CG&D
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cell Growth & Differentiation

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Juo, P.
Right arrow Articles by Blenis, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Juo, P.
Right arrow Articles by Blenis, J.
Cell Growth & Differentiation Vol. 10, 797-804, December 1999
© 1999 American Association for Cancer Research

FADD Is Required for Multiple Signaling Events Downstream of the Receptor Fas1

Peter Juo, Michele Sue-Ann Woo, Calvin J. Kuo, Paola Signorelli, Hans P. Biemann, Yusuf A. Hannun and John Blenis2

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]


    Abstract
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
To identify essential components of the Fas-induced apoptotic signaling pathway, Jurkat T lymphocytes were chemically mutagenized and selected for clones that were resistant to Fas-induced apoptosis. We obtained five cell lines that contain mutations in the adaptor FADD. All five cell lines did not express FADD by immunoblot analysis and were completely resistant to Fas-induced death. Complementation of the FADD mutant cell lines with wild-type FADD restored Fas-mediated apoptosis. Fas activation of caspase-2, caspase-3, caspase-7, and caspase-8 and the proteolytic cleavage of substrates such as BID, protein kinase C{delta}, 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
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Fas (APO-1/CD95) is a transmembrane receptor belonging to the TNF3 family of death receptors (1 , 2) . Cross-linking of Fas with Fas ligand or agonistic antibody (FasAb) results in the initiation of an apoptotic signal that ultimately leads to the demise of the cell (3 , 4) . Fas plays an important role in the immune system. The Fas ligand-Fas system is involved in the deletion of autoreactive T cells, the down-regulation of the immune response, and the maintenance of sites of immune privilege (2 , 5) . The importance of Fas in the immune system is underscored by the fact that mice and humans with mutations in Fas exhibit symptoms of autoimmune diseases (6, 7, 8) .

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-{kappa}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.


    Results
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Isolation of Cell Lines Deficient in FADD.
To identify essential components of the Fas-mediated apoptotic pathway, we undertook a genetic screen in mammalian cells. We initially isolated a Jurkat T lymphocyte subclone (A3) that was very sensitive to Fas-mediated apoptosis and had a low rate of spontaneous resistance [1/(2 x 107)]. We randomly mutagenized this cell line with the point mutagen, EMS, and the frameshift mutagen, ICR191, and selected the cells in the presence of FasAb (Fig. 1)Citation . 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)Citation . 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) .



View larger version (21K):
[in this window]
[in a new window]
 
Fig. 1. Mutagenesis scheme. Wild-type Jurkat cells (A3) were made neo resistant and then either left untreated or treated with the point mutagen, EMS, or the frameshift mutagen, ICR191. Cells were then plated in serial dilutions in 96-well plates in the presence of FasAb for 3–5 weeks. Cells in continuous passage for 6–8 months were also treated with FasAb to isolate spontaneous mutants. Twenty-nine Fas-resistant clones were isolated and expanded.

 
A variety of techniques were used to demonstrate that 23 of the Fas-resistant mutant cell lines contained mutations in the receptor Fas (data not shown). However, five cell lines contained mutations in the adaptor protein, FADD. RT-PCR analysis using primers specific for FADD indicated that all of the mutant cell lines contained correctly sized FADD RNA products (data not shown). Western blot analysis with antibodies to FADD indicated that in contrast to wild-type A3 cells, which express FADD, five mutant cell lines, I2.1, I5, I6.2, E1, and S4, do not express FADD at the protein level (Fig. 2A)Citation . 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. 2ACitation 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)Citation . 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. 2BCitation 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. 2BCitation and data not shown). To characterize signaling downstream of FADD, we focused on two FADD mutant cell lines, I2.1 and I6.2.



View larger version (33K):
[in this window]
[in a new window]
 
Fig. 2. Isolation and initial characterization of FADD mutant cell lines. A, five mutant cell lines do not express FADD protein. Lysates from wild-type (A3) and mutant cell lines were Western blotted for expression of FADD, caspase-8, DAXX, bcl-2, bcl-xL and MAPK. A3, wild-type; I4.1, Fas mutant. B, wild-type (A3), FADD (I2.1, I5, I6.2, E1, and S4), or Fas (I4.1) mutant cell lines were either left untreated (0, ) or treated with FasAb ({blacksquare}) 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.

 
Complementation of the FADD-deficient Cell Lines.
To investigate whether the FADD mutation is the only defect in the Fas pathway in these mutant cell lines, we complemented the mutant cell lines with wild-type FADD. Transfection of wild-type FADD but not empty vector into the FADD mutant cells rescued the ability of FasAb to induce apoptosis (Fig. 2C)Citation . FasAb induced apoptosis in the wild-type cells whether they were transfected with empty vector or wild-type FADD (Fig. 2C)Citation . 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)Citation . 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. 3BCitation ). Furthermore, FasAb activated proteases capable of cleaving the effector proteases caspase-3, caspase-7, and substrates PKC-{delta} and PARP in wild-type cells but not in FADD or Fas mutant cells (Fig. 3C)Citation . 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.



View larger version (55K):
[in this window]
[in a new window]
 
Fig. 3. FADD is required for Fas activation of multiple caspases. A, in vitro protease assays. Wild-type (A3), FADD (I2.1 and I6.2), or Fas (I4.1) mutant cells were either left untreated (0) or treated with FasAb for various amounts of time. Extracts were prepared and incubated with in vitro translated [35S]methionine-labeled substrates PARP or pro-caspase-2 for 1 h at 37°C. The reactions were analyzed by SDS-PAGE, followed by fluorography. Untreated substrate input is also shown (-). B and C, wild-type (A3), FADD (I2.1 and I6.2) or Fas (I4.1) mutant cells were either left untreated (0) or treated with FasAb for various amounts of time. Lysates were Western blotted with antibodies to: B, pro-caspase-2, pro-caspase-8, and BID; or C, pro-caspase-7, caspase-3, PKC-{delta}, PARP, and MAPK. Arrows, positions of the proteolytically cleaved fragments.

 
FADD and Caspase-8 Are Required for Fas-induced Ceramide Generation.
The sphingolipid ceramide is generated in response to multiple apoptotic stimuli and has been proposed to mediate the death signal (36) . Ceramide can activate several signaling proteins such as ceramide-activated protein kinase, PKC-{zeta}, 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)Citation . 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.



View larger version (18K):
[in this window]
[in a new window]
 
Fig. 4. FADD is required for Fas-induced increases in ceramide levels. Wild-type (•), FADD ({blacktriangleup}), or caspase-8 ({blacksquare}) mutant cells were either left untreated or treated with FasAb for various amounts of time. Total ceramide levels were determined and graphed as pmol of ceramide/nmol of total phospholipids. Data represent the average of two independent experiments.

 
FADD Is Required for Fas Activation of p38 and JNK.
We and others have shown previously that Fas cross-linking results in the activation of the stress kinases, p38 and JNK, in a caspase-dependent manner (41, 42, 43, 44, 45) . We determined whether FADD was required for Fas activation of p38 and JNK. Fas cross-linking of wild-type cells resulted in the activation of p38 and JNK. In contrast, Fas cross-linking was unable to activate p38 and JNK in the FADD or Fas mutant cell lines, suggesting that FADD is required for Fas activation of the stress kinases (Fig. 5A)Citation . 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)Citation . 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.



View larger version (32K):
[in this window]
[in a new window]
 
Fig. 5. FADD is required but not sufficient for Fas activation of p38 and JNK. A, wild-type (A3), FADD (I2.1 and I6.2), or Fas (I4.1) mutant cells were either left untreated or treated with FasAb for 4 h. p38 and JNK activity were determined in immune-complex kinase assays using GST-ATF2 as substrate. Phosphate incorporation was quantitated using a PhosphorImager and is presented as fold activation. B, wild-type Jurkat cells were either mock transfected (Lanes 1 and 4), transfected with FLAG-p38 alone (Lane 2), cotransfected with FLAG-p38 and HA-FADD (Lane 3), transfected with GST-JNK1 alone (Lane 5), or cotransfected with GST-JNK1 and HA-FADD (Lane 6). Immune complex kinase assays of FLAG-p38 or GST-JNK were performed against the substrate GST-ATF2 (top panels). Western blots of transfected FLAG-p38, GST-JNK, or HA-FADD are shown (bottom panels).

 

    Discussion
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
We used an unbiased forward genetic approach in mammalian cells to identify essential components of the Fas-mediated apoptotic signaling pathway. We obtained several Fas-resistant cell lines that contained mutations in the receptor Fas, the adaptor FADD, and the cysteine protease caspase-8. Complementation of wild-type FADD (Fig. 2C)Citation 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)Citation . 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)Citation . 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-{delta}, and PARP (Fig. 3)Citation . 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)Citation . 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)Citation , suggesting that these proteins mediate the Fas->ceramide 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.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Antibodies and Reagents.
Antibodies to caspase-7, caspase-8 (rat monoclonal), and BID were kindly provided by Junying Yuan (Harvard Medical School). Antibodies to DAXX (M112), PKC-{delta} (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 [{gamma}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 [{gamma}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.


    Acknowledgments
 
We thank Michael J. Robertson, Junying Yuan, Gerry Crabtree, Vishva M. Dixit, Vincent Cryns, and Honglin Li for advice and reagents. We thank Susan Reynolds and Richard Konz for technical assistance in FACS analysis and members of the Blenis lab for critical reading of the manuscript.


    Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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. Back

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 Back

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. Back

Received for publication 7/ 7/99. Revision received 9/ 7/99. Accepted for publication 9/ 8/99.


    References
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

  1. Peter M. E., Krammer P. H. Mechanisms of CD95 (APO-1/Fas)-mediated apoptosis. Curr. Opin. Immunol., 10: 545-551, 1998.[Medline]
  2. Nagata S. Apoptosis by death factor. Cell, 88: 355-365, 1997.[Medline]
  3. Trauth B. C., Klas C., Peters A. M. J., Matzku S., Moller P., Falk W., Debatin K-M., Krammer P. H. Monoclonal antibody-mediated tumor regression by induction of apoptosis. Science (Washington DC), 245: 301-305, 1989.[Abstract/Free Full Text]
  4. Yonehara S., Ishii A., Yonehara M. A cell-killing monoclonal antibody (Anti-Fas) to a cell surface antigen co-downregulated with the receptor of tumor necrosis factor. J. Exp. Med., 169: 1747-1756, 1989.[Abstract/Free Full Text]
  5. Abbas A. K. Die and let live: eliminating dangerous lymphocytes. Cell, 84: 655-657, 1996.[Medline]
  6. Watanabe-Fukunaga R., Brannan C. I., Copeland N. G., Jenkins N. A., Nagata S. Lymphoproliferation disorder in mice explained by defects in Fas antigen that mediates apoptosis. Nature (Lond.), 356: 314-317, 1992.[Medline]
  7. Rieux-Laucat F., Deist T. L., Hivroz C., Roberts I. A. G., Debatin K. M., Fischer A., deVillartay J. P. Mutations in Fas associated with human lymphoproliferative syndrome and autoimmunity. Science (Washington DC), 268: 1347-1349, 1995.[Abstract/Free Full Text]
  8. Fisher G. H., Rosenberg F. J., Straus S. E., Dale J. K., Middelton L. A., Lin A. Y., Strober W., Lenardo M. J., Puck J. M. Dominant-interfering Fas gene mutations impair apoptosis in a human autoimmune lymphoproliferative syndrome. Cell, 81: 935-946, 1995.[Medline]
  9. Chinnaiyan A. M., O’Rourke K., Tewari M., Dixit V. M. FADD, a novel death domain-containing protein, interacts with the death domain of Fas and initiates apoptosis. Cell, 81: 505-512, 1995.[Medline]
  10. Boldin M. P., Varfolomeev E. E., Pancer Z., Mett I. L., Camonis J. H., Wallach D. 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: 7795-7798, 1995.[Abstract/Free Full Text]
  11. Yang X., Khosravi-Far R., Chang H. Y., Baltimore D. Daxx, a novel Fas-binding protein that activates JNK and apoptosis. , 89: 1067-1076, 1997.
  12. Stanger B. Z., Leder P., Lee T-H., Kim E., Seed B. RIP. A novel protein containing a death domain that interacts with Fas/APO-1 (CD95) in yeast and causes cell death. Cell, 81: 513-523, 1995.[Medline]
  13. Chu K., Niu X., Williams L. T. A Fas-associated protein factor, FAF-1, potentiates Fas-mediated apoptosis. Proc. Natl. Acad. Sci. USA, 92: 11894-11898, 1995.[Abstract/Free Full Text]
  14. Sato T., Irie S., Kitada S., Reed J. C. FAP-1: a protein tyrosine phosphatase that associates with Fas. Science (Washington DC), 268: 411-415, 1995.[Abstract/Free Full Text]
  15. Okura T., Gong L., Kamitani T., Wada T., Okura I., Wei C-F., Chang H-M., Yeh E. T. H. Protection against Fas/APO-1- and tumor necrosis factor-mediated cell death by a novel protein, Sentrin. J. Immunol., 157: 4277-4281, 1996.[Abstract]
  16. Kischkel F. C., Hellbardt S., Behrmann I., Germer M., Pawlita M., Krammer P. H., Peter M. E. Cytotoxicity-dependent APO-1 (Fas/CD95)-associated proteins form a death-inducing signaling complex (DISC) with the receptor. EMBO J., 14: 5579-5588, 1995.[Medline]
  17. Varfolomeev E. E., Schuchmann M., Luria V., Chiannilkulchai N., Beckmann J. S., Mett I. L., Rebrikov D., Brodianski V. M., Kemper O. C., Kollet O., Lapidot T., Soffer D., Sobe T., Avraham K. B., Goncharov T., Holtmann H., Lonai P., Wallach D. Targeted disruption of the mouse caspase 8 gene ablates cell death induction by the TNF receptors, Fas/Apo1, and DR3 and is lethal prenatally. Immunity, 9: 267-276, 1998.[Medline]
  18. Muzio M., Chinnaiyan A. M., Kischkel F. C., O’Rourke K., Shevchenko A., Ni J., Scaffidi C., Bretz J. D., Zhang M., Gentz R., Mann M., Krammer P. H., Peter M. E., Dixit V. FLICE, a novel FADD-homologous ICE/CED-3-like protease, is recruited to the CD95 (Fas/APO-1) death-inducing signaling complex. Cell, 85: 817-827, 1996.[Medline]
  19. Medema J. P., Scaffidi C., Kischkel F. C., Shevchenko A., Mann M., Krammer P. H., Peter M. E. FLICE is activated by association with the CD95 death-inducing signaling complex (DISC). EMBO J., 16: 2794-2804, 1997.[Abstract]
  20. Juo P., Kuo C. J., Yuan J., Blenis J. Essential requirement for caspase-8/FLICE in the initiation of the Fas-induced apoptotic cascade. Curr. Biol., 8: 1001-1008, 1998.[Medline]
  21. Boldin M. P., Goncharov T. M., Goltsev Y. V., Wallach D. Involvement of MACH, a novel MORT1/FADD-interacting protease, in Fas/APO-1-and TNF receptor-induced death. Cell, 85: 803-815, 1996.[Medline]
  22. Kelliher M. A., Grimm S., Ishida Y., Kuo F., Stanger B. Z., Leder P. The death domain kinase RIP mediates the TNF-induced NF-{kappa}B signal. Immunity, 8: 297-303, 1998.[Medline]
  23. Yeh W-C., Pompa J. L., de la McCurrach M. E., Shu H-B., Elia A. J., Shahinian A., Ng M., Wakeham A., Khoo W., Mitchell K., El-Deiry W. S., Lowe S. W., Goeddel D. V., Mak T. W. FADD: essential for embryo development and signaling from some, but not all, inducers of apoptosis. Science (Washington DC), 279: 1954-1958, 1998.[Abstract/Free Full Text]
  24. Zhang J., Cado D., Chen A., Kabra N. H., Winoto A. Fas-mediated apoptosis and activation-induced T-cell proliferation are defective in mice lacking FADD/Mort1. Nature (Lond.), 392: 296-300, 1998.[Medline]
  25. Ting A. T., Pimentel-Muinos F. X., Seed B. RIP mediates tumor necrosis factor receptor 1 activation of NF-{kappa}B but not Fas/APO-1-initiated apoptosis. EMBO J., 15: 6189-6196, 1996.[Medline]
  26. Walsh C. M., Wen B. G., Chinnaiyan A. M., O’Rourke K., Dixit V. M., Hedrick S. M. A role for FADD in T cell activation and development. Immunity, 8: 439-449, 1998.[Medline]
  27. Newton K., Harris A. W., Bath M. L., Smith K. G. C., Strasser A. A dominant interfering mutant of FADD/MORT1 enhances deletion of autoreactive thymocytes and inhibits proliferation of mature T lymphocytes. EMBO J., 17: 706-718, 1998.[Abstract]
  28. Pellegrini S., John J., Shearer M., Kerr I. M., Stark G. R. Use of a selectable marker regulated by {alpha} interferon to obtain mutations in the signaling pathway. Mol. Cell. Biol., 9: 4605-4612, 1989.[Abstract/Free Full Text]
  29. John J., McKendry R., Pellegrini S., Flavell D., Kerr I. M., Stark G. R. Isolation and characterization of a new mutant human cell line unresponsive to {alpha} and ß interferons. Mol. Cell. Biol., 11: 4189-4195, 1991.[Abstract/Free Full Text]
  30. Muzio M., Stockwell B. R., Stennicke H. R., Salvesen G. S., Dixit V. M. An induced proximity model for caspase-8 activation. J. Biol. Chem., 273: 2926-2930, 1998.[Abstract/Free Full Text]
  31. Martin D. A., Siegel R. M., Zheng L., Lenardo M. J. Membrane oligomerization and cleavage activates the caspase-8 (FLICE/MACHa1) death signal. J. Biol. Chem., 273: 4345-4349, 1998.[Abstract/Free Full Text]
  32. Memon S. A., Hou J-Z., Moreno M. B., Zacharchuk C. M. Apoptosis induced by a chimeric Fas/FLICE receptor: lack of requirement for Fas- or FADD-binding proteins. J. Immunol., 160: 2046-2049, 1998.[Abstract/Free Full Text]
  33. Yang X., Chang H. Y., Baltimore D. Autoproteolytic activation of pro-caspases by oligomerization. Mol. Cell, 1: 319-325, 1998.[Medline]
  34. Li H., Zhu H., Xu C-J., Yuan J. Cleavage of BID by caspase 8 mediates the mitochondrial damage in the Fas pathway of apoptosis. Cell, 94: 491-501, 1998.[Medline]
  35. Luo X., Budhihardjo I., Zou H., Slaughter C., Wang X. Bid, a Bcl2 interacting protein, mediates cytochrome c release from mitochondria in response to activation of cell surface death receptors. Cell, 94: 481-490, 1998.[Medline]
  36. Hannun Y. A. Functions of ceramide in coordinating cellular stress responses to stress. Science (Washington DC), 274: 1855-1859, 1996.[Abstract/Free Full Text]
  37. Cifone M. G., DeMaria R., Roncaioli P., Rippo M. R., Azuma M., Lanier L. L., Santoni A., Testi R. Apoptotic signalling through CD95 (Fas/APO1) activates an acidic sphingomyelinase. J. Exp. Med., 180: 1547-1552, 1994.[Abstract/Free Full Text]
  38. Cifone M. G., Roncaioli P., Maria R. D., Camarda G., Santoni A., Ruberti G., Testi R. Multiple pathways originate at the Fas/APO-1 (CD95) receptor: sequential involvement of phosphatidylcholine-specific phospholipase C and acidic sphingomyelinase in the propagation of the apoptotic signal. EMBO J., 14: 5859-5868, 1995.[Medline]
  39. Gulbins E., Bissonnette R., Mahboubi A., Martin S., Nishioka W., Brunner T., Baier G., Baier-Bitterlich G., Byrd C., Lang F., Kolesnick R., Altman A., Green D. Fas-induced apoptosis is mediated via a ceramide-initiated Ras signaling pathway. Immunity, 2: 341-351, 1995.[Medline]
  40. Tepper C. G., Jayadev S., Liu B., Bielawska A., Wolff R., Yonehara S., Hannun Y. A., Seldin M. F. Role for ceramide as an endogenous mediator of Fas-induced cytotoxicity. Proc. Natl. Acad. Sci. USA, 92: 8443-8447, 1995.[Abstract/Free Full Text]
  41. Juo P., Kuo C. J., Reynolds S. E., Konz R. F., Raingeaud J., Davis R. J., Biemann H-P., Blenis J. Fas activation of the p38 mitogen-activated protein kinase signalling pathway requires ICE/CED-3 family proteases. Mol. Cell. Biol., 17: 24-35, 1997.[Abstract/Free Full Text]
  42. Cahill M. A., Peter M. E., Kischkel F. C., Chinnaiyan A. M., Dixit V. M., Krammer P. H., Nordheim A. CD95 (APO-1/Fas) induces activation of SAP kinases downstream of ICE-like proteases. Oncogene, 13: 2087-2096, 1996.[Medline]
  43. Huang S., Jiang Y., Li Z., Nishida E., Mathias P., Lin S., Ulevitch R. J., Nemerow G. R., Han J. Apoptosis signaling pathway in T cells is composed of ICE/Ced-3 family proteases and MAP kinase kinase 6b. Immunity, 6: 739-749, 1997.[Medline]
  44. Lenczowski J. M., Dominguez L., Eder A. M., King L. B., Zacharchuk C. M., Ashwell J. D. Lack of a role for Jun kinase and AP-1 in Fas-induced apoptosis. Mol. Cell. Biol., 17: 170-181, 1997.[Abstract/Free Full Text]
  45. Toyoshima F., Moriguchi T., Nishida E. Fas induces cytoplasmic apoptotic responses and activation of the MKK7-JNK/SAPK and MKK6–p38 pathways independent of CPP32-like proteases. J. Cell Biol., 139: 1005-1015, 1997.[Abstract/Free Full Text]
  46. Chang H. Y., Nishitoh H., Yang X., Ichijo H., Baltimore D. Activation of apoptosis signal-regulating kinase 1 (ASK1) by the adaptor protein DAXX. Science (Washington DC), 281: 1860-1863, 1998.[Abstract/Free Full Text]
  47. Liu Z-G., Hsu H., Goeddel D. V., Karin M. Dissection of TNF receptor 1 effector functions: JNK activation is not linked to apoptosis while NF{kappa}B activation prevents cell death. Cell, 87: 565-576, 1996.[Medline]
  48. Wajant H., Johannes F. J., Haas E., Siemienski K., Schwenzer R., Schubert G., Weiss T., Grell M., Scheurich P. Dominant-negative FADD inhibits TNFR60-, Fas/Apo1-, and TRAIL-R/Apo2L-mediated cell death but not gene induction. Curr. Biol., 8: 113-116, 1998.[Medline]
  49. Schwandner R., Wiegmann K., Bernardo K., Kreder D., Kronke M. TNF receptor death domain-associated proteins TRADD and FADD signal activation of acid sphingomyelinase. J. Biol. Chem., 273: 5916-5922, 1998.[Abstract/Free Full Text]
  50. Chinnaiyan A. M., Tepper C. G., Seldin M. F., O’Rourke K., Kischkel F. C., Hellbardt S., Krammer P. H., Peter M. E., Dixit V. M. FADD/MORT1 is a common mediator of CD95 (Fas/APO-1) and tumor necrosis factor receptor-induced apoptosis. J. Biol. Chem., 271: 4961-4965, 1996.[Abstract/Free Full Text]
  51. Chinnaiyan A. M., O’Rourke K., Yu G-L., Lyons R. H., Garg M., Duan D. R., Xing L., Gentz R., Ni J., Dixit V. M. Signal transduction by DR3, a death domain-containing receptor related to TNFR-1 and CD95. Science (Washington DC), 274: 990-992, 1996.[Abstract/Free Full Text]
  52. Wiegmann K., Schwandner R., Krut O., Yeh W-C., Mak T. W., Kronke M. Requirement of FADD for tumor necrosis factor-induced activation of acid sphingomyelinase. J. Biol. Chem., 274: 5267-5270, 1999.[Abstract/Free Full Text]
  53. Weiss A., Stobo J. D. Requirement for the coexpression of T3 and the T cell antigen receptor on a malignant human T cell line. J. Exp. Med., 160: 1284-1299, 1984.[Abstract/Free Full Text]
  54. Powell S. M., Petersen G. M., Krush A. J., Booker S., Jen J., Giardiello F. M., Hamilton S. R., Vogelstein B., Kinzler K. W. Molecular diagnosis of familial adenomatous polyposis. N. Engl. J. Med., 329: 1982-1987, 1993.[Medline]
  55. Li H., Bergeron L., Cryns V., Pasternack M. S., Zhu H., Shi L., Greenberg A., Yuan J. Activation of caspase-2 in apoptosis. J. Biol. Chem., 272: 21010-21017, 1997.[Abstract/Free Full Text]
  56. Zhang J., Alter N., Reed J. C., Borner C., Obeid L. M., Hannun Y. A. Bcl-2 interrupts the ceramide-mediated pathway of cell death. Proc. Natl. Acad. Sci. USA, 93: 5325-5328, 1996.[Abstract/Free Full Text]
  57. Bligh E. G., Dyer W. J. . Can. J. Biochem. Physiol., 37: 911-917, 1959.
  58. Preiss J., Loomis C. R., Bishop W. R., Stein R., Niedel J. E., Bell R. M. . J. Biol. Chem., 261: 8597-8600, 1986.[Abstract/Free Full Text]
  59. Veldhoven P. P. V., Bishop W. R., Bell R. M. . Anal. Biochem., 183: 177-189, 1989.[Medline]



This article has been cited by other articles:


Home page
Mol. Pharmacol.Home page
A. Ottosson-Wadlund, R. Ceder, G. Preta, K. Pokrovskaja, R. C. Grafstrom, M. Heyman, S. Soderhall, D. Grander, I. Hedenfalk, J. D. Robertson, et al.
Requirement of Apoptotic Protease-Activating Factor-1 for Bortezomib-Induced Apoptosis but Not for Fas-Mediated Apoptosis in Human Leukemic Cells
Mol. Pharmacol., January 1, 2013; 83(1): 245 - 255.
[Abstract] [Full Text] [PDF]


Home page
J Biol ChemHome page
S. C. Cazanave, J. L. Mott, S. F. Bronk, N. W. Werneburg, C. D. Fingas, X. W. Meng, N. Finnberg, W. S. El-Deiry, S. H. Kaufmann, and G. J. Gores
Death Receptor 5 Signaling Promotes Hepatocyte Lipoapoptosis
J. Biol. Chem., November 11, 2011; 286(45): 39336 - 39348.
[Abstract] [Full Text] [PDF]


Home page
Proc. Natl. Acad. Sci. USAHome page
J. V. Lu, B. M. Weist, B. J. van Raam, B. S. Marro, L. V. Nguyen, P. Srinivas, B. D. Bell, K. A. Luhrs, T. E. Lane, G. S. Salvesen, et al.
Complementary roles of Fas-associated death domain (FADD) and receptor interacting protein kinase-3 (RIPK3) in T-cell homeostasis and antiviral immunity
PNAS, September 13, 2011; 108(37): 15312 - 15317.
[Abstract] [Full Text] [PDF]


Home page
J Biol ChemHome page
A. J. Smith, H. Dai, C. Correia, R. Takahashi, S.-H. Lee, I. Schmitz, and S. H. Kaufmann
Noxa/Bcl-2 Protein Interactions Contribute to Bortezomib Resistance in Human Lymphoid Cells
J. Biol. Chem., May 20, 2011; 286(20): 17682 - 17692.
[Abstract] [Full Text] [PDF]


Home page
J Biol ChemHome page
X. W. Meng, M. P. Heldebrant, K. S. Flatten, D. A. Loegering, H. Dai, P. A. Schneider, T. S. Gomez, K. L. Peterson, S. A. Trushin, A. D. Hess, et al.
Protein Kinase C{beta} Modulates Ligand-induced Cell Surface Death Receptor Accumulation: A MECHANISTIC BASIS FOR ENZASTAURIN-DEATH LIGAND SYNERGY
J. Biol. Chem., January 8, 2010; 285(2): 888 - 902.
[Abstract] [Full Text] [PDF]


Home page
Mol. Cell. Biol.Home page
H. Chen, Y. Xia, D. Fang, D. Hawke, and Z. Lu
Caspase-10-Mediated Heat Shock Protein 90{beta} Cleavage Promotes UVB Irradiation-Induced Cell Apoptosis
Mol. Cell. Biol., July 1, 2009; 29(13): 3657 - 3664.
[Abstract] [Full Text] [PDF]


Home page
J. Immunol.Home page
E. Pericolini, E. Gabrielli, E. Cenci, M. De Jesus, F. Bistoni, A. Casadevall, and A. Vecchiarelli
Involvement of Glycoreceptors in Galactoxylomannan-Induced T Cell Death
J. Immunol., May 15, 2009; 182(10): 6003 - 6010.
[Abstract] [Full Text] [PDF]


Home page
BloodHome page
J. E. Karp, K. Flatten, E. J. Feldman, J. M. Greer, D. A. Loegering, R. M. Ricklis, L. E. Morris, E. Ritchie, B. D. Smith, V. Ironside, et al.
Active oral regimen for elderly adults with newly diagnosed acute myelogenous leukemia: a preclinical and phase 1 trial of the farnesyltransferase inhibitor tipifarnib (R115777, Zarnestra) combined with etoposide
Blood, May 14, 2009; 113(20): 4841 - 4852.
[Abstract] [Full Text] [PDF]


Home page
BloodHome page
C. P. Miller, S. Rudra, M. J. Keating, W. G. Wierda, M. Palladino, and J. Chandra
Caspase-8 dependent histone acetylation by a novel proteasome inhibitor, NPI-0052: a mechanism for synergy in leukemia cells
Blood, April 30, 2009; 113(18): 4289 - 4299.
[Abstract] [Full Text] [PDF]


Home page
J Biol ChemHome page
H. Z. Imtiyaz, X. Zhou, H. Zhang, D. Chen, T. Hu, and J. Zhang
The Death Domain of FADD Is Essential for Embryogenesis, Lymphocyte Development, and Proliferation
J. Biol. Chem., April 10, 2009; 284(15): 9917 - 9926.
[Abstract] [Full Text] [PDF]


Home page
J Biol ChemHome page
K. Kadohara, M. Nagumo, S. Asami, Y. Tsukumo, H. Sugimoto, M. Igarashi, K. Nagai, and T. Kataoka
Caspase-8 Mediates Mitochondrial Release of Pro-apoptotic Proteins in a Manner Independent of Its Proteolytic Activity in Apoptosis Induced by the Protein Synthesis Inhibitor Acetoxycycloheximide in Human Leukemia Jurkat Cells
J. Biol. Chem., February 27, 2009; 284(9): 5478 - 5487.
[Abstract] [Full Text] [PDF]


Home page
Proc. Natl. Acad. Sci. USAHome page
B. D. Bell, S. Leverrier, B. M. Weist, R. H. Newton, A. F. Arechiga, K. A. Luhrs, N. S. Morrissette, and C. M. Walsh
FADD and caspase-8 control the outcome of autophagic signaling in proliferating T cells
PNAS, October 28, 2008; 105(43): 16677 - 16682.
[Abstract] [Full Text] [PDF]


Home page
J. Immunol.Home page
T. Das, G. Sa, E. Paszkiewicz-Kozik, C. Hilston, L. Molto, P. Rayman, D. Kudo, K. Biswas, R. M. Bukowski, J. H. Finke, et al.
Renal Cell Carcinoma Tumors Induce T Cell Apoptosis through Receptor-Dependent and Receptor-Independent Pathways
J. Immunol., April 1, 2008; 180(7): 4687 - 4696.
[Abstract] [Full Text] [PDF]


Home page
BloodHome page
K. Janssen, S. Pohlmann, R. U. Janicke, K. Schulze-Osthoff, and U. Fischer
Apaf-1 and caspase-9 deficiency prevents apoptosis in a Bax-controlled pathway and promotes clonogenic survival during paclitaxel treatment
Blood, November 15, 2007; 110(10): 3662 - 3672.
[Abstract] [Full Text] [PDF]


Home page
J Biol ChemHome page
S. L. Osborn, S. J. Sohn, and A. Winoto
Constitutive Phosphorylation Mutation in Fas-associated Death Domain (FADD) Results in Early Cell Cycle Defects
J. Biol. Chem., August 3, 2007; 282(31): 22786 - 22792.
[Abstract] [Full Text] [PDF]


Home page
BloodHome page
D. Viemann, K. Barczyk, T. Vogl, U. Fischer, C. Sunderkotter, K. Schulze-Osthoff, and J. Roth
MRP8/MRP14 impairs endothelial integrity and induces a caspase-dependent and -independent cell death program
Blood, March 15, 2007; 109(6): 2453 - 2460.
[Abstract] [Full Text] [PDF]


Home page
BloodHome page
I. N. Lavrik, A. Golks, S. Baumann, and P. H. Krammer
Caspase-2 is activated at the CD95 death-inducing signaling complex in the course of CD95-induced apoptosis
Blood, July 15, 2006; 108(2): 559 - 565.
[Abstract] [Full Text] [PDF]


Home page
J. Immunol.Home page
H. Z. Imtiyaz, S. Rosenberg, Y. Zhang, Z. S. M. Rahman, Y.-J. Hou, T. Manser, and J. Zhang
The Fas-Associated Death Domain Protein Is Required in Apoptosis and TLR-Induced Proliferative Responses in B Cells
J. Immunol., June 1, 2006; 176(11): 6852 - 6861.
[Abstract] [Full Text] [PDF]


Home page
J Biol ChemHome page
E. Varfolomeev, H. Maecker, D. Sharp, D. Lawrence, M. Renz, D. Vucic, and A. Ashkenazi
Molecular Determinants of Kinase Pathway Activation by Apo2 Ligand/Tumor Necrosis Factor-related Apoptosis-inducing Ligand
J. Biol. Chem., December 9, 2005; 280(49): 40599 - 40608.
[Abstract] [Full Text] [PDF]


Home page
FASEB J.Home page
L. Thon, H. Mohlig, S. Mathieu, A. Lange, E. Bulanova, S. Winoto-Morbach, S. Schutze, S. Bulfone-Paus, and D. Adam
Ceramide mediates caspase-independent programmed cell death
FASEB J, December 1, 2005; 19(14): 1945 - 1956.
[Abstract] [Full Text] [PDF]


Home page
Mol. Pharmacol.Home page
J. Zhu, L. Xiong, B. Yu, and J. Wu
Apoptosis Induced by a New Member of Saponin Family Is Mediated through Caspase-8-Dependent Cleavage of Bcl-2
Mol. Pharmacol., December 1, 2005; 68(6): 1831 - 1838.
[Abstract] [Full Text] [PDF]


Home page
J Biol ChemHome page
C. Sandu, E. Gavathiotis, T. Huang, I. Wegorzewska, and M. H. Werner
A Mechanism for Death Receptor Discrimination by Death Adaptors
J. Biol. Chem., September 9, 2005; 280(36): 31974 - 31980.
[Abstract] [Full Text] [PDF]


Home page
J Biol ChemHome page
H. Z. Imtiyaz, Y. Zhang, and J. Zhang
Structural Requirements for Signal-induced Target Binding of FADD Determined by Functional Reconstitution of FADD Deficiency
J. Biol. Chem., September 9, 2005; 280(36): 31360 - 31367.
[Abstract] [Full Text] [PDF]


Home page
Proc. Natl. Acad. Sci. USAHome page
G. Chen, M. S. Bhojani, A. C. Heaford, D. C. Chang, B. Laxman, D. G. Thomas, L. B. Griffin, J. Yu, J. M. Coppola, T. J. Giordano, et al.
Phosphorylated FADD induces NF-{kappa}B, perturbs cell cycle, and is associated with poor outcome in lung adenocarcinomas
PNAS, August 30, 2005; 102(35): 12507 - 12512.
[Abstract] [Full Text] [PDF]


Home page
Proc. Natl. Acad. Sci. USAHome page
S. Kasibhatla, K. A. Jessen, S. Maliartchouk, J. Y. Wang, N. M. English, J. Drewe, L. Qiu, S. P. Archer, A. E. Ponce, N. Sirisoma, et al.
A role for transferrin receptor in triggering apoptosis when targeted with gambogic acid
PNAS, August 23, 2005; 102(34): 12095 - 12100.
[Abstract] [Full Text] [PDF]


Home page
J. Immunol.Home page
B. Trinite, C. Chauvin, H. Peche, C. Voisine, M. Heslan, and R. Josien
Immature CD4-CD103+ Rat Dendritic Cells Induce Rapid Caspase-Independent Apoptosis-Like Cell Death in Various Tumor and Nontumor Cells and Phagocytose Their Victims
J. Immunol., August 15, 2005; 175(4): 2408 - 2417.
[Abstract] [Full Text] [PDF]


Home page
J. Immunol.Home page
J. H. Li and J. S. Pober
The Cathepsin B Death Pathway Contributes to TNF Plus IFN-{gamma}-Mediated Human Endothelial Injury
J. Immunol., August 1, 2005; 175(3): 1858 - 1866.
[Abstract] [Full Text] [PDF]


Home page
Cancer Res.Home page
J.-Z. Qin, J. Ziffra, L. Stennett, B. Bodner, B. K. Bonish, V. Chaturvedi, F. Bennett, P. M. Pollock, J. M. Trent, M. J.C. Hendrix, et al.
Proteasome Inhibitors Trigger NOXA-Mediated Apoptosis in Melanoma and Myeloma Cells
Cancer Res., July 15, 2005; 65(14): 6282 - 6293.
[Abstract] [Full Text] [PDF]


Home page
J. Virol.Home page
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]


Home page
J Biol ChemHome page
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]


Home page
JCBHome page
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]


Home page
J Biol ChemHome page
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]


Home page
GutHome page
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]


Home page
J Biol ChemHome page
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]


Home page
J Biol ChemHome page
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]


Home page
JCBHome page
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]


Home page
J Biol ChemHome page
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]


Home page
J Biol ChemHome page
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]


Home page
J Biol ChemHome page
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]


Home page
J Biol ChemHome page
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]


Home page
J Biol ChemHome page
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]


Home page
Proc. Natl. Acad. Sci. USAHome page
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]


Home page
Clin. Cancer Res.Home page
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]


Home page
Cancer Res.Home page
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]


Home page
J Biol ChemHome page
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]


Home page
J Biol ChemHome page
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]


Home page
J Biol ChemHome page
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 27, 2003; 278(26): 24003 - 24010.
[Abstract] [Full Text] [PDF]


Home page
J Biol ChemHome page
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 21, 2003; 278(8): 5775 - 5785.
[Abstract] [Full Text] [PDF]


Home page
J Biol ChemHome page
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 21, 2003; 278(8): 5622 - 5629.
[Abstract] [Full Text] [PDF]


Home page
Mol. Cell. Biol.Home page
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]


Home page
J Biol ChemHome page
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 13, 2002; 277(37): 34343 - 34348.
[Abstract] [Full Text] [PDF]


Home page
EMBO J.Home page
M. R. Sprick, E. Rieser, H. Stahl, A. Grosse-Wilde, M. A. Weigand, and H. Walczak
Caspase-10 is recruited to and activated at the native TRAIL and CD95 death-inducing signalling complexes in a FADD-dependent manner but can not functionally substitute caspase-8
EMBO J., September 2, 2002; 21(17): 4520 - 4530.
[Abstract] [Full Text] [PDF]


Home page
J. Virol.Home page
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., May 15, 2002; 76(10): 5094 - 5107.
[Abstract] [Full Text] [PDF]


Home page
J Biol ChemHome page
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., December 7, 2001; 276(49): 46639 - 46646.
[Abstract] [Full Text] [PDF]


Home page
Cell Growth Differ.Home page
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]


Home page
J Biol ChemHome page
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 30, 2001; 276(48): 44944 - 44952.
[Abstract] [Full Text] [PDF]


Home page
J Biol ChemHome page
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 19, 2001; 276(42): 38748 - 38754.
[Abstract] [Full Text] [PDF]


Home page
J Biol ChemHome page
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 31, 2001; 276(35): 32585 - 32590.
[Abstract] [Full Text] [PDF]


Home page
Cell Growth Differ.Home page
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]


Home page
J Biol ChemHome page
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 18, 2000; 275(33): 25222 - 25230.
[Abstract] [Full Text] [PDF]


Home page
J Biol ChemHome page
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 18, 2000; 275(33): 25222 - 25230.
[Abstract] [Full Text] [PDF]


Home page
J Biol ChemHome page
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 31, 2001; 276(35): 32585 - 32590.
[Abstract] [Full Text] [PDF]


Home page
J Biol ChemHome page
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 30, 2001; 276(48): 44944 - 44952.
[Abstract] [Full Text] [PDF]


This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Juo, P.
Right arrow Articles by Blenis, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Juo, P.
Right arrow Articles by Blenis, J.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cell Growth & Differentiation