Fas-induced apoptosis was lamin B1. We then examined the eect of Bcl-2 on the then examined by incubating the cells with various processing of procaspases 8 and 3, the cleavage of concentrations of an agonistic anti-Fas antibody (CH- PARP, lamin B1, and DNA fragmentation. The 11). As shown in Figure 1c, half of the Jurkat cells Jurkat cell transformant clones (JB-2 and JB-3) that died within 12 h with 3 ng/ml of the anti-Fas overexpress Bcl-2 expressed procaspase 8 at a level antibody. On the other hand, 40 ng/ml of the anti- similar to the parental Jurkat cells (Figure 3a). When Fas antibody was necessary to kill the Bcl-2 these cells were treated with the anti-Fas antibody, the transformants, and more than 40% of the cells cleavage of procaspase 8 was signi®cantly retarded as remained alive even when they were incubated for compared with that observed in the parental Jurkat 12 h with 0.3 mg/ml of the antibody. These results cells. Most of the proform of caspase 8 disappeared indicated that Bcl-2 has a potent inhibitory eect on during a 2 h incubation with the anti-Fas antibody in Fas-induced apoptosis in human Jurkat cells. the parental Jurkat cells (Figure 2). In contrast, half of the caspase 8 proform remained intact after a 4 h treatment with the anti-Fas antibody in JB-2 and JB-3 Inhibition of the Fas-induced processing of caspase 8 by cells (Figure 3a). Similarly, the processing of Bcl-2 procaspase 3, the cleavage of PARP and lamin B1, The engagement of Fas with its ligand leads to the and the fragmentation of chromosomal DNA were activation of a cascade of caspases (Fraser and Evan, 1996; Nagata, 1997). Caspases exist as precursor forms in growing cells. When cells are activated to apoptosis, the precursor forms of caspases undergo proteolytic processing to mature forms (Nicholson and Thornberry, 1997). Among these, caspase 8 appears to be the ®rst protease that is activated, and this activation occurs in the DISC consisting of Fas, the adaptor molecule FADD/MORT1, and procaspase 8 (Kischkel et al., 1995). To examine whether caspase 8 is actually activated by the Fas engagement, a monoclonal antibody against human caspase 8 was developed, and used for Western blotting analysis. As shown in Figure 2, human procaspase 8 was detected as a doublet at 54 and 55 kDa, which agrees with the recent report that two dierent forms of human procaspase 8 are produced from two alternatively spliced mRNAs (Scadi et al., 1997). When Jurkat cells were treated with the anti-Fas antibody, procaspase 8 was quickly processed to the mature p18 protein through its intermediate p43, as reported previously (Medema et al., 1997). The time course for

Figure 3 Inhibition of the Fas-induced activation of caspases and DNA fragmentation by Bcl-2. (a) Eect of Bcl-2 overexpression on the Fas-induced activation of caspases. The parental Jurkat cells and its transformant clones (JB-2 and JB- 3) expressing Bcl-2 were either unstimulated (0 h) or stimulated for 4 h with 0.5 mg/ml of anti-Fas antibody (CH11; Lot 120). The cells were lysed with Laemmli's sample buer as described in Materials and methods, and the crude lysates from 56104 cells Figure 2 Processing of caspases and nuclear proteins during Fas- per lane were subjected to Western blotting analysis with induced apoptosis. Jurkat cells (56105 cells) were stimulated with antibodies against caspases 8 and 3, PARP, lamin B1, and a- 0.5 mg/ml of anti-Fas antibody (CH11; Lot 120) for the indicated tubulin used as probes. (b) Eect of the Bcl-2 overexpression on periods of time. The cells were immediately lysed with SDS as Fas-induced DNA fragmentation. Jurkat cells and the Bcl-2 described in Materials and methods, and the crude lysates from transformants (JB-2 and JB-3) were stimulated for 4 h with anti- 56104 cells per lane were subjected to Western blotting analysis Fas antibody, as above. The chromosomal DNA was prepared using antibodies against caspase 8, caspase 3, PARP, lamin B1, from unstimulated (0 h) or stimulated cells (4 h), and resolved on and a-tubulin as probes. Arrows indicate the proforms of caspases a 1.5% agarose gel. Sizes of the standard DNA markers are 8 and 3, intact PARP, lamin B1, and a-tubulin indicated on the left in base pairs (bp) Inhibition of Fas-induced apoptosis by Bcl-2 A Kawahara et al 2552 retarded in JB-2 and JB-3 cells (Figure 3a and b). Discussion These results indicated that the partial resistance of the Bcl-2-overexpressing cells to Fas-induced apoptosis Fas activation induces the recruitment of procaspase 8 was due in part to a defect in the processing of to the Fas receptor, and this association triggers the procaspase 8. caspase cascade that leads to apoptosis (Kischkel et al., 1995). In this report, we could detect the rapid FasL- induced association of caspase 8 with Fas in Jurkat Inhibition of Fas-induced recruitment of procaspase 8 to cells. On the other hand, Scadi et al. (1998) observed Fas by Bcl-2 little DISC formation in Jurkat cells, and suggested Processing of procaspase 8 occurs at the plasma that a caspase dierent from caspase 8 is activated at membrane, where it is recruited to the Fas cytoplasmic the initial stage of the Fas-induced apoptosis, which region via an adaptor, FADD/MORT1 (Medema et then damages mitochondria to activate caspase 3 and al., 1997). To examine the mechanism for the defect in 8. Since we have not studied the damage of procaspase 8 processing in the cells overexpressing Bcl- mitochondria during the Fas-induced apoptosis, we 2, Jurkat cells and JB-3 cells were treated for 5 min cannot rule out this possibility. However, we have with the soluble FasL at a concentration sucient to recently established a subline of Jurkat cells, which is kill about 80% of the Jurkat cells within 12 h. The de®cient in caspase 8. The cell line was completely procaspase 8 was immunoprecipitated with the anti- resistant to the Fas-induced apoptosis, and no caspase caspase 8 antibody, and analysed by Western blotting, 3 activation was observed by the Fas-engagement in probing with rabbit anti-Fas antibody. As shown in this cell line (manuscript in preparation), suggesting Figure 4, the Fas protein could be detected in the that caspase 8 is the primary caspase even in Jurkat immunoprecipitates from the Jurkat cells treated with cells. FasL, but not in the immunoprecipitates from the The overexpression of Bcl-2 in human Jurkat cells untreated Jurkat cells, indicating that procaspase 8 caused inecient recruitment of procaspase 8 to the quickly associated with Fas upon Fas engagement. In Fas receptor. We propose that this mechanism is contrast, the amount of procaspase 8 associated with responsible, at least in part, for the slow processing of Fas in JB-3 cells was very low after a 5 min treatment procaspases 8 and 3, and for the partial inhibition of with soluble FasL. On the other hand, when the Fas-induced apoptosis in Bcl-2-overexpressing cells. association of caspase 8 with Fas was examined after a These results agree with previous reports indicating

30 min treatment with the soluble FasL, less signi®cant that overexpression of Bcl-XL inhibits the activation of dierence was observed between the Jurkat and JB-3 caspase 3, thus preventing Fas-induced apoptosis cells (data not shown). These results indicated that Bcl- (Armstrong et al., 1996; Mandal et al., 1996). This 2 can inhibit or retard the recruitment of procaspase 8 mechanism is also consistent with the inecient DISC to the Fas receptor. formation observed in human T cells activated for a

short period, which express Bcl-XL and are resistant to Fas-induced apoptosis (Peter et al., 1997). In contrast to our results, Medema et al. (1998) and Srinivasan et al. (1998) indicated that the Fas-induced DISC formation as well as processing of procaspase 8

were not aected by Bcl-XL in human MCF7 breast carcinoma cells that were engineered to overexpress human Fas. These contradictory results are probably due to the dierences in the expression levels of Fas between Jurkat and MCF7 cells. If the inhibition of the DISC formation by Bcl-2 is due to the sequestering of procaspase 8 from the cytosol as discussed below, it is conceivable that the DISC formation is determined by a balance between the signals that recruit procaspase 8 to the Fas receptor and those that act to sequester it. Overexpression of Fas sensitizes cells to Fas-induced apoptosis (Clement and Stamenkovic, 1994), suggesting that increased clustering of Fas on the plasma membrane results in a stronger ability to recruit procaspase 8, which would overcome the sequestering of procaspase 8 by Bcl-2. Furthermore, it is possible Figure 4 Eect of Bcl-2 on the Fas-induced recruitment of caspase 8 to Fas. The parental Jurkat cells and the that the expression levels of other signaling molecules transformants overexpressing Bcl-2 (JB-3) were stimulated with such as FADD/MORT1 and caspase 8, relative to that 2.46103 units/ml of soluble FasL for 5 min. The cells were lysed of Bcl-2, could in¯uence the inhibitory function of Bcl- with 1% Triton X-100, and caspase 8 was immunoprecipitated 2 or Bcl-X on Fas-induced apoptosis. For example, if with the anti-caspase 8 antibody as described in Materials and L methods. The immunoprecipitates derived from 1.56107 cells/ the cells are abundant in procaspase 8, the eect of lane were then subjected to Western blotting analysis using Bcl-2 on the DISC formation would probably be small. rabbit anti-human Fas serum as the probe (upper panel). The In this regard, it would be necessary to compare the total cell lysates derived from 36105 cells/lane were also expression levels of Fas, FADD, and procaspase 8. analysed by Western blots probed with the anti-Fas antiserum Such analyses might clarify why the overexpression of (middle panel) or the anti-caspase 8 antibody (lower panel). Inhibition of Fas-induced recruitment of procaspase 8 to Fas Bcl-2 is eective in preventing Fas-induced apoptosis in was also observed in JB-2 cells some types of cells, but not in others. Inhibition of Fas-induced apoptosis by Bcl-2 A Kawahara et al 2553 How does the overexpression of Bcl-2 inhibit the concentrated 20-fold using an ultra®ltration membrane Fas-induced recruitment of procaspase 8 to the Fas (Centriprep 10; Amicon), and contained 36104 units/ml receptor? At least two possible mechanisms have been FasL activity. One unit of FasL activity represents the proposed for the inhibitory function of Bcl-2 in concentration of FasL that gave half-maximal cytotoxicity 4 apoptosis. Mitochondria damage induced by apoptotic against 7.5610 W4 cells in 100 ml as described (Tanaka et al., 1995). stimuli is known to cause the release of cytochrome C and/or apoptosis-inducing factor (AIF) to promote the cell-death program (Liu et al., 1996; Susin et al., 1997). Plasmid construction, and transformation

The pore-forming property of Bcl-XL suggests that Bcl- X prevents mitochondrial damage by enabling it to The expression plasmid for human Bcl-2 (pEF-Bcl2) has L been described (Itoh et al., 1993). The human Jurkat cell maintain its membrane potential and volume home- line (ATCC TIB152) was maintained in RPMI1640 ostasis (Heiden et al., 1997). In the second model, medium containing 10% fetal calf serum (Life Technolo- procaspase 8 is sequestered by Bcl-2 in the mitochon- gies). Jurkat cells (16107) were co-transfected by dria through Ced-4 protein, a cell-death regulator in C. electroporation (Suda et al., 1993) with 50 mgofthe elegans (Chinnaiyan et al., 1997; Spector et al., 1997; expression plasmid and 1 mgofpBSpacDPcarryingthe Wu et al., 1997). Although the ability of Apaf-1, the puromycin-resistant gene. The puromycin- or hygromycin- putative mammalian homolog of Ced-4, to bind to Bcl- resistant transformants were selected with 1 mg/ml puro- 2 and procaspase 8 has not yet been demonstrated, the mycin. The transformant clones expressing human Bcl-2 latter mechanism provides a likely explanation of the were selected by Western blotting analysis using anti- inhibitory activity of Bcl-2 on the DISC formation. human Bcl-2 antibody. Two independent transformant clones (JB-2 and JB-3) overexpressing human bcl-2 were Since the activation of procaspase 8 occurs only at the thus identi®ed. DISC on the plasma membrane, it is possible that the sequestering of procaspase to mitochondria inhibits its recruitment to the DISC, thus slowing down its Assay for cell viability and DNA fragmentation processing. Cell viability was assayed by using WST-1 methods Finally, Bcl-2 and Bcl-X are a multi-functional L essentially as described previously (Tanaka et al., 1995). proteins, and seem to inhibit the apoptotic process at In brief, cells (56104 cells in 100 ml) were stimulated with many stages by interacting with various proteins (Reed, various concentrations of anti-Fas antibody. Twelve hours 1997). In this regard, we cannot rule out the possibility later, 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophe- 2 that Bcl-2 and Bcl-XL inhibit Fas-induced apoptosis nyl) [H] tetrazolium monosodium salt (WST-1; Dojin downstream of the signaling cascade, in addition to Laboratories, Japan) and 1-methoxy-5-methylphenazinium their inhibitory function on the recruitment of methylsulfate were added to ®nal concentrations of 5.0 and procaspase 8 to the DISC. 0.2 mM, respectively, and incubated for 1 h. Cell viability was determined by measuring the absorbance with a test wavelength of 450 nm and a reference wavelength of 620 nm using an ELISA autoreader. DNA fragmentation Materials and methods in the apoptotic cells was determined as described previously (Enari et al., 1995). Antibodies and soluble human Fas ligand

To establish a mouse hybridoma producing anti-human Flow cytometric analysis and Western blotting caspase 8 antibody, the C-terminal portion of human caspase 8 (amino acids 176 ± 460) was produced in E. coli For ¯ow cytometry, approximately 16106 cells in 100 ml as a fusion protein with glutathione S-transferase. The wereincubatedonicefor30minwith2.5ng/mlofanti- fusion protein was used to immunize mice, and a Fas antibody (CH11) in staining buer (PBS containing hybridoma (clone 5F7) producing the anti-human caspase 2% FCS and 0.02% NaN3). After washing with staining 8 antibody was identi®ed. To produce anti-human Fas buer, the cells were stained on ice for 30 min with antibody, the cytoplasmic region of human Fas (amino ¯uorescein isothiocyanate (FITC)-conjugated anti-mouse acids 190 ± 333) was tagged with polyhistidine using the IgM antibody (200-fold dilution, Becton-Dickinson). Cells pQE vector (QIAGEN). The protein was produced in E. were then washed, and analysed using a FACScan (Becton coli and puri®ed on a QIAexpress Ni-NTA column Dickinson). (QIAGEN). Rabbits were immunized with the puri®ed For Western blotting analysis, cells (56105) were directly protein, and the serum was used as the anti-human Fas lysed by heating at 858C for 30 min in 100 ml of lysis buer serum. Mouse monoclonal antibodies against human Fas (2.5% SDS, 120 mM Tris-HCl [pH 6.8], 200 mM dithiothrei- (clone CH11) and human caspase 3 (clone 19) were tol, 20% glycerol), and mixed with 100 mlof26 Laemmli's purchased from Medical and Biological Laboratories sample buer. Proteins were separated by electrophoresis on (Nagoya, Japan) and Transduction Laboratories (Lexing- 10 ± 20% gradient or 10% polyacrylamide gels in the presence ton, KY, USA), respectively. Mouse monoclonal antibody of 0.1% SDS, and transferred to PVDF membranes against human PARP (clone C-2-10) was provided by Dr (Millipore). After blocking with 100% Block Ace (Dainihon GG Poirier. Mouse monoclonal antibodies against human Seiyaku, Japan) or PBST-milk (PBS containing 0.1% Tween lamin B1 (clone 101-B7) and a-tubulin (clone DMIA) were 20 and 5% non-fat dry milk), the membranes were incubated from Oncogene Science Inc. (Cambridge, MA, USA), and at 48C overnight with the respective primary antibody in the antibody against human Bcl-2 (clone 4D7) was from PBST-Block Ace (PBST containing 25% Block Ace) or PharMingen (San Diego, CA, USA). Horseradish (HRP)- PBST-milk. After washing three times with PBST, the conjugated goat anti-mouse and anti-rabbit IgG were membranes were incubated with HRP-conjugated anti- obtained from DAKO (Denmark) and Bio-Rad Labora- mouse IgG antibody or anti-rabbit IgG antibody in PBST- tories (Hercules, CA, USA), respectively. Block Ace or PBST-milk at room temperature for 1 h. After Soluble FasL was prepared by transfecting COS cells with washing with PBST, proteins recognized by the antibody the expression plasmid for mouse FasL (pEF-WX1) as were visualized by a chemiluminescence reaction (Renais- described previously (Suda et al., 1996). The medium was sance; DuPont NEN). Inhibition of Fas-induced apoptosis by Bcl-2 A Kawahara et al 2554 Immunoprecipitation with anti-caspase 8 antibody was washed ®ve times with lysis buer and subsequently dissolved in Laemmli's sample buer. Jurkat and its transformant cells (2.56107/10 ml) were stimulated with 2.46104 units of soluble FasL for 5 min. The cells were solubilized by incubation at 48Cfor30min in lysis buer (20 mM Tris-HCl [pH 7.5], 140 mM NaCl, 1% Triton X-100, 2 mM EDTA, 1 mM p-amidinophenyl Acknowledgements methanesulfonyl ¯uoride hydrochloride [pAPMSF], 50 mM We thank Dr GG Poirier and Y Tsujimoto for the anti-

NaF, 1 mM Na3VO4,10mg/ml leupeptin, 10 mg/ml aproti- PARP antibody and Bcl-2 cDNA, and Ms S Kumagai for nin, 10% glycerol). After removing insoluble materials by secretarial assistance. This work was supported in part by a centrifugation, the lysates were precleared with protein G- Grant-in-Aid from the Ministry of Education, Science and Sepharose by incubation for 1 h at 48C. Samples were then Culture in Japan. AK was supported by a postdoctoral incubated for 2 h at 48Cwith2mgofanti-caspase8 fellowship in Cancer Research of the Japan Society for the antibody and protein G-Sepharose. The immunoprecipitate Promotion of Science.

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