Mtd, a Novel Bcl-2 Family Member Activates Apoptosis in the Absence of Heterodimerization with Bcl-2 and Bcl-XL*

Naohiro Inohara, Daryoush Ekhterae, Irene GarciaDagger , Roberto Carrio§, Jesus Merino§, Andrew Merry, Shu Chen, and Gabriel Núñez

From the Department of Pathology and Comprehensive Cancer Center, The University of Michigan Medical School, Ann Arbor, Michigan 48109, the Dagger  Department of Pathology, Centre Medical Universitaire, 1211 Geneva 4, Switzerland, and the § Unidad de Immunologia, Departamento de Biologia Molecular, Universidad de Cantabria, Santander 39011, Spain

    ABSTRACT
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Abstract
Introduction
Procedures
Results & Discussion
References

We have identified and characterized Mtd, a novel regulator of apoptosis. Sequence analysis revealed that Mtd is a member of the Bcl-2 family of proteins containing conserved BH1, BH2, BH3, and BH4 regions and a carboxyl-terminal hydrophobic domain. In adult tissues, Mtd mRNA was predominantly detected in the brain, liver, and lymphoid tissues, while in the embryo Mtd mRNA was detected in the liver, thymus, lung, and intestinal epithelium. Expression of Mtd promoted the death of primary sensory neurons, 293T cells and HeLa cells, indicating that Mtd is a proapoptotic protein. Unlike all other known death agonists of the Bcl-2 family, Mtd did not bind significantly to the survival-promoting proteins Bcl-2 or Bcl-XL. Furthermore, apoptosis induced by Mtd was not inhibited by Bcl-2 or Bcl-XL. A Mtd mutant with glutamine substitutions of highly conserved amino acids in the BH3 domain retained its ability to promote apoptosis, further indicating that Mtd does not promote apoptosis by heterodimerizing with Bcl-2 or Bcl-XL. Mtd-induced apoptosis was not blocked by broad range synthetic caspase inhibitors z-VAD-fmk or a viral protein CrmA. Mtd is the first example of a naturally occurring Bcl-2 family member that can activate apoptosis independently of heterodimerization with survival-promoting Bcl-2 and Bcl-XL.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results & Discussion
References

Apoptosis, a morphologically distinguished form of programmed cell death, is critical not only during development and tissue homeostasis but also in the pathogenesis of a variety of diseases (1). Several regulatory components of the apoptotic pathway have been identified in various living organisms, including man (1, 2). bcl-2, the first member of an evolutionary conserved family of apoptosis regulatory genes, was initially isolated from the t(14;18) chromosomal translocation found in human B-cell follicular lymphomas and was subsequently shown to repress cell death triggered by a diverse array of stimuli (3-6). Several members of the family, including Bcl-2 and Bcl-XL, share conserved regions termed Bcl-2 homology domains 1, 2, 3 and 4 (BH1, BH2, BH3, and BH4) and function by repressing apoptosis (for review, see Ref. 7). The biochemical process by which Bcl-2 family members regulate cell death are poorly understood. The crystal structures of human and rat Bcl-XL have revealed a significant similarity to the pore-forming domains of diphtheria toxin and bacterial colicins, suggesting that Bcl-2 family members could function as channels for ions, proteins, or both (8, 9). Experiments with synthetic membranes have shown that recombinant Bcl-2, Bcl-XL, and Bax exhibit ion channel activity, suggesting that Bcl-2-related proteins could regulate apoptosis by regulating trafficking of molecules through intracellular membranes (10-12). However, the significance of these latter findings is unclear as there is no evidence that Bcl-2 or related proteins form ion-channels in vivo.

Analyses of the nematode cell death regulators CED-3, CED-4, and CED-9 has provided important insight into the biochemical mechanism that regulates apoptosis. CED-9, the nematode homologue of Bcl-2 and Bcl-XL, binds to CED-4 and represses cell death by interacting and inhibiting the killing activity of CED-3 through CED-4 (13-15). These studies predict that Bcl-2 and Bcl-XL regulate apoptosis at least in part by interacting with and inhibiting the activation of caspases through the putative mammalian CED-4 counterpart.

While proteins like Bcl-2 and Bcl-XL inhibit cell death, structurally related proteins, including Bax, Bak, Bad, Bid, Bik/Nbk, and Hrk activate apoptosis (16-23). Structural and functional analyses have revealed that proapoptotic proteins including Bax, Bak, and Hrk require the conserved BH3 region to interact with Bcl-2/Bcl-XL and activate apoptosis in transient assays (23-25). Moreover, NMR studies have demonstrated that the BH3 domain of Bak interacts with a hydrophobic cleft formed by the conserved BH1 and BH2 regions of Bcl-XL (26). To date, all death-promoting Bcl-2-related proteins heterodimerize with Bcl-2 and/or Bcl-XL, suggesting that these molecules promote cell death at least in part by interacting with and antagonizing Bcl-2 and Bcl-XL (16-29). In some systems, however, mutants of Bax and Bak can promote apoptosis in the absence of heterodimerization with Bcl-2 and Bcl-XL (30, 31).

Here we report the identification, cloning, and characterization of Mtd, a novel member of the Bcl-2 family. Expression of Mtd promotes cell death in rat primary neurons, 293T embryonic kidney cells, and HeLa cells. Unlike other Bcl-2-related proapoptotic proteins such as Bax and Bak, Mtd lacks binding activity to death antagonist Bcl-2 and Bcl-XL. Furthermore, apoptosis induced by Mtd does not require the conserved BH3 domain and is not inhibited by Bcl-2 and Bcl-XL nor by the broad range synthetic caspase inhibitors z-VAD-fmk and viral protein CrmA. To our knowledge, Mtd is the first example of a naturally occurring Bcl-2 family member that promotes apoptosis in the absence of direct interactions with survival-promoting Bcl-2 and Bcl-XL.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results & Discussion
References

Identification of the Mtd cDNA and Preparation of Expression Plasmids-- Partial nucleotide sequences of cDNAs homologous to Bcl-XL and Bcl-2 were identified in the expression sequence tag (EST)1 data base of GenBankTM using TBLASTN (NCBI). The predicted products of four EST cDNA clones 556544, 441963, 402040, and 553421 revealed significant homology to Bcl-XL and Bcl-2. The nucleotide sequence of the cDNAs was determined by dideoxy sequencing. The open reading frame of mouse Mtd was fused at the NH2 terminus with a Flag tag epitope sequence and cloned into pcDNA3 (Invitrogen) to generate pcDNA3-Flag-Mtd. The mammalian expression plasmids SFFV-HA-hBcl-XL, pcDNA3-beta -gal, pcDNA-crmA, and pcDNA3-caspase-8-AU1 have been described (15, 32, 33). The yeast expression plasmids pGBT9-hBcl-2, pGBT9-hBcl-XL, and pGBT9-hBak have been described (23). The plasmids pGAD10-Mtd and pGAD10-hBcl-2 were generated by fusing the Mtd and Bcl-2 coding regions with the GAL4 DNA-binding domain in the yeast pGAD10 vector. Mutations (L71Q/L74Q/L78Q) and Delta 91-213 were introduced into the Mtd amino acid sequence by site-directed mutagenesis using a polymerase chain reaction method. The authenticity of all constructs was confirmed by dideoxy sequencing.

Transfection, Expression, Immunoprecipitation, and Immunodetection of Tagged Proteins-- 5 × 106 293T cells were transfected with expression plasmids by calcium phosphate method as described previously (23). 5 µg of pcDNA3 or pcDNA3-Flag-Mtd was co-transfected with 10 µg of pSFFV-hBcl-2, pSFFV-HA-hBcl-XL, or pSFFV-neo. Total amount of DNA used was always 15 µg. After transfection, 293T cells were harvested at times shown in the figure legends and lysed with 0.2% Nonidet P-40 isotonic lysis buffer (16). One mg of soluble protein was incubated with 10 µg/ml anti-Flag polyclonal Ig, monoclonal anti-HA Ig, or control Ig overnight at 4 °C. Tagged proteins were immunoprecipitated with protein A-Sepharose 4B (Zymed Laboratories Inc.) and washed as described previously (23). Immunoprecipitates were subjected to 12% SDS-polyacrylamide electrophoresis and immunoblotted with anti-Flag, anti-Bcl-2, or anti-HA Ig.

Microinjection of DNA into Neurons-- Primary cultures of rat sympathetic neurons were prepared from superior cervical ganglia of newborn rats and prepared for microinjection as described (34). Individual neurons were injected in the nucleus (DNA solution at 100 ng/ml in 0.1 mM Tris-HCl, pH 7.2) with a mechanical Leitz manipulator until slight swelling was observed following a pression pulse of 0.1 atmospheres which took place after 2-4 s. Cell counts were performed 2 h later to determine the number of living cells (usually greater than 85%). Surviving cells were counted at 48 h after microinjection following staining with acridine orange (10 ng/ml). The percentage of microinjected neurons expressing Flag-Mtd was determined at 20 h postinjection in neurons kept in medium with or without NGF. Cells were fixed with 4% paraformaldehyde/phosphate-buffered saline for 20 min at room temperature and permeabilized with 0.1% Triton X-100 in phosphate-buffered saline. Neurons were stained with M2 anti-Flag monoclonal antibody (International Biotechnologies), followed by rhodamine-conjugated rat anti-mouse IgG (Boehringer Mannheim).

Cell Death and DNA Fragmentation Assays-- 293T and HeLa cells were transfected by the calcium phosphate methods with 0.2 µg of pcDNA3-beta -gal plus 0-1 µg of each pcDNA3-Flag-Mtd plus 0-3 µg of either pSFFV-hBcl-2, pSFFV-HA-h-Bcl-XL, or pSFFV-neo. 20 µM z-VAD-fmk (Enzyme Systems) was added at 8 h after transfection. At 24 h after transfection, cells were fixed and stained for beta -galactosidase as described previously (33). Percentage of apoptotic cells in triplicate cultures was determined by calculating the fraction of membrane blebbed blue cells from the total population of blue cells. In some experiments, cell death was measured by a reduction in the number of cells expressing beta -galactosidase relative to that obtained by transfection with the control plasmid. Staining of nuclei with acridine orange and ethidium bromide was performed as described previously (23). Genomic DNA was extracted and analyzed for DNA fragmentation as described (35).

In Situ Hybridization and Northern Blot Analysis-- Slides containing mouse embryo tissues were prepared as described (36). Each specimen was hybridized with a digoxigenin-labeled antisense RNA probe synthesized from a mouse Mtd cDNA template (nucleotides 189-1430) using an in vitro transcription kit (Promega). After washing with 0.2× SSC, samples were incubated with alkaline phosphatase-conjugated anti-digoxigenin antibody (Boehringer Mannheim) for 30 min, washed, and developed with alkaline phosphatase substrate. As a control, a sense Mtd RNA labeled probe was synthesized and used for hybridization as above. All slides were mounted with Crystal/mount (Biomeda Corp., Foster City, CA). For Northern blots, a fragment (nucleotides 189-1430) of the Mtd cDNA was radiolabeled by the random priming method using a commercial kit (Boehringer Mannheim) and applied for analysis of human multiple tissue poly(A)+ RNA blots (CLONTECH) according to the manufacturer's instructions.

    RESULTS AND DISCUSSION
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Abstract
Introduction
Procedures
Results & Discussion
References

Identification of Mtd, a Novel Bcl-2 Family Member-- To identify novel apoptosis-regulatory proteins, we screened the GenBankTM data base for cDNAs encoding proteins with homology to Bcl-2 and Bcl-XL sequences by using the TBLASTN program. Several ESTs containing overlapping nucleotide sequences with statistically significant amino acid homology to Bcl-2 and Bcl-XL were identified (p = 1.2 × 10-10). The longest cDNA (mouse EST clone 556544) was 1.4 kilobase pairs and analysis of its nucleotide sequence revealed an open reading frame that encoded a novel protein of 213 amino acids with a predicted relative molecular mass of 23,455 Da (Fig. 1). We designated this gene as mtd after matador, a Spanish word for killer (see below). Alignment analysis revealed that Mtd was a Bcl-2-related protein with predicted alpha 1-7 helices (8, 9) as well as BH1, BH2, BH3, and BH4 conserved homology regions and a putative COOH-terminal hydrophobic tail (Fig. 2A). In addition to Bcl-2, Mtd showed significant structural and amino acid homology with other Bcl-2 family members such as Bcl-XL, Bcl-w, Bax, Bak, and the Caenorhabditis elegans CED-9 homologue (Fig. 2B). Mtd is 22% identical to both Bcl-2 and Bcl-xL.


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Fig. 1.   Nucleotide and amino acid sequences of Mtd. The coding region is indicated with its amino acid sequence. The AATAAA polyadenylation signal and an ATTTA sequence motif for RNA destabilization are underlined.


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Fig. 2.   Structure, sequence, and alignment of Mtd with related proteins. A, schematic structure of Mtd. Conserved BH1 to BH4 regions and putative transmembrane (TM) region are shown by closed boxes. B, the amino acid sequence of Mtd is aligned with those of human Bak, Bax, Bcl-w, Bcl-2, and Bcl-XL and C. elegans CED-9. Hydrophobic residues are shown in reverse highlight. Negatively and positively charged residues are highlighted in light and dark gray, respectively. Proline and glycine residues (alpha /beta breaker) are in bold. Conserved residues among more than four proteins were indicated by dots. Residues that form the flexible loop in Bcl-2 (44 amino acids) and Bcl-XL (39 amino acids) are not included for simplicity. A transmembrane domain predicted by Kyte and Doolittle (41) method is indicated as TM. The entire nucleotide sequence of the Mtd cDNA is available as GenBankTM accession number AF027707. References for Bak, Bax, Bcl-w, Bcl-XL, Bcl-2, and CED-9 are given in the text.

Mtd mRNA Is Expressed in Embryonic and Adult Tissues-- We performed Northern blot analysis to assess the expression of mtd mRNA in various human tissues. Hybridization with a Mtd probe revealed a transcript of 2.5 kilobases in several adult tissues but more prominently in liver, brain, appendix, and lymphoid tissues (Fig. 3). Because several EST clones corresponding to Mtd sequences were derived from embryonic tissues, we evaluated the distribution of Mtd mRNA in mouse embryos by in situ hybridization. At stage E15 of development, intense labeling with an antisense Mtd probe was detected in the liver, thymus, lung, intestinal epithelium, and follicles of the whiskers and at a lower level in the developing cortical plate of the brain (Fig. 4, A-C). No significant labeling was detected when the embryonic tissues were hybridized with a sense Mtd control probe (Fig. 4D). At stage E13 of development, labeling with the Mtd probe was detected in the liver, thymus, lungs, intestinal structures, and intervertebral tissue.2 Thus, the expression of Mtd mRNA in embryonic and adult tissues is highly restricted and differs considerably from that reported for other Bcl-2 family members, including Bcl-2, Bcl-XL, and A1 (36, 37).


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Fig. 3.   Expression of Mtd in human adult tissues by Northern blot analysis. Poly(A)+ RNAs from various human tissues were hybridized with the Mtd cDNA as a probe.


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Fig. 4.   In situ hybridization analysis of Mtd expression in mouse embryos. A-C, Mtd labeling in parasagital sections of mouse embryo at stage E15 of development. A, Mtd labeling is observed in thymus (T), liver (Lv), lung (L), intestinal epithelia (IE), follicles of the whiskers (WF), and cortical plate (CP). B, high power of the thymus. C, high power of the liver. D, labeling with control sense Mtd probe.

Mtd Promotes the Death of Primary Neurons-- We constructed a Flag-tagged Mtd expression plasmid to begin to assess the product encoded by the Mtd cDNA and to elucidate its function. Transient transfection of the mtd cDNA into 293T cells induced the expression of a protein with the predicted molecular size of Mtd (Fig. 5A). Because mtd mRNA was expressed in the brain, we microinjected the Mtd expression plasmid into primary neurons isolated from dorsal root ganglia of neonatal rats. Twenty hours after injection, greater than 80% of the neurons that received DNA containing Flag-Mtd were stained with anti-Flag monoclonal antibody.3 In the presence of NGF, 88% ± 8 and 91% ± 5 of the neurons injected with empty vector DNA or with a Bcl-XL-producing vector were viable when the cells were assessed at day 3 postinjection (Fig. 5B). In contrast, 45% ± 6 of the neurons injected with the Mtd expression plasmid were viable (Fig. 5B, p < 0.01). Within 3 days of NGF deprivation, 30% of the neurons injected with vector alone survived, whereas only 18% of the cells injected with the Mtd expression plasmid were viable (Fig. 5B, p < 0.01). In control experiments, injection of a Bcl-XL expression construct rescued neurons from death induced by NGF withdrawal as reported previously (34).


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Fig. 5.   Mtd promotes apoptosis in primary sympathetic neurons from dorsal root ganglia. A, expression of Flag-tagged Mtd by immunoprecipitation/Western blot analysis. 293T cells were transfected with pcDNA3 or pcDNA3-Mtd-Flag expression plasmids and cellular lysates immunoprecipitated with anti-Flag antibody (f) or control IgG (c). Mtd protein were detected by immunoblot with anti-Flag antibody as described under "Experimental Procedures." IgH, immunoglobulin heavy chain. B, survival of sympathetic neurons after microinjection of Mtd and control plasmids. Results are shown as the mean percentage of surviving neurons ± S.D. at day 3 postinjection in the presence or absence of NGF.

Mtd Induces Apoptosis of Tumor Cells That Is Not Inhibited by Bcl-2 and Bcl-XL or the Synthetic Caspase Inhibitors z-VAD-fmk or CrmA-- To further characterize the proapoptotic activity of Mtd, we transiently transfected the Mtd-producing plasmid into 293T and HeLa carcinoma cells and subsequently observed the cells for features of apoptosis. The Mtd-transfected cells displayed morphological features of adherent cells undergoing apoptosis such as becoming rounded with plasma membrane blebbing, condensed nuclei and detached from the dish.3 In addition, Mtd induced nuclear condensation and fragmentation, a feature characteristic of apoptosis (Fig. 6A). Expression of Mtd induced significant killing activity when compared with control plasmid. At 24 h after transfection, about 25% of 293T cells underwent apoptosis compared with less than 1% with empty plasmid (Fig. 6B, p < 0.001). Unlike the proapoptotic activity induced by Bax (16), the killing promoted by Mtd was not inhibited by co-expression of Bcl-2 or Bcl-XL (Fig. 6B). In addition, the cell killing activity of caspase-8, but not that of Mtd, was inhibited by the broad spectrum caspase inhibitor z-VAD-fmk or the viral protein CrmA (Fig. 6B). Expression of Mtd induced oligonucleosomal fragmentation of genomic DNA, which was not inhibited by Bcl-XL (Fig. 6C). To further verify the proapoptotic activity of Mtd, HeLa cells were transfected with plasmids producing Mtd, Bax, or Bcl-2. Expression of Mtd and Bax killed HeLa cells when compared with empty vector or a plasmid producing Bcl-2 (Fig. 6D).


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Fig. 6.   Apoptosis induced by Mtd is not inhibited by Bcl-2, Bcl-XL, or caspases inhibitors. A, the nuclei of apoptotic cells were stained with acridine orange and ethidium bromide at 18 h after transfection with 2 µg of pcDNA3 (panel a) or pcDNA3-Mtd-Flag (panel b). Arrows show apoptotic cells with condensed and fragmented nuclei. B, 293T cells transfected with plasmids expressing pcDNA3-Flag-Mtd, pcDNA3-Flag-Mtd-BH3m (mt), or pcDNA3-Flag-Mtd Delta 91-213 (Delta ) in the presence or absence of the caspase inhibitor z-VAD-fmk or CrmA or co-transfected with Bcl-XL or Bcl-2 plasmids. Total amount of DNA used was always 4.2 µg. Numbers represent the amount of transfected plasmid in micrograms. A star indicates that z-VAD-fmk (20 µM) has been added to the cultures. At 24 h after transfection, apoptotic cells were counted as described under "Experimental Procedures." C, DNA fragmentation analysis of 293T cells transfected with the indicated plasmids. D, survival of HeLa cells after transfection with Mtd or control plasmids. Cells were transfected with 1.8 µg of each plasmid. At 24 h after transfection, the killing activity was determined as described under "Experimental Procedures."

A Mutant of Mtd with Replacement of Critical Residues in the BH3 Domain Retains Its Proapoptotic Activity-- The conserved BH3 domain of Bax/Bak mediates the binding to Bcl-2/Bcl-XL, and it appears essential for its proapoptotic activity in transient assays (24, 25). To determine if the BH3 domain of Mtd is necessary to induce apoptosis, we engineered a mutant form of Mtd (Mtd.BH3m) in which three critical hydrophobic amino acids (Leu71, Leu74, and Leu78) of the BH3 region were mutated to glutamine residues. These three residues in Bak have been shown to mediate the interaction with Bcl-XL through contacts with residues located in the hydrophobic pocket formed by the BH1/BH2 regions of Bcl-XL (26). The ability of Mtd.BH3m to induce apoptosis was similar to that of wild-type Mtd (Fig. 6B), indicating that a functional BH3 domain is dispensable for Mtd-mediated apoptosis. In contract, a Mtd mutant lacking the BH1, BH2, and COOH-terminal hydrophobic tail domain (Delta 91-213) did not induce apoptosis (Fig. 6B). Western blot analysis showed that both BH3m and Delta 91-213 mutants expressed in 293T cells.3

Mtd Lacks the Ability to Interact with Bcl-2, Bcl-XL, Bax, Bak, and Hrk-- We used a two-hybrid assay in yeast to determine the ability of Mtd to interact with Bcl-2 family members. In the two-hybrid assay, Mtd failed to interact with survival-promoting Bcl-2 and Bcl-XL or death-promoting Bax, Bak, and Hrk family members (Fig. 7A). In control experiments, Bcl-2 interacted with Bcl-XL and itself as it has been reported (Fig. 7A). To verify these results, we transiently co-transfected 293T cells with expression plasmids producing Flag-tagged Mtd and Bcl-XL. While control experiments showed that Bcl-XL interacts with endogenous Bax, Bcl-XL failed to immunoprecipitate Mtd (Fig. 7B), in agreement with the two-hybrid results. We conclude that Mtd does not bind significantly to survival-promoting Bcl-2 and Bcl-XL nor proapoptotic Bax, Bak, or Hrk. In addition, Mtd did not homodimerize, assessed by immunoprecipitation and two hybrid analyses.3


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Fig. 7.   Mtd fails to interact with Bcl-2, Bcl-XL, Bax, Bak, and Hrk. A, plasmids expressing Mtd or Bcl-2 fused to the GAL4 DNA-binding domain (upper) were co-transformed with plasmids expressing Bcl-2, Bcl-XL, Bax, Bak, or Hrk fused to the GAL4 transcriptional activation domain (lower). Growth of yeast in the absence of leucine, tryptophan, and histidine (His(-)) is indicative of protein-protein interaction. Growth in the presence of histidine (His(+)) is shown as a control. Expression of the GAL4-Mtd fusion protein in yeast transformed with control (lane 1) and Mtd (lane 2) plasmids are shown in the inset. B, 293T cells were transfected with plasmids producing HA-tagged Bcl-XL and/or Flag-tagged Mtd. After 16 h, lysates were immunoprecipitated with anti-HA antibody (h) or control IgG (c) and immunoprecipitates were immunoblotted with rabbit anti-Flag (to detect Mtd), anti-Bax, or anti-Bcl-XL antibodies. Control immunoprecipitation of the same lysates with mouse anti-Flag (f) or control IgG (c) followed by immunoblot with rabbit anti-Flag (indicated by Mtd, lower panel) is shown in the lower panel.

The results presented indicate that Mtd is a proapoptotic protein of the Bcl-2 family. In this respect, Mtd resembles Bax and Bak, two death-promoting Bcl-2 homologues, and Bad, Bid, Hrk, and Bik/Nbk, another set of more distantly related proteins of the Bcl-2 family. Proapoptotic members of the Bcl-2 family promote apoptosis at least in part by interacting with and inhibiting Bcl-2 and Bcl-XL and presumably other survival-promoting Bcl-2 family members (16-29). Mtd lacks the ability to bind to Bcl-2, Bcl-XL, and, thus, appears to promote apoptosis in the absence of heterodimerization with these death-repressing molecules. How does Mtd promote apoptosis? There are at least four nonexclusive possibilities. First, it is formally possible that Mtd interacts and antagonizes survival-promoting Bcl-2 family members other than Bcl-2 and Bcl-XL. However, a mutant form of Mtd lacking a functional BH3 domain remained active, indicating that it is unlikely that Mtd promotes cell death by binding to and inhibiting survival factors of the Bcl-2 family as the BH3 domain appears to be required for these interactions (23-27). These results are consistent with mutant analysis of Bax or Bak in stable cell lines, which showed that these proapoptotic Bcl-2 family members can promote apoptosis in the absence of heterodimerization with Bcl-2 and Bcl-XL (30, 31). A second possibility is that Mtd antagonizes Bcl-2/Bcl-XL function through competition or sequestration for cellular factors. A potential candidate is a mammalian homologue of the C. elegans CED-4, which regulates caspase activities (15). This hypothesis is consistent with the observation that functionally related Bax and Bik/Nbk can displace CED-4 from Bcl-XL (13). However it is unlikely that caspases play an important role in the mechanism of apoptosis induced by Mtd, because caspase inhibitors failed to block apoptosis induced by Mtd. The third possibility is that Mtd activates a death pathway distinct from that inhibited by Bcl-2 and Bcl-XL. Experimental evidence for Bcl-2/Bcl-XL-independent cell death pathways has been revealed in certain cells during CD95- and Bax- induced apoptosis (38, 39). Finally, we cannot exclude that Mtd promotes cell death by mechanisms unrelated to those involving interaction with cellular factors. For example, as it has been suggested for Bax, Mtd could promote apoptosis by forming pores to allow ions or small molecules to cross intracellular membranes (12).

Previous work with the proapoptotic Bax suggested that it activates cell death by inducing two separate caspase-dependent and -independent pathways, each of which is sufficient for apoptosis (39). Similarly Mtd could promote cell death by engaging both caspase-dependent and -independent mechanisms. Because apoptosis induced by Mtd was not inhibited by the broad range synthetic caspase inhibitors z-VAD-fmk or CrmA, the results suggest that apoptosis induced by Mtd is at least in part caspase-independent. While we cannot formally exclude a role for an as yet uncharacterized member of the caspase family that is not subject to inhibition by the broad spectrum inhibitor z-VAD-fmk, the results argue that death agonists like Bax and Mtd can induce cell death by caspase-independent mechanisms. The latter may involve several mechanisms, including alterations in mitochondrial membrane potential and generation of reactive oxygen species (40).

    ACKNOWLEDGEMENTS

We thank Juan A. Garcia-Porrero and Jaime Jimeno for help with the analysis of mouse embryos; Marie Dominique Vesin for technical work, V. Dixit for pcDNA3-crmA; and Mary A. Benedict, Maribel González-Garcia, Luis del Peso, Yuanming Hu, and Dayang Wu for their critical review of this manuscript.

    Note Added in Proof

While this manuscript was being reviewed, cloning of bok, a rat bcl-2 family member was reported (42). The protein sequence of Bok is identical to that of mouse Mtd.

    FOOTNOTES

* This work was supported in part by Grant CA-64556 from the National Institutes of Health (to G. N.), Grant 96/104 from the Comisión Interministerial de Ciencia y Tecnología, Spanish Ministry of Education and Science (to J. M.), a postdoctoral fellowship from the National Institutes of Health (to D. E.), Spanish Ministry of Education and Science Grant FP93-5058921 (to R. C), and Grants 31-42275.94 and 31.37516.93 from the Swiss National Foundation (to I. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF027707.

Recipient of Research Career Development Award CA-64421 from the National Institutes of Health. To whom correspondence should be addressed: Dept. of Pathology, University of Michigan Medical School, Ann Arbor, MI 48109. Tel.: 313-764-8514; Fax: 313-647-9654; E-mail: gabriel.nunez{at}umich.edu.

1 The abbreviations used are: EST, expression sequence tag; HA, hemagglutinin; NGF, nerve growth factor.

2 R. Carrio, J. Merino, and G. Núñez, unpublished results.

3 N. Inohara and G. Núñez, unpublished results.

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Abstract
Introduction
Procedures
Results & Discussion
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