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Molecular and Cellular Biology, October 1998, p. 6052-6062, Vol. 18, No. 10
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

E1B 19,000-Molecular-Weight Protein Interacts with and Inhibits CED-4-Dependent, FLICE-Mediated Apoptosis

Jeonghoon Han,1 Hershel D. Wallen,2 Gabriel Nuñez,2 and Eileen White1,3,*

Center for Advanced Biotechnology and Medicine1 and Department of Molecular Biology and Biochemistry and Cancer Institute of New Jersey,3 Rutgers University, Piscataway, New Jersey 08854, and Department of Pathology and Comprehensive Cancer Center, University of Michigan Medical School, Ann Arbor, Michigan 481092

Received 11 May 1998/Returned for modification 6 July 1998/Accepted 17 July 1998

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Genetic studies of the nematode Caenorhabditis elegans (C. elegans) have identified several important components of the cell death pathway, most notably CED-3, CED-4, and CED-9. CED-4 directly interacts with the Bcl-2 homologue CED-9 (or the mammalian Bcl-2 family member Bcl-xL) and the caspase CED-3 (or the mammalian caspases ICE and FLICE). This trimolecular complex of CED-4, CED-3, and CED-9 is functional in that CED-9 inhibits CED-4 from activating CED-3 and thereby inhibits apoptosis in heterologous systems. The E1B 19,000-molecular weight protein (E1B 19K) is a potent apoptosis inhibitor and the adenovirus homologue of Bcl-2-related apoptosis inhibitors. Since E1B 19K and Bcl-xL have functional similarity, we determined if E1B 19K interacts with CED-4 and regulates CED-4-dependent caspase activation. Binding analysis indicated that E1B 19K interacts with CED-4 in a Saccharomyces cerevisiae two-hybrid assay, in vitro, and in mammalian cell lysates. The subcellular localization pattern of CED-4 was dramatically changed by E1B 19K, supporting the theory of a functional interaction between CED-4 and E1B 19K. Whereas expression of CED-4 alone could not induce cell death, coexpression of CED-4 and FLICE augmented cell death induction by FLICE, which was blocked by expression of E1B 19K. Even though E1B 19K did not prevent FLICE-induced apoptosis, it did inhibit CED-4-dependent, FLICE-mediated apoptosis, which suggested that CED-4 was required for E1B 19K to block FLICE activation. Thus, E1B 19K functions through interacting with CED-4, and presumably a mammalian homologue of CED-4, to inhibit caspase activation and apoptosis.

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Programmed cell death (PCD) or apoptosis is a genetically controlled and evolutionarily conserved mechanism of the cell to commit suicide. It is widely recognized that apoptosis plays a critical role in organ development, tissue homeostasis, and disease processes (24, 46). Inappropriate regulation of apoptosis can lead to neurodegenerative disorders, abnormal development, and cancer. Therefore, identifying and understanding the mechanism of action of components of the apoptotic machinery are fundamentally important in a variety of physiological settings.

Genetic analysis of the nematode Caenorhabditis elegans has identified three important components of the cell death pathway, which are also conserved in vertebrates (14, 21). CED-3 is an effector for inducing PCD and is a member of the family of aspartate-specific cysteine proteases known as caspases (20, 56). Another important component of cell death regulation machinery in C. elegans is CED-4, which also induces PCD (42, 54). Genetic studies have indicated that CED-4 requires CED-3 to induce PCD and that CED-4 regulates the activity of CED-3, suggesting that CED-4 may function upstream of CED-3 (42). Recent biochemical and functional data have indicated that CED-4 induces the proteolytic activation of CED-3 (5, 41, 51), indicating that CED-4 may function at a key regulatory step in the process of PCD. A mammalian relative of CED-4, Apaf-1, which activates caspase-9 in a cytochrome c- and dATP-dependent manner has recently been identified (27, 57). A third component of the C. elegans pathway is CED-9. However, CED-9 is an inhibitor of apoptosis and blocks CED-3-induced cell death, which indicates that it acts upstream of CED-3 (22). Furthermore, CED-4 is required for the inhibition of CED-3-induced cell death by CED-9, suggesting that CED-9 regulates CED-3 through CED-4 (41, 42). Mammalian counterparts of CED-9 are the antiapoptotic Bcl-2 family members which include Bcl-2, Bcl-xL, and the adenoviral E1B 19,000-molecular-weight protein (E1B 19K) (26, 46). Bcl-2, Bcl-xL, and E1B 19K block the activation of caspases, which is consistent with the function of CED-9 in nematodes (2, 25, 32, 35, 37). Therefore, the basic PCD machinery in both C. elegans and mammals includes effectors (caspases such as CED-3), activators (so far represented by CED-4 and Apaf-1), and inhibitors (antiapoptotic Bcl-2 family members represented by CED-9, Bcl-2, Bcl-xL, and E1B 19K) to regulate apoptosis. Precisely how these proteins accomplish this regulation at the biochemical level has been emerging only recently.

In vitro, in Saccharomyces cerevisiae two-hybrid assays, and in overexpression experiments with mammalian cells, CED-4 can interact directly with CED-9 and CED-3 (6, 41, 44, 52). Furthermore, CED-4 activates CED-3 processing and accelerates CED-3-induced apoptosis (5, 41, 51). CED-9, in turn, prevents CED-4 from activating CED-3, thereby inhibiting CED-3 processing (5, 41, 51). In mammalian cells, exogenously expressed CED-4 also interacts with Bcl-xL and FLICE or ICE (6). These data suggest that the function of these three main components of the cell death machinery may be regulated by direct interaction and that this mechanism is conserved between nematodes and mammals.

Bcl-2 family members play a critical role in the regulation of apoptosis in a variety of different settings. Bcl-2-homologous proteins are divided into two categories according to functional activity, i.e., antiapoptotic and proapoptotic proteins (26, 36), suggesting that the regulation of apoptosis in mammals is more complex than in C. elegans. A common feature of the regulation by Bcl-2 family members is homo- and heterodimerization between antiapoptotic and proapoptotic proteins to either induce or inhibit apoptosis (30). It has been demonstrated that Bcl-xL, an antiapoptotic protein, interacts with CED-4 but that Bax and Nbk (also called Bik), proapoptotic proteins, do not (6). It has been proposed that Bcl-xL interacts with and inhibits the function of CED-4, or perhaps mammalian counterparts of CED-4, and that Bax and Nbk/Bik antagonize Bcl-xL but not CED-4, thereby inducing cell death (6). Whether this simplistic model in which the death regulators interact directly with each other is sufficient to explain the mechanism of cell death control remains to be determined.

The function of the adenoviral Bcl-2 homologue E1B 19K appears to be very similar to that of other Bcl-2 family members. Bcl-2, for example, will substitute for E1B 19K during adenovirus infection of human cells and will cooperate with E1A to transform rodent cells (10, 33, 45). In addition, E1B 19K, Bcl-xL, and Bcl-2 have sequence homology, particularly within Bcl-2-homologous regions 1 and 3 (3, 10, 12, 17, 53), and block apoptosis induced by p53 (9, 13, 38, 40). Bcl-2, Bcl-xL, and E1B 19K also bind to Bax, Nbk/Bik, and Bak (46). Therefore, E1B 19K shows functional and sequence homology with Bcl-2 and Bcl-xL and interacts with some of the same cellular proteins, suggesting that these Bcl-2 family members may act by similar mechanisms to inhibit apoptosis.

To extend the observations on the protein interactions between E1B 19K, Bcl-2, and Bcl-xL, we tested E1B 19K for the ability to bind to and regulate CED-4-dependent caspase activation. E1B 19K interacted with CED-4 in a yeast two-hybrid assay, in vitro, and in cell lysates. Subcellular localization of CED-4 was dramatically altered in E1B 19K-expressing cells and CED-4 was colocalized with E1B 19K. In functional assays, CED-4 alone did not induce cell death but rather potentiated cell death induction by FLICE, which was inhibited by E1B 19K expression. Thus, E1B 19K shares the ability of Bcl-xL to bind to and inhibit CED-4-dependent FLICE activation and thereby apoptosis.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Plasmid construction. The standard PCR technique was used to generate yeast fusion plasmids (pAS-CED-4, pGAD-19K, and pGBT9-ratBax). The PCR product of CED-4 (51) was digested with XmaI and PstI and ligated into the pAS vector. E1B 19K was cloned into the pGAD vector by using EcoRI and XhoI sites. Rat Bax (rBax) was cloned into pGBT9 by using EcoRI and BamHI sites. Missense mutants of E1B 19K (pm7, pm44, pm51, pm87, and pm102) (10, 11, 50) were cloned by PCR in pGAD at EcoRI and XhoI sites. pACT-CED-9, pGAD-ratBax, and pGAD-hNbk were previously described (16, 17, 52).

E1B 19K in pGEX4T-1 was previously described (16). All of E1B 19K deletion mutants (mutants with deletions of N30 [Delta N30], C93, C70, and C36, and mutants containing amino acids 30 to 146, 30 to 93, and 64 to 146) were subcloned from the pGBT9 vector (34) into the pGEX4T-1 vector by using EcoRI and SalI sites. The BamHI site from the pGBT9 vector remained intact. Human Bcl-2 in pGEX4T-3 was cloned by standard PCR methods by using EcoRI and XhoI sites without the transmembrane region of human Bcl-2 at the C terminus (deletion of 13 amino acids). rBax in pGEX4T-1 was also cloned at EcoRI and XhoI sites without the transmembrane region of rBax at the C terminus (deletion of 19 amino acids).

pcDNA3-E1B 19K and pcDNA3-Myc-ratBax mammalian expression vectors were previously described (16). pcDNA3-AU1-FADD (7) and pcDNA3-HA-FLICE (29) are cytomegalovirus (CMV) expression vectors that express an N-terminal AU1-tagged human FADD and a C-terminal hemagglutinin (HA)-tagged human FLICE, respectively. Both expression vectors were generously provided by Vishva Dixit (University of Michigan, Ann Arbor). pcDNA3-Flag-Bcl-xL (43) is a CMV-driven expression vector expressing the human Bcl-xL protein with a Flag epitope tag at the N terminus. Wild-type CED-4 and mutant CED-4 proteins (those with the mutations Delta N86, Delta C473, Delta C328, Delta C401, I258N, and DD250-251AA) were cloned into pcDNA3 at a KpnI site and have a Myc tag at their N termini (52). E1B 19K mutant CMV expression vectors (pm7, pm44, pm51, pm87, and pm102) were previously described (10, 11, 50).

Two-hybrid system. Binding ability for each combination of interacting proteins was analyzed with the YGH1 strain (ura3-25 his3-200 ade2-101 lys-2,801 trp1-901 leu2-3 Canr gal4-542 gal80-538 LYS2::gal1uas-gal1tata-HIS3 URA3::gal1-lacZ) (18). Transformations were performed by standard lithium acetate procedures, with 4 µg of plasmid DNA and 20 µg of sheared, denatured salmon sperm DNA being used for each transformation. Transformants were plated on yeast dropout plates lacking leucine and tryptophan. Transformants were assayed for beta -galactosidase activity by a filter-based assay (18).

Protein interaction assays. For fusion protein binding assays, BL21 DE3 was transformed with the glutathione S-transferase (GST) fusion plasmids indicated in the figures, and expression was induced with 0.5 mM isopropyl-beta -D-thiogalactopyranoside. A 100-µl aliquot of culture was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to evaluate fusion protein induction. The remaining culture was sonicated on ice by using seven short bursts at 10 s each (Fischer Sonic Dismembranator 3000) and clarified by centrifugation, and the supernatant was resuspended in 50% (vol/vol) glutathione-Sepharose beads (Pharmacia Biotech, Piscataway, N. J.). An aliquot of the protein-bound beads was analyzed by SDS-PAGE to ensure that equal amounts of pure fusion proteins were present.

In vitro binding assays for CED-4 and E1B 19K interaction were performed by incubating equal amounts of GST or GST-E1B 19K fusion protein (immobilized on glutathione-Sepharose beads) with in vitro-translated CED-4 protein diluted in 0.5 ml of buffer (50 mM Tris [pH 7.5], 150 mM NaCl, 0.2% Nonidet P-40 [NP-40]). The mixture was incubated for 2 h at 4°C, washed three times with buffer, and resuspended in 2× Laemmli buffer. All samples were boiled for 5 min, and proteins were resolved by SDS-12% PAGE. Gels were fixed in 50% methanol and 10% glacial acetic acid for 2 h and dried.

To detect the interaction of E1B 19K and CED-4 in cell lysates, pcDNA3-Myc-CED-4 was transfected into COS cells. Twenty-four hours posttransfection the transfected cells were washed with phosphate-buffered saline and lysed in 1 ml of cold NETN lysis buffer (20 mM Tris [pH 8.0], 100 mM NaCl, 1 mM EDTA, 0.2% NP-40) containing protease inhibitors (0.1 mM phenylmethylsulfonyl fluoride, 10 mM benzamidine, 0.1 mg of bacitracin per ml, 1.0 µg of pepstatin A per ml, 10 mM sodium bisulfite) for 20 min. The cell lysate was centrifuged at 10,000 × g for 10 min to remove cellular debris. The lysate was then incubated with GST alone and GST-19K bound to glutathione-Sepharose beads for 2 h and washed as described above. Samples were resolved by SDS-17% PAGE. The precipitated CED-4 protein was detected by Western blot analysis with an anti-Myc monoclonal antibody (Oncogene Science, Inc., Cambridge, Mass.).

All mutant CED-4 proteins were tested for binding to E1B 19K and the protein with amino acids 64 to 146 by the GST system. Equal amounts of GST, GST-19K, and the protein with amino acids 64 to 146 (3 µg) were incubated with in vitro-transcribed mutant CED-4 proteins (with the mutations Delta N86, Delta C473, Delta C328, Delta C401, I258N, and DD250-251AA) diluted in 0.5 ml of the NETN buffer. The mixtures were incubated for 2 h and washed. Samples were resolved by SDS-12% PAGE.

The E1B 19K deletion mutant proteins (the proteins with the mutations Delta N30, Delta C93, Delta C70, and Delta C36 and the proteins with amino acids 30 to 146, 30 to 93, and 64 to 146) in pGEX4T-1 were used in a binding assay to determine their abilities to bind to rBax and CED-4. In vitro-translated rBax or CED-4 was incubated with an equal amount of each E1B 19K deletion mutant protein and immobilized on glutathione-Sepharose beads in 0.5 ml of the NETN buffer. After 2 h of incubation, the mixtures were washed and resolved by SDS-20% PAGE.

rBax antagonization of the E1B 19K and CED-4 interaction was performed with GST-19K, GST-Bax, GST-Bcl-2, and in vitro-translated CED-4. GST-Bax and GST-Bcl-2 proteins in the amounts 2.5, 5, and 7.5 µg were added to 2.5-µg amounts of GST-19K and in vitro-translated CED-4 in the binding assay. These mixtures were incubated in 0.5 ml of the NETN buffer for 2 h and washed with the same buffer. The precipitates were resolved by SDS-20% PAGE.

Indirect immunofluorescence. COS cells were electroporated with the pcDNA3-Myc-CED-4 and pcDNA3-19K. Cells were fixed with methanol 24 h posttransfection and double-labeled with an anti-Myc monoclonal antibody (Oncogene Science, Inc.) at a 1:5 dilution and an anti-E1B 19K polyclonal antibody (p21) (49) at a 1:200 dilution. Antibodies were visualized with goat anti-mouse rhodamine-conjugated and goat anti-rabbit fluorescein-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, Inc., West Grove, Pa.).

Functional assays. To examine CED-4 and E1B 19K functional relationships in transient-expression assays, 6 µg of pcDNA3-Myc-CED-4 and 2 µg of pCMVbeta -gal carrying the gene expressing beta -galactosidase from the CMV promoter were cotransfected with 18 µg of pcDNA3-19K or pcDNA3-Flag-Bcl-xL into HeLa cells by electroporation as previously described (9). The amount of transfected DNA from the pcDNA3-AU1-FADD and pcDNA3-HA-FLICE plasmids was also fixed at 6 µg. The transfected cells were incubated for 24 h and 72 h. Percentages of blue cells were assessed as described previously (17).

CED-4 mutant proteins (the Delta N86, Delta C473, Delta C328, Delta C401, I258N, and DD250-251AA proteins) were used to define the regulatory region of CED-4 for augmenting FLICE-induced apoptosis. Six micrograms of CED-4, 6 µg of CED-4 mutant proteins, 6 µg of FLICE, and 18 µg of E1B 19K were used for transfection into HeLa cells. For each combination, 2 µg of the pCMVbeta -gal construct was included. The combined DNA was transfected into HeLa cells and incubated for 24 h. Percentages of blue cells were assessed as described previously (17).

Missense E1B 19K mutant proteins (pm7, pm44, pm51, pm87, and pm102) were assayed for inhibition of rBax-induced apoptosis and CED-4-dependent, FLICE-mediated apoptosis. Eighteen micrograms of pcDNA3-19K was cotransfected with 6 µg of pcDNA3-rBax or with 6 µg of pcDNA3-Myc-CED-4 and 6 µg of pcDNA3-HA-FLICE into HeLa cells. Two micrograms of the pCMVbeta -gal construct was also included. The transfected cells were incubated for 24 h, and the percentages of blue cells were assessed as described previously (17).

Western blotting. Cell extracts for Western blot analysis were prepared from subconfluent cultures, and 25 µg of protein from each cell line was analyzed by SDS-PAGE and semidry blotting onto nitrocellulose membranes by standard procedures. Immune complexes were detected by enhanced chemiluminescence according to the specifications of the manufacturer (Amersham Corp., Arlington Heights, Ill.).

To check the expression of CED-4 mutant proteins, all CED-4 mutant constructs were transfected into COS cells and 24 h posttransfection the expression of CED-4 mutant proteins was detected by Western blot analysis with an anti-Myc monoclonal antibody (Oncogene Science, Inc.).

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

E1B 19K interacts with CED-4 in yeast. To determine the ability of E1B 19K to bind to CED-4, yeast two-hybrid assays were performed. The CED-4 cDNA was cloned in frame with the yeast GAL4 DNA-binding domain of the pAS vector. E1B 19K was fused to the GAL4 activation domain in the pGAD vector as previously described (16). The binding activity was assessed by an X-Gal (5-bromo-4-chloro-3-indolyl-beta -D-galactopyranoside) filter assay. CED-4 interacted with E1B 19K, although it showed less binding ability than CED-9 with CED-4 (Table 1). The association of CED-4 with E1B 19K was specific in that CED-4 was unable to interact with the other Bcl-2 family members, Bax, and Nbk/Bik (Table 1). These results suggest that E1B 19K, as well as CED-9, interacts specifically with CED-4. Since proapoptotic Bcl-2 homologues such as Bax and Nbk/Bik do not interact with CED-4, they may function by interacting with and inhibiting antiapoptotic Bcl-2-like proteins and preventing their association.

                              
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  		TABLE 1.   Interaction of the E1B 19K protein with CED-4 in yeasta

E1B 19K associates with CED-4 in vitro and in cell lysates. A GST fusion protein system was employed to confirm the binding specificity of CED-4 with E1B 19K in yeast. GST alone and GST-19K fusion proteins were immobilized on glutathione-Sepharose beads and purified. Equal amounts of GST fusion proteins (3 µg) were mixed with in vitro-transcribed and -translated and [35S]methionine-labeled CED-4 in buffer containing 0.2% NP-40. In vitro-translated CED-4 bound to GST-19K fusion protein, but it did not interact with GST alone (Fig. 1A). These results indicate that E1B 19K can specifically interact with CED-4 in vitro, which serves as an independent confirmation of results obtained from the yeast two-hybrid system.


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  		FIG. 1.   Interaction of E1B 19K with CED-4 in vitro and in cell lysates. (A) In vitro-translated CED-4 associates with the GST-19K fusion protein. GST fusion proteins were immobilized on glutathione-Sepharose beads and incubated with in vitro-translated CED-4 protein in a buffer containing 0.2% NP-40. The first lane in panel A shows in vitro-translated CED-4 protein. (B) Interaction of the cellular CED-4 protein with GST-19K. pcDNA3-Myc-CED-4 was transfected into COS cells, and cold cell lysates prepared from the transfected cells were incubated with GST fusion proteins. The precipitated proteins from the GST fusion proteins were analyzed by SDS-PAGE and by Western blotting with an anti-Myc monoclonal antibody against a Myc tag on CED-4.

To substantiate the physiological relevance of the CED-4 and E1B 19K interaction, the interaction in mammalian cells was assessed. The E1B 19K protein, however, is insoluble in mammalian cells, necessitating harsh detergent conditions to extract the E1B 19K protein for immunoprecipitation (47, 48). In order to circumvent this problem, the E1B 19K protein was immobilized on glutathione-Sepharose beads as a GST fusion protein (GST-19K) and then incubated with unlabeled lysates prepared from CED-4-transfected COS cells. Cellular proteins bound to the GST-19K fusion protein were analyzed by an immunoblotting assay with an anti-Myc monoclonal antibody directed against a Myc epitope tag on CED-4. CED-4 was specifically detected only when it was incubated with GST-19K fusion protein and not with GST alone (Fig. 1B). These results demonstrate that E1B 19K can associate with CED-4 specifically in the context of a whole-cell extract, supporting the findings obtained from the two-hybrid system and the in vitro binding assays.

E1B 19K alters the subcellular localization of CED-4. To assess the subcellular localization of CED-4 in the absence and in the presence of E1B 19K protein, double-label indirect immunofluorescence experiments were performed. The CED-4 expression vector (pcDNA3-Myc-CED-4) was transfected with or without the E1B 19K expression vector (pcDNA3-E1B 19K) into COS cells, and the cells were fixed at 24 h posttransfection and stained with a monoclonal antibody specific for the Myc epitope on CED-4 and a polyclonal antibody directed against the E1B 19K protein. CED-4 displayed a diffuse, cytoplasmic localization pattern, indicating that it was localized in the cytosol as previously described (52) (Fig. 2A). As previously reported, the E1B 19K protein is associated with the cytoplasmic and nuclear membranes and with the insoluble nuclear lamina (34, 47, 48). Coexpression of CED-4 and E1B 19K resulted in a dramatic change of the localization of CED-4 (Fig. 2B). The CED-4 protein was relocalized to perinuclear membranes at locations corresponding to the intracellular distribution of the E1B 19K protein (Fig. 2C). Approximately 85% of the CED-4- and E1B 19K-expressing cells displayed this colocalization pattern, indicating that E1B 19K expression altered the subcellular localization of CED-4.


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  		FIG. 2.   Subcellular localization patterns of CED-4 in the presence and absence of E1B 19K. COS cells were transfected with pcDNA3-Myc-CED-4 and/or pcDNA3-19K expression vectors and processed for double-label indirect immunofluorescence 24 h posttransfection. The cells were stained with an anti-Myc monoclonal antibody against a Myc tag on CED-4 and an anti-E1B 19K polyclonal antibody directed against the E1B 19K protein (p21 antibody). (A) CED-4 expression alone. The transfected cells were stained with an anti-Myc monoclonal antibody. (B and C) Coexpression of CED-4 and E1B 19K. (B) An anti-Myc monoclonal antibody against a Myc tag on CED-4 was used to stain the transfected cells. (C) An anti-19K polyclonal antibody directed against the E1B 19K protein was used for staining.

E1B 19K inhibits CED-4-dependent, FLICE-mediated apoptosis. The E1B 19K protein blocks Fas and tumor necrosis factor alpha pathways (15, 19, 50), where an adaptor molecule, FADD, recruits FLICE to the death receptor complex to induce apoptosis (1, 8). FADD-induced apoptosis can be blocked by E1B 19K, whereas FLICE-induced apoptosis cannot (32) (Fig. 3). However, when FADD and FLICE are coexpressed, E1B 19K inhibits FADD-dependent FLICE activation and cell death (32) (Fig. 3). This demonstrates that E1B 19K is able to block FLICE-induced apoptosis in the presence of FADD. The mechanism by which E1B 19K blocks FLICE-induced cell death through FADD may be functionally analogous to the C. elegans model system for regulating apoptosis. Through a direct interaction of E1B 19K with CED-4, it is conceivable that E1B 19K may block FLICE-induced apoptosis through CED-4, which parallels the inhibition of FADD-mediated FLICE activation by E1B 19K. Thus, we sought to determine if E1B 19K could regulate CED-4-dependent FLICE-mediated apoptosis in similar assays.


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  		FIG. 3.   E1B 19K inhibits the ability of CED-4 to potentiate FLICE-induced apoptosis. The pcDNA3-Myc-CED-4, pcDNA3-AU1-FADD, and pcDNA3-HA-FLICE constructs were cotransfected by electroporation with pcDNA3-19K or pcDNA3-Flag-Bcl-xL into HeLa cells as indicated. pCMVbeta -gal was included for each combination of transfection. The percentage of blue viable cells in each combination was determined relative to that in the vector control (Vec). At 24 (A) and 72 (B) h posttransfection, a beta -galactosidase assay was performed.

To examine a functional relationship between CED-4 and E1B 19K, HeLa cells were transiently transfected with CED-4, FLICE, FADD, and E1B 19K expression vectors. The transfection of CED-4 into HeLa cells did not induce cell death (41, 52) (Fig. 3). In support of this observation, a genetic study of C. elegans has suggested that CED-4 requires CED-3 to implement cell death (42). To investigate the function of CED-4 in the presence of FLICE, a mammalian counterpart of CED-3, CED-4 and FLICE expression vectors were cotransfected with pCMVbeta -gal to express beta -galactosidase in HeLa cells, and the percentages of blue viable cells were assessed at 24 and 72 h posttransfection.

FADD expression induced cell death at 24 h posttransfection, but E1B 19K or Bcl-xL blocked FADD-induced apoptosis (Fig. 3) (32). FLICE expression alone did not induce substantial cell death at 24 h posttransfection but did so dramatically at 72 h posttransfection. Thus, FLICE expression was not inhibited by E1B 19K or Bcl-xL (Fig. 3). E1B 19K or Bcl-xL, however, blocked FLICE-induced apoptosis when FLICE was coexpressed with FADD (Fig. 3) (32). Although CED-4 alone did not induce cell death, coexpression of CED-4 and FLICE strongly induced cell death compared to that induced by FLICE alone at 24 h posttransfection (Fig. 3), indicating that CED-4 stimulates the ability of FLICE to induce cell death in mammalian cells. Importantly, the cotransfection of E1B 19K or Bcl-xL with CED-4 and FLICE blocked CED-4-dependent, FLICE-mediated apoptosis (Fig. 3), indicating that CED-4 is required for E1B 19K or Bcl-xL to inhibit FLICE-induced apoptosis. Thus, CED-4 alone cannot induce cell death but CED-4 potentiates FLICE-induced apoptosis. However, E1B 19K or Bcl-xL inhibits CED-4-dependent, FLICE-mediated apoptosis and CED-4 is necessary for E1B 19K or Bcl-xL to block FLICE-induced apoptosis. These results remarkably parallel the requirement of FADD for E1B 19K to inhibit FLICE activation and apoptosis (32).

Mutational analysis of CED-4 binding and function. To determine the binding specificity of CED-4 for E1B 19K, we used a series of mutant forms of CED-4 in GST fusion protein binding assays. The Delta C328, Delta C401, and I258N proteins are three CED-4 mutant proteins that display a loss-of-function phenotype in C. elegans (Fig. 4A) (55). The Delta C328 and Delta C401 proteins have stop codons at residues 328 and 401, respectively (Fig. 4A). The I258N protein has an amino acid substitution of Asn for Ile at residue 258 (Fig. 4A). We also generated three other CED-4 mutant proteins, the Delta N86, Delta C473, and DD250-1AA proteins (Fig. 4A). The Delta N86 protein has the first 86 N-terminal amino acids deleted and thus does not contain the caspase recruitment domain (CARD). The Delta C473 protein contains a stop codon at position 473 but retains the CARD and the Apaf-1-homologous region (57). The DD250-251AA protein introduces two point mutations of Asp to Ala at residues of 250 and 251 in the second P-loop site (57) (Fig. 4A).


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  		FIG. 4.   Mapping of the E1B 19K binding region in CED-4. (A) Schematic representation of CED-4 and corresponding mutant proteins. P-loops represent locations of predicted ATP-binding sites. Amino acids 1 to 88 have CARD homology. Amino acids 89 to 435 are homologous to part of Apaf-1, which is a mammalian homologue of CED-4. (B) Binding of the CED-4 mutant proteins to E1B 19K. GST fusion proteins, GST alone, and GST-19K were immobilized on glutathione-Sepharose beads and were incubated with each in vitro-translated wild-type and CED-4 mutant protein in the buffer containing 0.2% NP-40. The mixtures were incubated for 2 h and washed three times with the NETN buffer. Samples were resolved by SDS-12% PAGE.

For binding assays with GST fusion proteins, GST alone or GST-19K was incubated for 2 h with each in vitro-translated CED-4 mutant protein in buffer containing 0.2% NP-40. Unlike GST alone, the GST-19K fusion protein precipitated wild-type CED-4 (Fig. 4B). All of the CED-4 mutant proteins also interacted with GST-19K, but not with GST alone, although the Delta N86 and DD250-251AA proteins displayed dramatically reduced binding abilities (Fig. 4B). It has been reported that the Delta C328, Delta C401, and I258N mutant proteins also interact with CED-9 (51) as they did with E1B 19K (Fig. 4). These results suggest that there may be two binding sites in CED-4 for E1B 19K, one located in the CARD and the other located between residues 87 and 327 in CED-4 in the region homologous to Apaf-1. Since FADD has a death effector domain (DED) which is structurally related to the CARD, the requirement of the CARD of CED-4 for efficient E1B 19K binding may be related.

CED-4 mutant proteins were then evaluated for functional activity in mammalian cells (Fig. 5). All CED-4 constructs were transfected into COS cells, and at 24 h posttransfection, the expression of the proteins was detected by conventional Western blot analysis with an anti-Myc monoclonal antibody. With the exception of the Delta N86 protein, all CED-4 mutant proteins were highly expressed, at levels comparable to the level of expression of wild-type CED-4 (Fig. 5A).


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  		FIG. 5.   Functional analysis of CED-4 mutant proteins and inhibition by E1B 19K. (A) Expression levels of CED-4 mutant proteins. Each CED-4 mutant construct was transfected into COS cells, and 24 h posttransfection the levels of expression of wild-type and mutant CED-4 proteins were detected by Western blot analysis with an anti-Myc monoclonal antibody directed against the epitope tag. (B) Abilities of wild-type and CED-4 mutant proteins to activate FLICE-induced apoptosis. Wild-type CED-4, CED-4 mutant protein (with mutations Delta N86, Delta C473, Delta C328, Delta C401, I258N, and DD250-251AA), FLICE, and/or E1B 19K expression vectors were cotransfected with pCMVbeta -gal into HeLa cells as indicated in the figure. The transfected cells were incubated for 24 h. The percentage of blue viable cells in each transfection was determined relative to that in the vector control (Vec). -, no CED-4 or CED-4 mutant DNA added.

Wild-type CED-4 does not induce cell death by itself (51) but rather augments FLICE-induced apoptosis (Fig. 3). To characterize the ability of mutant forms of CED-4 to stimulate FLICE-induced apoptosis, each CED-4 mutant expression vector was cotransfected with the FLICE and pCMVbeta -gal vectors into HeLa cells. The percentages of blue cells were assessed by a beta -galactosidase assay 24 h posttransfection. Wild-type CED-4 displayed potent killing activity only in the presence of FLICE, as was indicated by the low percentage of blue cells (Fig. 5B). The Delta N86, Delta C328, Delta C401, I258N, and DD250-251AA CED-4 mutant proteins were defective in their ability to augment FLICE-induced apoptosis (Fig. 5B). It has been shown that the CED-4 Delta C328, Delta C401, and I258N mutant proteins display a loss-of-function phenotype in C. elegans (55), which is consistent with the results with HeLa cells. The Delta C473 protein, however, retained FLICE-dependent cell killing activity (Fig. 5B). Since E1B 19K interacted with the Delta C473 protein, it is not surprising that E1B 19K inhibited the apoptotic effect of the Delta C473 protein (Fig. 5B). Therefore, only the C-terminal 77 amino acids may be dispensable for stimulating FLICE-induced apoptosis.

Mutational analysis of E1B 19K binding and function. It has been demonstrated that E1B 19K interacts with Bax and inhibits Bax-induced apoptosis (4, 16). In this report, it has been shown that E1B 19K also binds to CED-4 and blocks CED-4-dependent, FLICE-mediated apoptosis. We performed a GST fusion protein binding assay using deletion mutant proteins of E1B 19K to map the sequence requirement in E1B 19K for interaction with CED-4 and Bax (Fig. 6). Wild-type E1B 19K interacted with both Bax and CED-4, whereas GST alone did not bind to either Bax or CED-4 (Fig. 6B). The E1B 19K protein has a moderately conserved N terminus which includes BH3, a highly conserved central region which includes BH1, and a poorly conserved C terminus (10, 50). Deletion of the E1B 19K N terminus in proximity to BH3 ablated Bax binding but not binding to CED-4 (Fig. 6A and B). Deletion of the C-terminal half (or more) of E1B 19K did not permit binding to either CED-4 or Bax (Fig. 6A and B). Whether this deletion caused the E1B 19K protein to be misfolded is not known. However, smaller E1B 19K fragments containing only BH3 (the Delta C70 protein and the protein with amino acids 19 to 57) bound both CED-4 and Bax, suggesting that BH3 is a binding site on E1B 19K for both (Fig. 6A and B). Whereas BH3 appeared to be the only sequence determinant on E1B 19K required for Bax interaction, an E1B 19K mutant protein (the protein containing amino acids 64 to 146) which lacked BH3 but contained the central conserved region including BH1 interacted with CED-4 more robustly than full-length E1B 19K (Fig. 6A and B). Thus, BH3 serves as the only E1B 19K binding site for Bax whereas CED-4 interacts independently with two regions of E1B 19K: BH3 and the central conserved region.


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  		FIG. 6.   Mapping of the sequence determinants within E1B 19K required for interaction with Bax and CED-4. (A) A schematic representation of the E1B 19K protein and deletion mutants. Among the various adenovirus serotypes, amino acids 1 to 81 have 44% identity, amino acids 82 to 115 make up the most conserved region with 58% identity, and amino acids 116 to 176 make up the least conserved region with 22% identity (10). Brackets indicate BH1 and BH3. The Delta N30 protein is deleted from the amino-terminal direction. The Delta C93, Delta C70, and Delta C36 proteins are deleted from the carboxy-terminal direction. The proteins with amino acids 30 to 146, 30 to 93, 64 to 146, and 19 to 57 are deleted from both directions. Wild-type E1B 19K and deletion mutants were tested for interaction with Bax and CED-4 in a GST fusion protein binding assay. +++++, very strong interaction; +++, strong interaction; ++, moderate interaction; -, no interaction. (B) GST fusion protein binding assay of deletion mutants of E1B 19K binding to Bax and CED-4. In vitro-translated rBax or CED-4 was incubated with the same amount of each E1B 19K deletion mutant and immobilized on glutathione-Sepharose beads in 0.5 ml of the NETN buffer. After 2 h of incubation, the mixtures were washed and resolved by SDS-20% PAGE. (C) The E1B 19K deletion mutant with amino acids 64 to 146 interacts with all CED-4 mutants. Equal amounts of GST and GST plus the deletion mutant protein with amino acids 64 to 146 were incubated with each in vitro-translated CED-4 mutant in the NETN buffer for 2 h. The mixtures were washed and resolved by SDS-12% PAGE. WT, wild type.

As the E1B 19K mutant protein containing amino acids 64 to 146 interacted with CED-4 more strongly than the wild-type E1B 19K protein, we tested its ability to bind to a series of CED-4 mutants to map the region of CED-4 to which it bound (Fig. 6C). The mutant protein containing amino acids 64 to 146 strongly interacted with wild-type CED-4 and all CED-4 mutant proteins (Fig. 6C). These data indicate that the binding site of the mutant protein containing amino acids 64 to 146 in CED-4 is also located from amino acids 87 to 327 of CED-4. Interestingly, no diminution in binding to the Delta N86 protein was observed with the protein with amino acids 64 to 146 (Fig. 6C), which was observed with the wild-type E1B 19K protein (Fig. 6B). Perhaps BH3 of E1B 19K interacts with the N-terminal CARD of CED-4 and destabilizes E1B 19K binding to CED-4.

Five previously characterized missense mutant proteins of the E1B 19K protein (pm7, pm44, pm51, pm87, and pm102) were also used to define the genetic requirements within E1B 19K for binding to CED-4 and Bax and for inhibition of Bax and CED-4-dependent, FLICE-mediated apoptosis. Functional experiments with the E1B 19K deletion mutant proteins named above are not possible because they do not produce stable proteins. The two-hybrid system was used to determine the abilities of the five 19K missense mutant proteins to bind to Bax or CED-4. pm7, pm44, and pm102 maintained the ability to interact with Bax as previously reported (16) (Fig. 7A). However, substitution of either phenylalanine for serine at position 51 (pm51) or glycine for alanine at position 87 (pm87) in the E1B 19K protein resulted in loss of the ability to interact with Bax in yeast (10, 16) (Fig. 7A). Loss of Bax binding with pm51 is consistent with the role of BH3 in the E1B 19K interaction with Bax. pm87 has an alanine substitution in the glycine residue, which is conserved in the BH1 of all Bcl-2 family members. This glycine residue is in a pivotal location adjacent to the hydrophobic cleft which serves as the BH3 binding pocket and may thereby affect Bax binding (28, 39). Wild-type CED-4 interacted with E1B 19K, whereas pm7, pm44, pm87, and pm102 interacted weakly and pm51 did not interact with CED-4 (Fig. 7A). These results support the findings with the deletion mutant proteins, suggesting that CED-4 has two binding sites on E1B 19K but that Bax requires only BH3. This is, again, exemplified by binding of pm87 to CED-4 but not to Bax. Thus, there may be two binding sites on CED-4 for E1B 19K (CARD and amino acids 87 to 327) and two binding sites on E1B 19K for CED-4 (BH3 and amino acids 64 to 146, including BH1).


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  		FIG. 7.   Mapping of E1B 19K binding to and inhibition of CED-4 and Bax. (A) Schematic representation of E1B 19K protein, locations of missense mutations, and binding abilities of missense E1B 19K mutant proteins to CED-4 and Bax. Wild-type and missense mutant proteins of E1B 19K (pm7, pm44, pm51, pm87, and pm102) in the pGAD vector, Bax in pGBT9, and CED-4 in pAS were used in yeast two-hybrid assays to detect interaction. Each combination of plasmid expressing the GAL4 DNA-binding domain (pAS and pGBT9) and the GAL4 activation domain (pGAD) was cotransformed into the YGH1 yeast strain as indicated. The binding strength was assessed by an X-Gal assay. Strong interaction is indicated by +++, moderate interaction is indicated by ++, weak interaction is indicated by +, and no interaction is indicated by -. (B) Functional assay for E1B 19K inhibition of CED-4- and Bax-induced apoptosis. Mutant proteins of E1B 19K were cotransfected with pcDNA3-Myc-ratBax or pcDNA3-Myc-CED-4 and pcDNA3-HA-FLICE into HeLa cells as indicated. For each combination, 2 µg of the pCMVbeta -gal plasmid was also cotransfected. Twenty-four hours posttransfection, viability of cells was assessed by a beta -galactosidase assay. The percentage of blue cells in the each transfection was determined relative to that in the vector control. -, no E1B 19K OR E1B 19K mutant DNA added.

To evaluate the functional activity of the E1B 19K missense mutant proteins, HeLa cells were cotransfected with Bax or CED-4 and FLICE expression vectors (Fig. 7B). Twenty-four hours posttransfection, the percentages of blue viable cells were assessed by a beta -galactosidase assay. Expression levels of E1B 19K mutant proteins in HeLa cells were measured by Western blot analysis (data not shown) and, as previously reported, were similar to that of wild-type E1B 19K (10). Wild-type E1B 19K showed antiapoptotic activity by efficiently blocking both Bax-induced and CED-4 dependent, FLICE-mediated apoptosis, as was indicated by the high percentage of blue cells (Fig. 7B). pm7 and pm44 displayed a reduced ability to block Bax-induced apoptosis but retained the ability to inhibit CED-4-dependent, FLICE-mediated apoptosis (Fig. 7B). However, pm51 did not retain any survival-promoting function for either Bax or CED-4-FLICE pathways (Fig. 7B). Alternatively, pm87 could not block apoptosis by Bax but retained most of its ability to inhibit apoptosis by CED-4 and FLICE (Fig. 7B). Since pm87 binds to CED-4 but not to Bax, E1B 19K binding appears to correlate with E1B 19K function. pm102 retained the same level of antiapoptotic activity as wild-type E1B 19K for both CED-4 and Bax (Fig. 7B). Thus, the pm87 mutation, which alters the absolutely conserved glycine residue in BH1, discriminates between CED-4 and Bax binding, suggesting that the E1B 19K protein binds to Bax and CED-4 differently. These different binding profiles directly correlate with the ability of the E1B 19K protein to inhibit Bax- and CED-4-dependent apoptosis (Fig. 7B).

Bax disrupts the interaction between E1B 19K and CED-4. Since the requirements within E1B 19K for interaction with Bax and CED-4 overlapped within BH3, we tested the ability of Bax to antagonize the interaction of E1B 19K with CED-4 (Fig. 8). GST-19K and in vitro-translated CED-4 were used in a GST fusion protein binding assay with or without GST-Bax or GST-Bcl-2. GST-19K precipitated with CED-4, although GST alone did not (Fig. 8). Given that Bcl-2 does not interact with E1B 19K and CED-4, addition of GST-Bcl-2 protein in the GST-19K and CED-4 binding assay did not change the binding ability of E1B 19K to CED-4 (Fig. 8). However, as the amount of GST-Bax protein was increased, the binding ability of E1B 19K to CED-4 was decreased (Fig. 8). This result indicates that Bax and CED-4 cannot interact with E1B 19K at the same time. Therefore, Bax can antagonize the interaction of E1B 19K with CED-4. By preventing E1B 19K from interacting with CED-4, Bax may promote caspase activation and thereby apoptosis.


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  		FIG. 8.   Bax disrupts the interaction between CED-4 and E1B 19K. GST-Bax or GST-Bcl-2 in the amounts 2.5, 5, and 7.5 µg was used in binding assays with GST-19K (2.5 µg) and in vitro-translated CED-4. These mixtures were incubated in the NETN buffer for 2 h and washed with the same buffer. The precipitates were resolved by SDS-20% PAGE.

E1B 19K inhibits FLICE processing. We have shown that E1B 19K blocks CED-4- and FLICE-induced apoptosis (Fig. 3). To address whether E1B 19K blocks FLICE processing, HeLa cells were transiently transfected with FLICE or FLICE plus CED-4 in the presence or absence of E1B 19K (Fig. 9). Whole-cell extracts were produced 24 h posttransfection and immunoblotted with an anti-HA polyclonal antibody to detect the epitope on the FLICE N terminus. The transfection of the pcDNA3 negative control vector and CED-4 could not produce processed FLICE (Fig. 9). Since overexpression of FLICE alone did not induce efficient cell death at 24 h posttransfection, the inactive zymogen form of FLICE was still detectable (Fig. 9). However, full-length FLICE disappeared when FLICE was cotransfected with CED-4, indicating that CED-4 activated FLICE processing (Fig. 9). Coexpression of E1B 19K with FLICE and CED-4 inhibited FLICE processing, as was indicated by the abundance of unprocessed full-length FLICE (Fig. 9). Therefore, CED-4 activates FLICE processing and E1B 19K blocks FLICE activation through CED-4, which is consistent with inhibition of CED-4-dependent, FLICE-mediated apoptosis by E1B 19K.


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  		FIG. 9.   E1B 19K blocks FLICE activation. HeLa cells were transfected with the pcDNA3 control vector, FLICE, and CED-4 plus FLICE in the presence or absence of E1B 19K at the same concentrations used in the viability assay (Fig. 3). Twenty-four hours posttransfection, whole-cell lysates were prepared, resolved by SDS-PAGE, and then immunoblotted with an anti-HA polyclonal antibody to recognize pro-FLICE (66 kDa).

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

We have demonstrated that CED-4 interacts with the E1B 19K protein but not with either Bax or Nbk/Bik. E1B 19K expression altered the cytosolic localization of CED-4 to that of its own perinuclear membrane localization pattern. CED-4 expression alone did not induce cell death, but CED-4 stimulated the killing effect of FLICE, suggesting that CED-4 activates FLICE. E1B 19K blocked this CED-4-dependent, FLICE-mediated apoptosis, and this E1B 19K inhibition of CED-4 cosegregated with the ability of E1B 19K to bind to CED-4. Thus, E1B 19K most likely interacts with a mammalian homologue of CED-4 to prevent caspase activation and apoptosis, as Bcl-xL binds to Apaf-1 and inhibits caspase-9 activation (23, 31). These observations are reminiscent of inhibition of FADD-dependent FLICE activation by E1B 19K (32). The E1B 19K protein may prevent caspase activation and thereby apoptosis generally by inhibiting activators (CED-4) or adapters (FADD).

Recently published data have shown that CED-4 alone induces cell death (6, 31). However, expression of CED-4 alone did not induce cell death in our experiments. Genetic analysis has also shown that CED-4 requires CED-3 to induce cell death (42). In support of this observation, several other groups have recently reported that CED-4 alone does not induce cell death in 293T cells, MCF-7 breast carcinoma cells, and Sf-21 cells (41, 51). The discrepancy in CED-4 activities may result from the use of different cell lines and methods used for transfection.

Mutation analysis of both E1B 19K and CED-4 has indicated that there are two interaction regions on E1B 19K for CED-4 (BH3 and amino acids 64 to 146) as well as two interaction regions on CED-4 for E1B 19K (CARD and amino acids 87 to 327) (Fig. 10). Amino acids 87 to 327 make up the region of CED-4 which is homologous to Apaf-1 (Fig. 4). Whether E1B 19K also interacts with and inhibits caspase activation by Apaf-1 remains to be determined. Removal of the CARD of CED-4 (Delta N86) greatly diminished interaction with E1B 19K (Fig. 6B), suggesting that the CARD is one binding site for E1B 19K. Interestingly, FADD contains a DED that is a specific type of CARD (1, 29). E1B 19K efficiently blocks FADD-dependent FLICE activation (32), which remarkably parallels E1B 19K inhibition of CED-4-dependent FLICE activation. As CED-4 and FADD contain a CARD or a CARD-like DED, respectively, direct interaction of E1B 19K with CARD-like domains may be the mechanism of E1B 19K inhibition. Whereas direct interaction of E1B 19K with CED-4 can be demonstrated, similar binding experiments with FADD have been more difficult. Perhaps a conformational change in FADD is required for E1B 19K binding to the DED of FADD (32). Direct demonstration of trimolecular complex formation between E1B 19K, CED-4, and FLICE has also been difficult to demonstrate in vivo due to the insolubility of the E1B 19K protein demonstrated in immunoprecipitation assays.


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  		FIG. 10.   Model for E1B 19K-Bax-CED-4 interactions. See the text for explanation. aa, amino acids.

Comparison of the E1B 19K binding profile for Bax and CED-4 suggests that E1B 19K binding to each is different. BH3 of E1B 19K was sufficient to interact with both full-length Bax and CED-4 (Fig. 6B). Also, a 29-amino-acid fragment of Bax encompassing BH3 is necessary and sufficient to interact with E1B 19K (16). However, the E1B 19K mutant protein with amino acids 64 to 146 which did not contain BH3 but did contain the central conserved region interacted only with CED-4 and not Bax (Fig. 6B). Furthermore, the requirements for interaction with Bax disrupted the interaction between CED-4 and E1B 19K. This suggests that the binding sites for CED-4 and Bax on E1B 19K overlap and that Bax binding to E1B 19K displaces CED-4 from E1B 19K (Fig. 10).

This study demonstrated that E1B 19K interacts with and inhibits CED-4-dependent, FLICE-mediated apoptosis, suggesting that E1B 19K requires CED-4 to block FLICE-induced apoptosis. Thus, these data suggest that E1B 19K functions upstream of the locus of caspase activation and regulates apoptosis by controlling caspase activation in addition to inhibiting proapoptotic Bcl-2 family members such as Bax and Bak. This dual mechanism of E1B 19K allows it to act as a potent antiapoptotic protein, leading to a complete inhibition of caspase activation and thereby apoptosis.

    ACKNOWLEDGMENTS

We thank Vishva Dixit for providing the FADD and the FLICE in pcDNA3. We also thank A. Thomas, K. Degenhardt, A. Gaur, D. Perez, Y. Shen, A. Cuconati, R. Sundararajan, and G. Kasof for helpful comments and suggestions.

This work was supported by a grant from the NIH (CA53370) to E.W.

    FOOTNOTES

* Corresponding author. Mailing address: Center for Advanced Biotechnology and Medicine, 679 Hoes Ln., Piscataway, NJ 08854. Phone: (732) 235-5329. Fax: (732) 235-5795. E-mail: ewhite{at}mbcl.rutgers.edu.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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