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Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115 [P. J., M. S-A. W., C. J. K., J. B]; Dana Farber Cancer Institute and Brigham and Women’s Hospital, Boston, Massachusetts 02115 [C. J. K.]; Medical University of South Carolina, Charleston, South Carolina 29403 [P. S., Y. A. H.]; and Genzyme Corporation, Cambridge, Massachusetts 02139 [H. P. B]
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Abstract |
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 TOP Abstract IntroductionResultsDiscussionMaterials and MethodsReferences |
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, and poly(ADP-ribose) polymerase were completely defective in the FADD mutant cell lines. In addition, Fas activation of the stress kinases p38 and c-Jun NH2 kinase and the generation of ceramide in response to Fas ligation were blocked in the FADD mutant cell lines. These data indicate that FADD is essential for multiple signaling events downstream of Fas. |
Introduction |
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TOPAbstractIntroductionResultsDiscussionMaterials and MethodsReferences |
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The intracellular domain of Fas contains a protein-protein interaction motif of about 100 amino acids called the death domain. Using yeast two-hybrid and other techniques, several proteins have been shown to interact with the intracellular domain of Fas, including FADD (9
, 10)
, DAXX (11)
, RIP (12)
, FAF-1 (13)
, FAP-1 (14)
, and Sentrin (15)
. In addition, the adaptor FADD was biochemically shown to be recruited to the death-inducing signaling complex upon receptor cross-linking (16)
. FADD is also capable of interacting with the cysteine protease caspase-8, which resides at the apex of the cysteine protease cascade and is required for Fas-induced apoptosis (1
, 16, 17, 18, 19, 20, 21)
. FADD and RIP have recently been homozygously deleted in mice (22, 23, 24)
. RIP does not appear to be required for Fas-mediated apoptosis because RIP-deficient cells are completely sensitive to Fas-induced death. However, RIP is involved in TNF activation of nuclear factor-
B (22
, 25)
. FADD-deficient mice die in utero and exhibit abdominal hemorrhage and defects in heart development (23)
. FADD null cells are completely resistant to Fas-induced death, indicating that FADD plays an important role in Fas-mediated apoptosis (23
, 24)
. Studies from FADD-deficient mice and transgenic mice expressing a dominant-negative allele of FADD also indicate that FADD is required for T-cell activation-induced proliferation (24
, 26
, 27)
. Other Fas-interacting proteins, such as DAXX, FAP-1, and FAF-1, have also been implicated in Fas-mediated apoptosis (11
, 13
, 14)
; however, the physiological relevance of these proteins is not yet clear because gene knockouts or somatic cell mutants have not been generated.
We used an unbiased forward genetic approach to identify essential components of the Fas-induced apoptotic signaling pathway. We randomly mutagenized Jurkat T lymphocytes and selected for cell lines resistant to Fas-mediated apoptosis. We obtained cell lines with defects in Fas, FADD, or caspase-8. Previous studies of T cells from FADD null mice or mice expressing dominant-negative FADD did not biochemically characterize FADD-regulated signal transduction. In this study, we used the FADD-deficient Jurkat cells to investigate the role of FADD in multiple signaling events downstream of Fas.
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Results |
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TOPAbstractIntroductionResultsDiscussionMaterials and MethodsReferences |
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. Stark and colleagues (28
, 29)
have successfully used this mutagenesis technique to isolate mammalian cells with recessive mutations (see "Materials and Methods"). We obtained 29 Fas-resistant clones, suggesting that these cell lines contained defects in the Fas signaling pathway; five mutant cell lines were obtained after EMS mutagenesis, 20 mutant cell lines were obtained after ICR191 mutagenesis, and four mutant cell lines were spontaneously generated after continuous passage (Fig. 1)
. We have identified the defect in all 29 mutant cell lines; 23 cell lines contain mutations in the receptor Fas, five cell lines contain mutations in the adaptor FADD, and one cell line contains a mutation in the cysteine protease caspase-8/FLICE. We have previously described the caspase-8 mutant cell line (20)
.
|
. As a control, we show that Fas-resistant cell line I4.1, which contains a mutation in the death domain of Fas, expresses wild-type levels of FADD (Fig. 2A
and data not shown). In addition, all five FADD mutant cell lines express wild-type levels of caspase-8, DAXX, bcl-2, bcl-xL, and MAPK (Fig. 2A)
. The FADD mutant cell lines all express wild-type levels of Fas as determined by surface FACS analysis and RT-PCR (data not shown). Wild-type cells (A3), FADD, or Fas mutant cells were treated with FasAb for various amounts of time, and the extent of cell death was determined using trypan blue dye exclusion and DNA ladders (Fig. 2B
and data not shown). Whereas wild-type cells died over time and generated DNA ladders, the FADD and Fas mutant cell lines were completely resistant to Fas-mediated apoptosis (Fig. 2B
and data not shown). To characterize signaling downstream of FADD, we focused on two FADD mutant cell lines, I2.1 and I6.2.
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. FasAb induced apoptosis in the wild-type cells whether they were transfected with empty vector or wild-type FADD (Fig. 2C)
. These data suggest that mutant FADD is the sole genetic lesion in the Fas pathway in these mutant cells and provides genetic evidence that FADD is required for Fas-induced death.
FADD Is Required for Fas Activation of Multiple Caspases.
We used the FADD-deficient cell lines to dissect signaling events downstream of Fas. Through its death effector domain, FADD is capable of recruiting and oligomerizing the cysteine protease caspase-8, which can subsequently initiate the caspase cascade (18, 19, 20, 21
, 30, 31, 32, 33)
. DAXX may also be recruited to the Fas death domain, providing a FADD-independent mechanism of coupling Fas to the cysteine protease cascade (11)
. Although our study and data from others indicate that FADD is required for Fas-induced death (23
, 24)
, careful biochemical characterization of signaling downstream of FADD has not yet been carried out. Wild-type, FADD, or Fas mutant cell lines were treated with FasAb for various amounts of time, and cell extracts were prepared. We tested the ability of Fas-activated proteases in wild-type and mutant cell extracts to cleave PARP and pro-caspase-2 in vitro. PARP and pro-caspase-2 were proteolytically cleaved in extracts from FasAb treated wild-type but not FADD or Fas mutant cells (Fig. 3A)
. We next examined the ability of FasAb to activate multiple caspases in vivo by using immunoblot analysis. FasAb treatment of wild-type cells but not FADD or Fas mutant cells resulted in the proteolytic cleavage and presumed activation of caspases with long prodomains, such as caspase-2 and caspase-8, and the caspase-8 substrate BID (Refs. 34
, 35
; Fig. 3B
). Furthermore, FasAb activated proteases capable of cleaving the effector proteases caspase-3, caspase-7, and substrates PKC- and PARP in wild-type cells but not in FADD or Fas mutant cells (Fig. 3C)
. These data suggest that FADD is essential for Fas activation of multiple caspases and that a parallel pathway linking Fas to the caspase cascade through DAXX does not appear to function in Jurkat cells in the absence of FADD.
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, and ceramide-activated protein phosphatase; however, the precise mechanism by which ceramide induces apoptosis is not yet clear (36)
. Fas receptor cross-linking has been shown to result in an increase in sphingomyelinase activity and intracellular ceramide (36, 37, 38, 39, 40)
. Several studies have suggested that ceramide plays an important role in mediating the Fas death signal (36, 37, 38, 39, 40)
. However, the origin of the signal emanating from Fas that leads to increased ceramide levels is not known. We sought to determine whether FADD and caspase-8 are required for Fas-induced increases in endogenous ceramide levels. FasAb treatment of wild-type but not FADD or caspase-8 mutant cells resulted in an increase in intracellular ceramide (Fig. 4)
. These results indicate that FADD and caspase-8 mediate the Fas
ceramide signal and are required for the generation of ceramide in response to Fas ligation.
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. However, cotransfection of FADD and p38 or FADD and JNK did not result in the activation of the stress kinases, suggesting that overexpression of FADD is not sufficient to activate the stress kinases (Fig. 5B)
. In contrast, osmotic shock was still capable of activating transfected p38 and JNK (data not shown). These data suggest that FADD is required for Fas activation of p38 and JNK but is not sufficient for their activation.
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Discussion |
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TOPAbstractIntroductionResultsDiscussionMaterials and MethodsReferences |
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or caspase-8 (20)
back into the FADD or caspase-8 mutant cell lines, respectively, restored Fas-mediated apoptosis, suggesting that these are the only defects in the Fas pathway in these cells. Because we used an unbiased genetic screen and obtained cell lines with specific mutations in FADD and caspase-8, this study supports recent results from mouse knockout studies by providing genetic evidence that FADD and caspase-8 are required for Fas-mediated apoptosis (23
, 24)
. We did not obtain mutations in other proteins reported to associate with Fas. This may be attributable simply to the fact that our mutagenesis was not saturating. However, it is also possible that other proteins implicated in the Fas pathway are not essential for Fas-mediated apoptosis or have redundant functions downstream of FADD and caspase-8.
Five mutant cell lines do not express FADD by immunoblot analysis but appear to express wild-type levels of several other proteins, including DAXX and caspase-8 (Fig. 2A)
. Recent studies have suggested that DAXX can bind Fas and mediate a FADD-independent apoptotic signal (11
, 46)
. All five FADD-deficient cell lines are completely resistant to Fas-mediated apoptosis, indicating that in Jurkat cells, there are no other major parallel pathways. FADD null lymphocytes and embryonic fibroblasts from homozygously deleted mice are completely deficient in Fas-induced death, supporting the conclusion that there are no other major parallel Fas-death pathways (23
, 24)
. DAXX has also been implicated in mediating Fas activation of JNK and p38 (11
, 46)
. We show that Fas activation of p38 and JNK is defective in our FADD-deficient cell lines and that transfection of FADD alone is not sufficient to activate p38 and JNK (Fig. 5)
. These results are consistent with other studies that have shown that FADD is not sufficient to activate the stress kinases (11
, 47)
. However, our data indicate that although FADD is not sufficient, it is required for Fas activation of p38 and JNK. Hence, DAXX may function downstream of FADD and caspase-8, cooperate with FADD, or be more relevant in other cell types.
In this study, we have used our FADD-deficient cell lines to dissect signaling events downstream of Fas. To analyze FADD-dependent signaling, other groups have overexpressed a dominant-negative version of FADD consisting of the death domain alone (47, 48, 49, 50, 51)
. Use of this reagent is complicated by the fact that the FADD death domain may bind the Fas death domain and block the recruitment of other Fas death domain-interacting proteins. Experiments from FADD null cells showed that FADD was required for Fas-induced death and heart development (23
, 24) . However, detailed biochemical characterization of signaling events downstream of Fas were not performed in these cells. We now show that FADD is required for Fas activation of multiple caspases, including caspase-2, caspase-3, caspase-7, and caspase-8 and for cleavage of substrates BID, PKC-, and PARP (Fig. 3)
. These results suggest that in Jurkat cells, parallel signaling pathways that might emanate from Fas are not sufficient to activate caspases.
FADD binds and recruits caspase-8 to the receptor complex, which ultimately results in its activation (18
, 19
, 21)
. We provide direct evidence that FADD is required for Fas activation of caspase-8 because Fas-induced processing of caspase-8 and cleavage of the caspase-8 substrate BID are defective in the FADD mutant cell lines (Fig. 3B)
. Fas cross-linking has been shown to result in an increase in intracellular ceramide levels that may potentially mediate apoptosis, but the origin of this signal emanating from the receptor complex has remained obscure (37, 38, 39, 40)
. We now show that FADD and caspase-8 are required for Fas-induced increases in intracellular ceramide (Fig. 4)
, suggesting that these proteins mediate the Fasceramide signal. The increase in ceramide may be mediated by acid sphingomyelinases because a recent study showed that TNF-induced activation of acid sphingomyelinases requires FADD (52)
. However, the mechanism by which caspase-8 regulates ceramide levels remains unclear.
In conclusion, the isolation of cell lines containing mutations in FADD in a genetic screen provides strong evidence that FADD is required for Fas-induced death, supporting recently published data from FADD knockout mice (23 , 24) . Furthermore, we have used these FADD-deficient cell lines to show that FADD is required for multiple signaling events downstream of Fas. We show that FADD is required for Fas activation of caspase-2, caspase-3, caspase-7, and caspase-8, suggesting that other signaling pathways emanating from Fas are not sufficient to activate these caspases. We also show that increases in intracellular ceramide in response to Fas ligation require FADD and caspase-8. Finally, we show that although FADD is not sufficient to activate the stress kinases, p38 and JNK, it is required for Fas activation of these kinases.
These Fas-, FADD-, and caspase-8-deficient cell lines provide useful reagents for studying the physiological role of these proteins in the absence of overexpression. In particular, it will be interesting to determine the role of FADD and caspase-8 in apoptosis induced by other members of the death receptor family and other receptor-independent inducers of apoptosis.
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Materials and Methods |
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TOPAbstractIntroductionResultsDiscussionMaterials and MethodsReferences |
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(C20), caspase-2 (Ich-1L C20), bcl-xL (S-18), and bcl-2 (100) were purchased from Santa Cruz Biotechnology. Caspase-3 antibodies were purchased from PharMingen. Anti-Fas monoclonal antibody (CH11) was purchased from Kamiya Biomedical Co. (Thousand Oaks, CA). Antibodies to FADD were purchased from PharMingen and Transduction Labs. Antibodies to PARP (C2–10) were obtained from G. G. Poirier (CHUL Research Center, Quebec, Canada). [35S]Methionine and [
32P]ATP were obtained from Amersham (Cleveland, OH) and New England Nuclear (Boston, MA), respectively.
Mutagenesis.
For EMS mutagenesis, Jurkat subclone A3 was either left untreated (1 x 108) or treated (1 x 108) with EMS (Sigma) at 200 µg/ml for 24 h (53)
. Cells were allowed to recover for 5 days prior to selection in FasAb. To obtain one cell/well, cells were plated in serial dilutions into 96-well plates in the presence of FasAb (CH11; Kamiya). After 2–4 weeks, surviving clones were expanded and further characterized. ICR191 mutagenesis was performed as described (28
, 29)
. Briefly, Jurkat subclone A3 was either left untreated (1 x 108) or treated (1 x 108) with ICR191 (Polysciences, Inc.) at 2 µg/ml for 2 h. Cells were exposed to ICR191 for 3 cycles to increase the frequency of mutagenizing both alleles (28
, 29)
. After the initial treatment, mutagen was washed out, and cells were allowed to recover for 15 days. Cells were then treated again with ICR191 and allowed to recover for 2 days before the final treatment. Cells were then plated in serial dilutions into 96-well plates in the presence of FasAb as described above (CH11; Kamiya).
RT-PCR and in Vitro Transcription and Translation.
RNA was isolated from cells using Trizol (Life Technologies, Inc.). Reverse-transcription was performed using oligo(dT) primers (Superscript; Life Technologies, Inc.), followed by PCR using primers specific for Fas. The 5' primers also contained a T7 RNA polymerase promoter overhang (54)
. To visualize the protein products, the PCR products were purified using Gene Clean (Bio 101), transcribed (T7 polymerase), translated, and labeled with [35S]methionine (Amersham) in vitro using a TNT kit (Promega). Labeled proteins were separated by SDS-PAGE.
Transfection and Complementation.
Jurkat cells were transfected as described (20)
. FADD was subcloned into pRSV using HindIII and XbaI after PCR using pcDNA3-FADD as a template (gift from V. Dixit, University of Michigan Medical School, Ann Arbor, MI). After 1-h recovery, cells were spun over a Ficoll-Paque gradient (Pharmacia) to remove dead cells attributable to the transfection procedure. Transfections were divided in half and either left untreated or treated with FasAb (ascites, 7C11 at 1:1000; gift from M. J. Robertson, Indiana University School of Medicine) for 12 h. Cells were then washed in FACS wash (0.5% BSA in PBS) and stained with anti-CD8-FITC (Collaborative Labs) at a 1:50 dilution. Propidium iodide (Sigma) was added 5 min prior to FACS analysis at 40 µg/ml to detect dead cells. Cells were analyzed on a FACScaliber (Becton Dickinson) using Cell Quest software.
Western Blot Analysis.
This was performed as previously described (20)
.
In Vitro Protease and Protein Kinase Assays.
Protease assays were performed as described by Li et al. (55)
. Briefly, Jurkat cells were concentrated to 2 x 107 cells/ml in complete medium and either left untreated or treated with 250 ng/ml anti-FasAb (CH11) for various times. Cells were washed twice with ice-cold RPMI and resuspended at 4 x 105 cells/µl in extraction buffer [10 mM HEPES (pH 7), 2 mM MgCl2, 50 mM NaCl, 5 mM EGTA (pH 7.6), 40 mM ß-glycerophosphate, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, and 1 µg/ml pepstatin]. Cells were lysed by four cycles of freeze/thawing in a dry ice/ethanol bath. Extracts were centrifuged at 14,000 x g for 15 min at 4°C and stored at -80°C. Substrates pcDNA3-procaspase-2 and pBSII-PARP were in vitro transcribed, translated, and labeled with [35S]methionine using TNT (Promega) for 90 min at 30°C. Five µl of cell extract were incubated with 0.5–1.5 µl of the translation reaction for 1 h at 37°C. An equal volume of 2x sample buffer was added to stop the reaction. Cleavage products were analyzed by SDS-PAGE and fluorography.
Ceramide Assays.
Jurkat cells (2 x 106 cells) were treated with FasAb for various amounts of time, washed twice in ice-cold PBS, and rapidly frozen in a methanol-dry ice bath. Ceramide levels were determined as described previously (56)
. Briefly, lipids were extracted using the Bligh and Dyer method (57)
. Ceramide levels were evaluated using the Escherichia coli diacylglycerol kinase assay (58
, 59)
. Lipids were incubated at room temperature for 30 min in the presence of ß-octylglucoside/dioleoyl-phosphatidyl glycerol micelles, 2 mM DTT, 5 µg of proteins from diacylglycerol kinase membranes, and 2 mM ATP mixed with [32P]ATP in a final volume of 100 µl. The reaction products were separated by TLC in chloroform:acetone:methanol:acetic acid:water (50:20:15:10:5), and the radioactivity associated with phosphatidic acid and ceramide-P was measured by liquid scintillation. Ceramide and diacylglycerol levels were quantified using external standards and normalized to phospholipids.
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Acknowledgments |
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Footnotes |
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1 This work was partially supported by Grant CA46595 (to J. B.) and Grant GM-43825 (to Y. A. H.) from the NIH and a Hoechst Roussel Marion Exploratory Award (to J. B.). J. B. is an established investigator of the American Heart Association. P. J. was supported in part by a National Science Foundation Predoctoral Fellowship. C. J. K. was supported by the Dana-Farber Cancer Institute, Brigham and Women’s Hospital, and a grant from Howard Hughes Medical Institute to the Department of Cell Biology. 
2 To whom requests for reprints should be addressed, at Department of Cell Biology, 240 Longwood Avenue, Harvard Medical School, Boston, MA 02115. Phone: (617) 432-4848; Fax: (617) 432-1144; E-mail: jblenis{at}hms.harvard.edu
3 The abbreviations used are: TNF, tumor necrosis factor; Ab, antibody; RT-PCR, reverse transcription-PCR; MAPK, mitogen-activated protein kinase; FACS, fluorescence-activated cell sorter; PARP, poly(ADP-ribose) polymerase; PKC, protein kinase C; JNK, c-Jun NH2 kinase; EMS, ethyl methane sulfonate.
Received for publication 7/ 7/99. Revision received 9/ 7/99. Accepted for publication 9/ 8/99.
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References |
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TOPAbstractIntroductionResultsDiscussionMaterials and MethodsReferences |
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B signal. Immunity, 8: 297-303, 1998.[Medline]
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 E. A. Acheampong, Z. Parveen, L. W. Muthoga, V. Wasmuth-Peroud, M. Kalayeh, A. Bashir, R. Diecidue, M. Mukhtar, and R. J. Pomerantz Molecular Interactions of Human Immunodeficiency Virus Type 1 with Primary Human Oral Keratinocytes J. Virol., July 1, 2005; 79(13): 8440 - 8453. [Abstract] [Full Text] [PDF]
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D. Milhas, O. Cuvillier, N. Therville, P. Clave, M. Thomsen, T. Levade, H. Benoist, and B. Segui Caspase-10 Triggers Bid Cleavage and Caspase Cascade Activation in FasL-induced Apoptosis J. Biol. Chem., May 20, 2005; 280(20): 19836 - 19842. [Abstract] [Full Text] [PDF]
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 F. Henkler, E. Behrle, K. M. Dennehy, A. Wicovsky, N. Peters, C. Warnke, K. Pfizenmaier, and H. Wajant The extracellular domains of FasL and Fas are sufficient for the formation of supramolecular FasL-Fas clusters of high stability J. Cell Biol., March 28, 2005; 168(7): 1087 - 1098. [Abstract] [Full Text] [PDF]
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T.-G. Jin, A. Kurakin, N. Benhaga, K. Abe, M. Mohseni, F. Sandra, K. Song, B. K. Kay, and R. Khosravi-Far Fas-associated Protein with Death Domain (FADD)-independent Recruitment of c-FLIPL to Death Receptor 5 J. Biol. Chem., December 31, 2004; 279(53): 55594 - 55601. [Abstract] [Full Text] [PDF]
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 J Doering, B Begue, M J Lentze, F Rieux-Laucat, O Goulet, J Schmitz, N Cerf-Bensussan, and F M Ruemmele Induction of T lymphocyte apoptosis by sulphasalazine in patients with Crohn's disease Gut, November 1, 2004; 53(11): 1632 - 1638. [Abstract] [Full Text] [PDF]
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C.-F. Lin, C.-L. Chen, W.-T. Chang, M.-S. Jan, L.-J. Hsu, R.-H. Wu, M.-J. Tang, W.-C. Chang, and Y.-S. Lin Sequential Caspase-2 and Caspase-8 Activation Upstream of Mitochondria during Ceramideand Etoposide-induced Apoptosis J. Biol. Chem., September 24, 2004; 279(39): 40755 - 40761. [Abstract] [Full Text] [PDF]
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B. M. Murphy, E. M. Creagh, and S. J. Martin Interchain Proteolysis, in the Absence of a Dimerization Stimulus, Can Initiate Apoptosis-associated Caspase-8 Activation J. Biol. Chem., August 27, 2004; 279(35): 36916 - 36922. [Abstract] [Full Text] [PDF]
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S. Kreuz, D. Siegmund, J.-J. Rumpf, D. Samel, M. Leverkus, O. Janssen, G. Hacker, O. Dittrich-Breiholz, M. Kracht, P. Scheurich, et al. NF{kappa}B activation by Fas is mediated through FADD, caspase-8, and RIP and is inhibited by FLIP J. Cell Biol., August 2, 2004; 166(3): 369 - 380. [Abstract] [Full Text] [PDF]
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L. R. Thomas, A. Henson, J. C. Reed, F. R. Salsbury, and A. Thorburn Direct Binding of Fas-associated Death Domain (FADD) to the Tumor Necrosis Factor-related Apoptosis-inducing Ligand Receptor DR5 Is Regulated by the Death Effector Domain of FADD J. Biol. Chem., July 30, 2004; 279(31): 32780 - 32785. [Abstract] [Full Text] [PDF]
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M. Filippova, L. Parkhurst, and P. J. Duerksen-Hughes The Human Papillomavirus 16 E6 Protein Binds to Fas-associated Death Domain and Protects Cells from Fas-triggered Apoptosis J. Biol. Chem., June 11, 2004; 279(24): 25729 - 25744. [Abstract] [Full Text] [PDF]
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F. K.-M. Chan, J. Shisler, J. G. Bixby, M. Felices, L. Zheng, M. Appel, J. Orenstein, B. Moss, and M. J. Lenardo A Role for Tumor Necrosis Factor Receptor-2 and Receptor-interacting Protein in Programmed Necrosis and Antiviral Responses J. Biol. Chem., December 19, 2003; 278(51): 51613 - 51621. [Abstract] [Full Text] [PDF]
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X. W. Meng, J. Chandra, D. Loegering, K. Van Becelaere, T. J. Kottke, S. D. Gore, J. E. Karp, J. Sebolt-Leopold, and S. H. Kaufmann Central Role of Fas-associated Death Domain Protein in Apoptosis Induction by the Mitogen-activated Protein Kinase Kinase Inhibitor CI-1040 (PD184352) in Acute Lymphocytic Leukemia Cells in Vitro J. Biol. Chem., November 21, 2003; 278(47): 47326 - 47339. [Abstract] [Full Text] [PDF]
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N. Harper, M. A. Hughes, S. N. Farrow, G. M. Cohen, and M. MacFarlane Protein Kinase C Modulates Tumor Necrosis Factor-related Apoptosis-inducing Ligand-induced Apoptosis by Targeting the Apical Events of Death Receptor Signaling J. Biol. Chem., November 7, 2003; 278(45): 44338 - 44347. [Abstract] [Full Text] [PDF]
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A. Algeciras-Schimnich, E. M. Pietras, B. C. Barnhart, P. Legembre, S. Vijayan, S. L. Holbeck, and M. E. Peter Two CD95 tumor classes with different sensitivities to antitumor drugs PNAS, September 30, 2003; 100(20): 11445 - 11450. [Abstract] [Full Text] [PDF]
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 L. Molto, P. Rayman, E. Paszkiewicz-Kozik, M. Thornton, L. Reese, J. C. Thomas, T. Das, D. Kudo, R. Bukowski, J. Finke, et al. The Bcl-2 Transgene Protects T Cells from Renal Cell Carcinoma-mediated Apoptosis Clin. Cancer Res., September 15, 2003; 9(11): 4060 - 4068. [Abstract] [Full Text] [PDF]
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E. A. Choi, H. Lei, D. J. Maron, J. M. Wilson, J. Barsoum, D. L. Fraker, W. S. El-Deiry, and F. R. Spitz Stat1-dependent Induction of Tumor Necrosis Factor-related Apoptosis-inducing Ligand and the Cell-Surface Death Signaling Pathway by Interferon {beta} in Human Cancer Cells Cancer Res., September 1, 2003; 63(17): 5299 - 5307. [Abstract] [Full Text] [PDF]
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N. M. Storey, M. Gomez-Angelats, C. D. Bortner, D. L. Armstrong, and J. A. Cidlowski Stimulation of Kv1.3 Potassium Channels by Death Receptors during Apoptosis in Jurkat T Lymphocytes J. Biol. Chem., August 29, 2003; 278(35): 33319 - 33326. [Abstract] [Full Text] [PDF]
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N. Harper, M. Hughes, M. MacFarlane, and G. M. Cohen Fas-associated Death Domain Protein and Caspase-8 Are Not Recruited to the Tumor Necrosis Factor Receptor 1 Signaling Complex during Tumor Necrosis Factor-induced Apoptosis J. Biol. Chem., July 3, 2003; 278(28): 25534 - 25541. [Abstract] [Full Text] [PDF]
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S.-W. Ryu, S.-J. Lee, M.-Y. Park, J.-i. Jun, Y.-K. Jung, and E. Kim Fas-associated Factor 1, FAF1, Is a Member of Fas Death-inducing Signaling Complex J. Biol. Chem., June 20, 2003; 278(26): 24003 - 24010. [Abstract] [Full Text] [PDF]
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N. Holler, A. Tardivel, M. Kovacsovics-Bankowski, S. Hertig, O. Gaide, F. Martinon, A. Tinel, D. Deperthes, S. Calderara, T. Schulthess, et al. Two Adjacent Trimeric Fas Ligands Are Required for Fas Signaling and Formation of a Death-Inducing Signaling Complex Mol. Cell. Biol., February 15, 2003; 23(4): 1428 - 1440. [Abstract] [Full Text] [PDF]
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T. Vanden Berghe, M. Kalai, G. van Loo, W. Declercq, and P. Vandenabeele Disruption of HSP90 Function Reverts Tumor Necrosis Factor-induced Necrosis to Apoptosis J. Biol. Chem., February 14, 2003; 278(8): 5622 - 5629. [Abstract] [Full Text] [PDF]
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G. V. Denis, Q. Yu, P. Ma, L. Deeds, D. V. Faller, and C.-Y. Chen Bcl-2, via Its BH4 Domain, Blocks Apoptotic Signaling Mediated by Mitochondrial Ras J. Biol. Chem., February 14, 2003; 278(8): 5775 - 5785. [Abstract] [Full Text] [PDF]
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 B. Neumeister, M. Faigle, K. Lauber, H. Northoff, and S. Wesselborg Legionella pneumophila induces apoptosis via the mitochondrial death pathway Microbiology, November 1, 2002; 148(11): 3639 - 3650. [Abstract] [Full Text] [PDF]
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L. R. Thomas, D. J. Stillman, and A. Thorburn Regulation of Fas-associated Death Domain Interactions by the Death Effector Domain Identified by a Modified Reverse Two-hybrid Screen J. Biol. Chem., September 6, 2002; 277(37): 34343 - 34348. [Abstract] [Full Text] [PDF]
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D. L. Bolton, B.-I. Hahn, E. A. Park, L. L. Lehnhoff, F. Hornung, and M. J. Lenardo Death of CD4+ T-Cell Lines Caused by Human Immunodeficiency Virus Type 1 Does Not Depend on Caspases or Apoptosis J. Virol., April 16, 2002; 76(10): 5094 - 5107. [Abstract] [Full Text] [PDF]
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 H. Qi, P. Juo, J. Masuda-Robens, M. J. Caloca, H. Zhou, N. Stone, M. G. Kazanietz, and M. M. Chou Caspase-mediated Cleavage of the TIAM1 Guanine Nucleotide Exchange Factor during Apoptosis Cell Growth Differ., December 1, 2001; 12(12): 603 - 611. [Abstract] [Full Text] [PDF]
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F. C. Kischkel, D. A. Lawrence, A. Tinel, H. LeBlanc, A. Virmani, P. Schow, A. Gazdar, J. Blenis, D. Arnott, and A. Ashkenazi Death Receptor Recruitment of Endogenous Caspase-10 and Apoptosis Initiation in the Absence of Caspase-8 J. Biol. Chem., November 30, 2001; 276(49): 46639 - 46646. [Abstract] [Full Text] [PDF]
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Z. Han, P. Pantazis, J. H. Wyche, N. Kouttab, V. J. Kidd, and E. A. Hendrickson A Fas-associated Death Domain Protein-dependent Mechanism Mediates the Apoptotic Action of Non-steroidal Anti-inflammatory Drugs in the Human Leukemic Jurkat Cell Line J. Biol. Chem., October 12, 2001; 276(42): 38748 - 38754. [Abstract] [Full Text] [PDF]
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C.-Y. Chen, P. Juo, J. S. Liou, C.-Q. Li, Q. Yu, J. Blenis, and D. V. Faller The Recruitment of Fas-associated Death Domain/Caspase-8 in Ras-induced Apoptosis Cell Growth Differ., June 1, 2001; 12(6): 297 - 306. [Abstract] [Full Text] [PDF]
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I. Rivera-Walsh, M. E. Cvijic, G. Xiao, and S.-C. Sun The NF-kappa B Signaling Pathway Is Not Required for Fas Ligand Gene Induction but Mediates Protection from Activation-induced Cell Death J. Biol. Chem., August 11, 2000; 275(33): 25222 - 25230. [Abstract] [Full Text] [PDF]
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D. Siegmund, D. Mauri, N. Peters, P. Juo, M. Thome, M. Reichwein, J. Blenis, P. Scheurich, J. Tschopp, and H. Wajant Fas-associated Death Domain Protein (FADD) and Caspase-8 Mediate Up-regulation of c-Fos by Fas Ligand and Tumor Necrosis Factor-related Apoptosis-inducing Ligand (TRAIL) via a FLICE Inhibitory Protein (FLIP)-regulated Pathway J. Biol. Chem., August 24, 2001; 276(35): 32585 - 32590. [Abstract] [Full Text] [PDF]
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M. Gomez-Angelats and J. A. Cidlowski Protein Kinase C Regulates FADD Recruitment and Death-inducing Signaling Complex Formation in Fas/CD95-induced Apoptosis J. Biol. Chem., November 21, 2001; 276(48): 44944 - 44952. [Abstract] [Full Text] [PDF]
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) or treated with FasAb (
) for various amounts of time. Cell death was monitored by trypan blue dye exclusion and DNA laddering (data not shown). Bars, SD. C, complementation of FADD mutant cell lines I2.1 and I6.2. Wild-type (A3) or FADD mutant (I2.1 and I6.2) cells were transfected with a surface marker, CD8, and empty pRSV vector (1:3 ratio) or CD8 and pRSV-FADD (1:3 ratio). Cells were either left untreated or treated with FasAb for 12 h. Cells were stained with anti-CD8-FITC and propidium iodide. Transfected, viable cells were identified by FACS on the basis of CD8 expression and propidium iodide exclusion. The number of viable, CD8-positive cells is expressed as a percentage relative to the untreated controls. Data represent the means; bars, SD; n = 3.
), or caspase-8 (