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[Cancer Research 61, 1138-1143, February 1, 2001]
© 2001 American Association for Cancer Research

Regular Articles

Signaling Events Triggered by Tumor Necrosis Factor-related Apoptosis-inducing Ligand (TRAIL): Caspase-8 Is Required for TRAIL-induced Apoptosis1

Dai-Wu Seol2, Jianrong Li3, Mi-Hyang Seol, Sang-Youel Park, Robert V. Talanian and Timothy R. Billiar

Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261 [D-W. S., J. L., M-H. S., S-Y. P., T. R. B.], and BASF Bioresearch Corporation, Worcester, Massachusetts 01605 [R. V. T.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a TNF family member and potent apoptosis inducer. In contrast to TNF-{alpha} or Fas ligand, relatively little is known about the signaling events activated by TRAIL. In particular, the initial caspase(s) required for TRAIL-induced apoptosis remains to be determined. Caspase-3-like protease but not caspase-1-like protease (YVADase) activity rapidly increased in HeLa cells in response to TRAIL treatment. The increase in protease activity correlated with the profile of apoptotic cell death that was inhibited by the pan-caspase inhibitor Z-VAD-fmk. In response to TRAIL, caspase-8, an initiator caspase in death receptor-mediated apoptosis, was activated within 1 h in association with Bid cleavage, cytochrome c release, caspase-3 activation, and DNA fragmentation factor 45 cleavage. Z-IETD-fmk, a caspase-8 inhibitor, completely blocked caspase-8 activation and resulted in inhibition of caspase-3 (a caspase-3-like protease) activation and apoptotic cell death. Overexpression of a caspase-8 dominant negative mutant inhibited apoptosis induced by TRAIL. Caspase-8-deficient Jurkat cells were resistant to both TRAIL- and Fas-induced apoptosis, whereas wild-type Jurkat cells were susceptible to both TRAIL- and Fas-induced apoptosis. The caspase-8-reintroduced caspase-8-deficient Jurkat cells acquired normal susceptibility to both TRAIL and agonistic Fas antibody. Reverse transcription-PCR and sequence analyses have revealed that these caspase-8-deficient Jurkat cells express wild-type caspase-10. Therefore, our data indicate that caspase-8 is required for TRAIL-induced apoptosis and suggest that caspase-10 may play a minor role, if any, in TRAIL-induced apoptosis.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Apoptosis is an evolutionarily conserved and genetically regulated biological process that plays an important role in the homeostasis of multicellular organisms (1, 2, 3) . Aberrations of this process can be detrimental to organisms. TNF4 family members such as TNF-{alpha}, FasL, and TRAIL have been identified as important inducers of ligand-induced apoptosis.

TNF-R1 activation elicits the recruitment of a cellular adaptor protein, TNF-R1-associated death domain-containing protein (4) , to the activated TNF-R1. TNF-R1-associated death domain-containing protein recruits other cellular proteins such as FADD (5) , TNF-R-associated factor 2 (5) , and receptor-interacting protein (6) . FADD interacts directly with procaspase-8, leading to its proteolytic activation (7, 8, 9) . TNF-R-associated factor 2 and receptor-interacting protein stimulate the signal pathways leading to activation of nuclear factor {kappa}B and Jun kinase, which have been shown to inhibit apoptosis in some cell types (10 , 11) . Activation of nuclear factor {kappa}B also leads to induction of the genes involved in proinflammatory and immune reactions (12, 13, 14, 15) . These observations indicate that TNF-{alpha} activates both apoptotic and antiapoptotic signal pathways. Thus, TNF-{alpha} induces limited apoptosis in a number of cell systems unless the antiapoptotic signal pathway is blocked (16, 17, 18) .

Unlike TNF-R1, the activated Fas recruits only FADD as an adaptor molecule (19, 20, 21, 22) . As a result, Fas activates only the apoptotic signaling pathways. This probably explains the observation that FasL has stronger apoptotic activity than TNF-{alpha}.

Once activated by TNF-{alpha} or FasL, caspase-8 initiates caspase cascades leading to cleavage of cytosolic, cytoskeletal, and nuclear proteins and DNA. Studies have demonstrated that the Bcl-2 family member Bid is cleaved by caspase-8. The cleaved Bid was shown to induce cytochrome c release from mitochondria (23 , 24) . Once released, cytochrome c binds to Apaf-1 and participates in caspase-9 activation (25 , 26) . The activated caspase-9 is then able to activate caspase-3 (25) , which in turn liberates a DNase termed CAD (caspase-activated DNase) from an inhibitor of CAD (ICAD/DFF-45) by cleaving the ICAD protein (27, 28, 29) . This process leads to DNA degradation, a hallmark event in apoptosis.

TRAIL, a recently identified TNF family member, also activates cognate receptor molecules through trimerization (30 , 31) , similar to other TNF family members. DR4/TRAIL-R1 (32) and DR5/TRAIL-R2 (33, 34, 35) are intact TRAIL-Rs through which apoptotic signals are transmitted into the cytoplasm, whereas DcR1 and DcR2 are truncated TRAIL-Rs in which the cytoplasmic regions containing the death domains are deleted. Thus, overexpression of DcR1 and DcR2 blocks functions of DR4 and DR5 in TRAIL-induced apoptosis (32 , 34, 35, 36) , probably by competing with DR4 or DR5 for TRAIL.

Although TRAIL is a TNF family member (37 , 38) , it has some notable differences from TNF-{alpha} and FasL. Unlike Fas, whose expression is limited to certain tissues, TRAIL-Rs are widely expressed (37 , 38) , thus most tissues and cell types may be TRAIL targets. TRAIL has a unique selectivity for triggering apoptosis in tumor cells and may be less active against normal cells. Hence, in contrast to FasL or agonistic Fas antibody, which induces fulminant massive liver damage (39 , 40) when introduced systemically, TRAIL exhibited no detectable cytotoxicity in mice (41) and monkeys (42) . HIV-1infected T cells were also shown to be more susceptible than uninfected T cells to TRAIL (43) . These features have focused considerable attention on TRAIL as a potential therapeutic to treat human cancers and AIDS.

Despite the physiological and therapeutic importance of TRAIL, relatively little is known about the death signaling events for TRAIL. In particular, the adaptor molecule(s) for TRAIL action is still unclear. As a result, the initiator caspase(s) required for TRAIL-induced apoptosis remains to be determined. Here we address the initiator caspase required for TRAIL-induced apoptosis and the death signal events triggered by TRAIL.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Viability Assay.
HeLa cells were purchased from American Type Culture Collection and maintained in F-12K culture medium. Wild-type, caspase-8-deficient, and caspase-8-reintroduced caspase-8-deficient Jurkat cells (44) were obtained from Dr. S. Nagata (Osaka University, Osaka, Japan) and maintained in RPMI 1640. To determine cell viability, HeLa or Jurkat cells plated in 12-well were exposed to the indicated amount of purified recombinant TRAIL (45) . Viable cells were stained with crystal violet for HeLa cells or MTT for Jurkat cells followed by spectrophotometric analysis as described previously (45) . To examine the involvement of caspases in apoptosis, HeLa cells plated in 12-well were preincubated with Ac-YVAD-cho (200 µM; Alexis, San Diego, CA), Ac-DEVD-cho (200 µM; Alexis), Z-VAD-fmk (100 µM; Alexis), or Z-IETD-fmk (100 µM; Enzyme Systems Products, Livermore, CA) for 1 h, exposed to TRAIL (100 ng/ml) for the indicated time, stained with crystal violet, and assayed for viability.

Measurement of DEVDase or YVADase Activity.
To prepare whole cell lysates, HeLa cells incubated with TRAIL (100 ng/ml) for the indicated time were collected and sonicated in cell lysis buffer (100 µg/ml phenylmethylsulfonyl fluoride, 2 µg/ml leupeptin, and 1 mM sodium orthovanadate in PBS). DEVDase or YVADase activity was measured as described previously (46) .

Detection of Cytochrome c Release.
HeLa cells incubated with TRAIL (100 ng/ml) for the indicated time were harvested and resuspended in ice-cold buffer C (PBS and 250 mM sucrose). After homogenization using a Dounce homogenizer, the cytosolic fraction was collected and loaded onto a 15% SDS gel. The released cytochrome c was detected by Western blotting.

Western Blotting.
Whole cell lysates prepared as described above were resolved on a 15% SDS gel. Western blotting analyses were performed as described previously (45 , 47 , 48) using antibody against human caspase-8 (PharMingen), Bid (provided by Dr. X. Yin, University of Pittsburgh, Pittsburgh, PA), caspase-3 (Santa Cruz Biotechnology), DFF-45/ICAD (Millennium Biotechnology, CA), or Bad (Santa Cruz Biotechnology).

Transient Transfection and Apoptosis Assay.
HeLa cells plated in 12-well were cotransfected with 200 ng of ß-galactosidase expression plasmid and 1 µg of the control vector or the caspase-8 DN expression plasmid (8) using Lipofectin (Life Technologies, Inc.) as described previously (45) . Thirty-six h after transfection, cells were exposed to TRAIL (100 ng/ml) for 3 h and stained with X-gal. Apoptotic cells showing typical apoptotic morphology were counted under the microscope as described previously (45) .

RT-PCR and Capase-10 Sequence Analyses.
Total RNA was isolated from caspase-8-deficient Jurkat cells, and RT-PCR was performed to detect caspase-10 expression using the primer sets (see Fig. 5Citation ) as described previously (45) . The RNA integrity and RT-PCR were monitored by actin expression. The PCR products resolved in a 1% agarose gel were cut out, subcloned to pcDNA3 plasmid (Invitrogen), and subjected to automated DNA sequencing.



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  		Fig. 5. Wild-type caspase-10 expression in caspase-8-deficient Jurkat cells. A, total RNA isolated from caspase-8-deficient Jurkat cells was subjected to RT-PCR (Lanes 1 and 2) as described previously (45) . For caspase-10/b PCR (Lane 3) as a positive control, pCMV-Caspase-10/b plasmid was used as a PCR template. As an internal control for RT-PCR, ß-actin cDNA was amplified by PCR under the same experimental conditions. PCR products were run in a 1% agarose gel and visualized by ethidium bromide staining. The arrows mark the expected sizes of caspase-10/a, caspase-10/b, and caspase-10/d. B, the RT-PCR products (Lane 1) indicated by arrows in A were cut out from the agarose gel, subcloned to pcDNA3, and subjected to automated DNA sequencing. The top panel schematically presents the caspase-10/a domains and the mutation sites (53) . The bottom panel presents the wild-type nucleotide and amino acid sequence corresponding to the mutation sites of caspase-10/a as well as the DNA sequencing results of caspase-10/a and caspase-10/d.

 

   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
TRAIL Induces Apoptosis by Activating Caspases.
To identify signaling molecules involved in TRAIL-induced apoptosis, we first tested the susceptibility of HeLa, HepG2, Hep3B, A431, A549, MCF-7, PANC-1, and SK-OV-3 cells to TRAIL-induced apoptosis. Concentrations of TRAIL protein ranging from 25 to 200 ng/ml were used. HeLa cells were found to be the most susceptible (data not shown) and were chosen for further study. We examined time-dependent apoptosis in response to TRAIL (Fig. 1A)Citation . TRAIL (100 ng/ml) induced apoptosis in approximately 50% of the cells within 2 h and >75% of the cells within 5 h.



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  		Fig. 1. TRAIL induces apoptosis by activating caspases. A, HeLa cells plated at 1.0 x 105 cells/12-well were incubated with TRAIL (100 ng/ml) for the indicated time. Viable cells were stained with crystal violet, followed by spectrophotometric analysis as described previously (45) . Viability of control cells was set at 100%, and viability relative to the control is presented. Bar, SE. The experiments were performed in triplicate at least twice. B, 100 µg of whole cell lysates prepared from HeLa cells that were treated with TRAIL (100 ng/ml) for the indicated time were analyzed for DEVDase or YVADase activity as described previously (46) . C, HeLa cells plated in 12-well were preincubated for 1 h with Ac-YVAD-cho (200 µM), Ac-DEVD-cho (200 µM), or Z-VAD-fmk (100 µM) and exposed to TRAIL (100 ng/ml) for the indicated time. Cell viability was determined as described in A. Bar, SE.

 
Studies have demonstrated that caspases play key roles in apoptosis. To determine whether caspase activation is a required signal event for TRAIL-induced apoptosis and whether increases in caspase activity coincided with apoptotic cell death, we measured caspase activity such as DEVDase and YVADase activity. TRAIL increased DEVDase approximately 5-fold by 2 h. No increase in YVADase activity was observed (Fig. 1B)Citation . As expected, Ac-YVAD-cho, a peptide inhibitor for YVADase, did not block TRAIL-induced apoptosis. Ac-DEVD-cho, a peptide inhibitor for DEVDase, also failed to block apoptosis triggered by TRAIL (Fig. 1C)Citation , as reported previously (49) . In contrast, Z-VAD-fmk, a pan-caspase inhibitor, completely blocked TRAIL-induced apoptosis, indicating that caspase activation is a required signal event for TRAIL-induced apoptosis. These results suggest that in addition to DEVDase, other caspases may also be involved in TRAIL-induced apoptosis. However, we cannot rule out the possibility that the failure of Ac-DEVD-cho to inhibit TRAIL-induced apoptosis resulted from poor uptake of the inhibitor by HeLa cells.

Death Signaling Pathway Activated by TRAIL.
Because both TNF-R1- and Fas-mediated apoptotic signals commonly activate caspase-8 as an initiator caspase, we investigated whether TRAIL also activates caspase-8. As shown in Fig. 2Citation , TRAIL activated caspase-8 within 1 h as determined by the disappearance of procaspase-8. We next examined the downstream components of the caspase-8-initiated death signaling pathway. Rapid activation of caspase-8 coincided with cleavage of Bid (23 , 24 , 50) , a recently identified caspase-8 substrate. Studies have demonstrated that the processed Bid induces the release of cytochrome c from the mitochondria (23 , 24 , 50) , which in turn leads to the activation of caspase-3 (25) . Indeed, Bid cleavage coincided with caspase-3 activation and cleavage of DFF-45/ICAD (Fig. 2)Citation , a caspase-3 substrate (27, 28, 29) . As expected, Bad, an apoptogenic Bcl-2 family member that is not susceptible to cleavage by caspases, remained intact (Fig. 2)Citation . These data indicate that TRAIL activates the death signal pathway that is commonly activated by other death receptors and induces apoptosis, in part, by activating a signaling pathway linked to cytochrome c release.



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  		Fig. 2. TRAIL activates caspase-8 and its downstream signaling molecules. Whole cell lysates (40 µg of protein) prepared from HeLa cells that were treated with TRAIL (100 ng/ml) for the indicated time were fractionated on a 15% SDS gel, and activation/cleavage of caspase-8, Bid, caspase-3, or DFF-45/ICAD was analyzed by Western blotting. Equal loading of the samples was demonstrated by Bad expression. NS, nonspecific bands. The figure is representative of experiments that were performed at least twice.

 
Blockade of Caspase-8 Activation Inhibits TRAIL-induced Apoptosis.
The cleavage of procaspase-8 in response to TRAIL suggested that caspase-8 might be required for TRAIL-induced apoptosis. Therefore, the requirement for caspase-8 was examined next. Z-IETD-fmk, a caspase-8 inhibitor, completely blocked TRAIL-induced apoptosis (Fig. 3A)Citation . Western blotting analysis revealed that this peptide inhibitor prevented the disappearance of procaspase-8 (Fig. 3B)Citation . Furthermore, blockade of caspase-8 activation by the inhibitor resulted in failure of downstream caspase-3 activation (Fig. 3B)Citation . To examine the functional role of caspase-8 in a more specific manner in TRAIL signaling, a caspase-8 DN, in which the cysteine residue of the active site was mutated, was used to prevent caspase-8 activation. Transient transfection of this caspase-8 DN also inhibited TRAIL-induced apoptosis (Fig. 3C)Citation . These results suggest that caspase cascades triggered by TRAIL may be signaled only through caspase-8.



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  		Fig. 3. Blockade of caspase-8 impairs TRAIL-induced apoptosis. A, HeLa cells plated in 12-well were preincubated for 1 h with Z-IETD-fmk (100 µM) and exposed to TRAIL (100 ng/ml) for 3 h. Cell viability was determined as described in Fig. 1Citation A. Bar, SE. The experiments were performed in triplicate at least twice. B, HeLa cells plated in 12-well were preincubated for 1 h with Z-IETD-fmk (100 µM) and treated with TRAIL (100 ng/ml) for 3 h, and whole cell lysates were prepared. Forty µg of each whole cell lysate were fractionated on a 15% SDS gel, and activation/cleavage of caspase-8 or caspase-3 was analyzed by Western blotting. NS, a nonspecific band that can be considered as a loading control. C, HeLa cells plated at 1.0 x 105 cells/12-well were transiently transfected with 0.2 µg of pCMVß-gal plus 1 µg of vector or caspase-8 DN expression plasmid. Thirty-six h after transfection, cells were exposed to TRAIL (100 ng/ml) for 3 h and stained with X-gal. Apoptotic cells were counted under the microscope as described previously (45) . Bar, SE. The experiments were performed in triplicate at least twice.

 
Caspase-8 Deficiency Impairs TRAIL-induced Apoptosis.
To further address whether caspase-8 is required as an initiator caspase for TRAIL action, we used caspase-8-deficient Jurkat cells. As shown in Fig. 4ACitation , TRAIL induced apoptosis effectively in wild-type Jurkat cells, but not in caspase-8-deficient Jurkat cells (44) . These results were repeatable with another caspase-8-deficient Jurkat cell line (Ref. 22 ; data not shown). As expected, wild-type Jurkat cells were also susceptible to agonistic Fas antibody, whereas caspase-8-deficient cells were not, suggesting that other cellular components involved in apoptotic signaling may remain intact in the caspase-8-deficient Jurkat cells. To further rule out the possibility that other genetic variations rather than caspase-8 deletion might affect the susceptibility of the cells to TRAIL, we examined TRAIL-induced apoptosis in caspase-8-reintroduced caspase-8-deficient Jurkat cells (44) . The reconstituted caspase-8 fully restored the apoptotic response of the cells to both TRAIL and agonistic Fas antibody (Fig. 4B)Citation .



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  		Fig. 4. Caspase-8 is required for TRAIL-induced apoptosis. A, wild-type and caspase-8-deficient Jurkat cells were exposed to agonistic Fas antibody (100 ng/ml) or TRAIL (100 ng/ml) for 12 h, and cell viability was analyzed by MTT assay as described previously (45) . Viability of control cells was set at 100%, and cell death relative to the control is presented. Bar, SE. The experiments were performed in triplicate at least twice. B, caspase-8-deficient Jurkat cells into which caspase-8 was reintroduced were treated with agonistic Fas antibody (100 ng/ml) or TRAIL (100 ng/ml) for 12 h. Cell viability was analyzed by MTT assay and presented as described in A. The experiments were performed in triplicate at least twice.

 
Caspase-10 is the most similar to caspase-8 in sequence and structural features (51 , 52) . Hence, caspase-10 has been considered an initiator caspase and suggested to be involved in apoptosis induced by death receptor activation. In fact, cells from patients with autoimmune lymphoproliferative syndrome type II have genetically inherited caspase-10 mutations, resulting in partially impaired apoptosis in response to TRAIL, TNF-{alpha}, and FasL (53) . These findings suggested that caspase-10 might be a common initiator caspase for death receptor-mediated apoptosis. Thus, we investigated whether caspase-10 is also required for TRAIL-induced apoptosis. To address this, we first examined whether caspase-10 is expressed in caspase-8-deficient Jurkat cells. RT-PCR analysis identified caspase-10/a and caspase-10/d isoforms (52) of caspase-10 (Fig. 5A)Citation . Next we searched for any mutations in caspase-10/a and caspase-10d because specific mutations identified in autoimmune lymphoproliferative syndrome type II patients partially impaired apoptotic response. Nucleotide sequence analyses revealed no mutations at the described mutation sites (Fig. 5B)Citation . The presence of wild-type caspase-10 in the caspase-8-deficient, TRAIL-resistant cells suggests that caspase-10 may not be important for TRAIL-induced apoptosis in these cells.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Because TRAIL is a potent apoptosis inducer and has tumor selectivity (41 , 42) , it is considered a promising cancer therapy. However, the death signaling events triggered by TRAIL are not fully understood. Caspase-8 has been identified as an initiator caspase activated by death receptors such as TNF-R1 and Fas but not by TRAIL-Rs. The overall structure of caspase-10 is similar to that of caspase-8 (51 , 52) . As expected, it was shown that caspase-10 was recruited to the activated TNF-R1 and Fas in a FADD-dependent manner (51) . Thus, caspase-10 is believed to act as an initiator caspase in death receptor-mediated apoptosis.

We demonstrated here that caspase-8 was required for TRAIL-induced apoptosis. A blockade of caspase-8 activation in HeLa cells and deletion of caspase-8 in Jurkat cells prevented activation of downstream death signaling, resulting in a failure of the cells to undergo apoptosis in response to TRAIL. This impairment of apoptosis resulting from caspase-8 deficiency was not complemented by the natural expression level of wild-type caspase-10 in Jurkat cells. Previous studies have demonstrated that the cowpox serpin, CrmA, effectively blocks TRAIL-R (32 , 33) and Fas-mediated apoptosis (54 , 55) , suggesting that CrmA-sensitive caspase(s) plays an essential role in TRAIL-R- and Fas-mediated signaling. Most importantly, caspase-8 but not caspase-10 was identified to be potently inhibited by CrmA, although both caspase-8 and caspase-10 were able to activate/cleave various downstrean executioner caspases (9) . These findings suggest that caspase-8 but not caspase-10 may be a key initiator caspase in TRAIL-R- and Fas-mediated apoptosis. These findings support our data showing that caspase-8-deficient Jurkat cells are resistant to apoptosis induced by both TRAIL and agonistic Fas antibody, even if the cells express wild-type caspase-10. Therefore, it is suggested that caspase-10 may play a minor role, if any, as an initiator caspase in TRAIL- and FasL-induced apoptosis. Instead, caspase-10 may play a more important role in certain conditions such as granzyme B-mediated apoptosis (56) and development (52) , based on the caspase-10 expression pattern during development.

Similar to FasL, TRAIL induced Bid cleavage and subsequent signaling events including caspase-3 activation and DFF-45 cleavage. Based on our findings, caspase-8 activation is believed to be a common proximal signaling event triggered by TRAIL-R, TNF-R1, and Fas.

Two TRAIL-Rs (DR4 and DR5) that can transmit apoptosis-inducing activity of TRAIL have been identified to date (32, 33, 34, 35) . However, it is unknown whether both receptors are identical in all biological features. For example, the question of whether both receptors activate caspase-8 through the same adaptor molecule(s) remains to be answered. Past studies have demonstrated that Fas signal is transmitted through FADD. Ectopically expressed FADD-DN or FADD knockout impaired Fas-induced apoptosis. Unlike Fas signaling, the involvement of FADD in TRAIL signaling is highly controversial (32 , 33 , 57, 58, 59, 60, 61) . However, our findings that caspase-8 is required for TRAIL, combined with the observation that cells from FADD knockout mice are resistant to Fas-induced apoptosis but still fully susceptible to TRAIL (62) , suggest that a FADD-independent pathway exists for TRAIL-Rs to activate caspase-8. Although TRAIL signaling appears similar to Fas signaling, TRAIL induces apoptosis more efficiently than FasL, as tested using various tissue-derived cell lines such as HeLa, HepG2, Hep3B, A431, A549, MCF-7, PANC-1 and SK-OV-3 (see the text).5 Cultured hepatocytes were also more easily killed by TRAIL (63) than by agonistic Fas antibody, despite the findings that hepatocytes express high levels of Fas (64 , 65) . Thus, it is tempting to hypothesize that the powerful apoptosis-inducing activity of TRAIL may be related to the unique adaptor molecule(s) that interacts with DR4 and/or DR5. Therefore, ongoing studies are attempting to identify the TRAIL-R adaptor(s) that lead to caspase-8 activation.


   ACKNOWLEDGMENTS
 
We thank Dr. V. Dixit for the caspase-8 DN expression plasmid, Dr. X. Yin for Bid antibody, and Drs. S. Nagata and J. Blenis for Jurkat cells.


   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 Supported by Competitive Medical Research Fund (to D-W. S.) and NIH Grants GM44100 and GM53789 (to T. R. B.). Back

2 To whom requests for reprints should be addressed, at BST W1503, Department of Surgery, University of Pittsburgh School of Medicine, 200 Lothrop Street, Pittsburgh, PA 15261. Phone: (412) 624-6740; Fax: (412) 624-1172; E-mail: seold+{at}pitt.edu Back

3 J. L. is the recipient of Postdoctoral National Research Service Award GM19866 from the USPHS. Back

4 The abbreviations used are: TNF, tumor necrosis factor; TRAIL, TNF-related apoptosis-inducing ligand; FADD, Fas-associated death domain-containing protein; TNF-R, TNF receptor; TRAIL-R, TRAIL receptor; FasL, Fas ligand; DEVDase, caspase-3-like protease; YVADase, caspase-1-like protease; DFF, DNA fragmentation factor; RT-PCR, reverse transcription-PCR; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; DN, dominant negative. Back

5 D-W. Seol, M-H. Seol, S-Y. Park, T. R. Billiat. p53 status does not affect TRAIL-induced apoptosis, manuscript in preparation. Back

Received 5/29/00. Accepted 11/30/00.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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