Cleavage of Bax Enhances Its Cell Death Function

Experimental Cell Research 256, 375–382 (2000) doi:10.1006/excr.2000.4859, available online at http://www.idealibrary.com on

Cleavage of Bax Enhances Its Cell Death Function David E. Wood and Elizabeth W. Newcomb 1 Department of Pathology, New York University School of Medicine and Kaplan Comprehensive Center, 550 First Avenue, New York, New York 10016

Members of the Bcl-2 family of proteins are key regulators of apoptosis. Some of these proteins undergo posttranslational modification, such as phosphorylation or proteolysis, that serves to alter their function. Caspases are known to cleave Bid, a proapoptotic family member, as well as Bcl-2 and Bcl-X L, two prosurvival family members, which activate their cytotoxic activity resulting in the release of cytochrome c from mitochondria. Previously we showed that Bax was cleaved by calpain rather than by caspases from fulllength 21 kDa to generate a cleavage fragment of 18 kDa. Since cleavage of Bid serves to activate its cytotoxic activity, we wanted to determine if the p18 form of Bax exhibited increased cytotoxicity compared to p21 Bax. Using a transient transfection system in human embryonic kidney 293T cells we show that the p18 form of Bax displays a more potent ability to induce cell death. The pancaspase inhibitor Z-VAD-fmk completely blocked apoptosis induced by p21 Bax but only partially inhibited apoptosis induced by p18 Bax. Cyclosporin A, an inhibitor of the mitochondrial permeability transition (PT) pore, had no effect on Bax-mediated apoptosis of 293T cells suggesting that apoptosis was independent of the PT. Thus cleavage of p21 Bax during apoptosis to the p18 form may serve to increase the intrinsic cytotoxic properties of this proapoptotic molecule and enhance its cell death function at the mitochondria. © 2000 Academic Press

INTRODUCTION

Members of the Bcl-2 family of proteins are key regulators of apoptosis. The family is characterized by proteins that inhibit (e.g., Bcl-2) or promote apoptosis (e.g., Bax) [1]. Three functions have been assigned to various Bcl-2 family members: (a) homodimerization with themselves and heterodimerization with other family members; (b) interactions with other types of proteins that are non-Bcl-2 family members; and (c) formation of pores or channels [2]. The relative importance of these functions in regulating apoptosis re1 To whom correspondence and reprint requests should be addressed. Fax: (212) 263-8211. E-mail: [email protected].

mains to be determined, but it may be dependent on the types of Bcl-2 members present in a cell and the type of stimuli used to trigger apoptosis in that cell type [3]. Recent work has demonstrated that several of the Bcl-2 family members undergo posttranslational modifications that serve to alter their functions in some fashion. One such modification is phosphorylation. For example, the proapoptotic member Bad is phosphorylated in the presence of growth factors causing it to dissociate from Bcl-X L at the mitochondria and releasing it into the cytosol where it complexes with the protein 14-3-3 thereby abrogating its ability to promote apoptosis [4]. Conversely, dephosphorylation of Bad by calcineurin causes Bad to dissociate from 14-3-3 in the cytosol and translocate to the mitochondria thereby restoring the ability of Bad to promote apoptosis [5]. Bcl-2 is also phosphorylated, in its loop region, during apoptosis caused by the microtubule-stabilizing agent paclitaxel disrupting its prosurvival function [6]. In addition to phosphorylation, several family members are targets for proteolysis. For example, Bid, a proapoptotic family member found in the cytosol, can be cleaved by caspase-8 resulting in the translocation of the C-terminal cleavage fragment of Bid to the mitochondria where it causes the release of cytochrome c [7, 8]. Bcl-2 and Bcl-X L are also targets for caspases in some circumstances [9, 10]. Interestingly, cleavage of these two proteins is an activating event converting them from prosurvival molecules into proapoptotic molecules capable of causing the release of cytochrome c from the mitochondria [9 –11]. Previously we have shown that Bax is also a target for proteolysis, but is cleaved by calpain rather than by caspases in HL-60 lymphoid cells [12, 13]. Proteolysis of full-length 21-kDa Bax (p21) in drug-treated HL-60 cells generates a cleavage fragment of 18 kDa (p18) that has been observed in a variety of different cell types induced to undergo apoptosis by several different types of stimuli. These include alphavirus infection of rat embryo (R6) fibroblasts, staurosporine treatment of MN9D dopaminergic cells and SH-SY5Y neuroblastoma cells, inducible Bax expression in yeast cells, and

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ionizing radiation of SK-NSH neuroblastoma cells [11, 14 –17]. In our model of drug-induced apoptosis of HL-60 cells, Bax cleavage occurs as a late event dependent upon caspase-mediated activation of calpain [13]. Since cleavage of Bid, another cytosolic proapoptotic family member, activates its cytotoxic activity we wanted to determine if the p18 form of Bax exhibited heightened cytotoxicity compared to p21 Bax. Using a well-established transient transfection system in human embryonic kidney (HEK) 2 293T cells, we compared the cytotoxic activities of wild-type p21 Bax to the p18 form of Bax. Both Bax proteins were able to initiate apoptosis. However, the p18 form of Bax displayed a more potent ability to induce cell death. Apoptosis induced by p21 Bax was completely blocked by the pan caspase inhibitor Z-VAD-fmk. In contrast, Z-VAD-fmk could only partially block apoptosis induced by the p18 form of Bax. Finally, apoptosis induced by p21 or p18 Bax in 293T cells was not inhibited by cyclosporin A (CsA), an inhibitor of the mitochondrial permeability transition pore (PTP) [18]. Our results suggest that Bax-mediated apoptosis of 293T cells does not require opening of the PTP and the accompanying collapse of the mitochondrial inner membrane potential (⌬⌿ m) [18]. Thus cleavage of p21 Bax during apoptosis to the p18 form may serve to increase the intrinsic cytotoxic properties of this proapoptotic molecule and enhance its cell death function at the mitochondria. MATERIALS AND METHODS Cell culture. HEK293T cells, transformed with the SV40 large T antigen, were cultured in DMEM supplemented with 10% heatinactivated fetal bovine serum and 100 U/ml penicillin, 100 ␮g/ml streptomycin. Cells were split every 3 days to ensure logarithmic growth. Bax expression constructs. Human full-length p21 Bax cDNA cloned into pcDNA3 was used for subcloning into the EcoRI site of the mammalian expression vector pcDNA3.1 (Invitrogen, Carlsbad, CA). The truncated p18 Bax cDNA, Bax-t, was generated by PCR using pcDNA3-Bax as a template and the following primers, a 5⬘ primer with the sequence 5⬘-GCAGGGGAATTCGGGGGGGAGGCAC-3⬘ and the 3⬘ primer for the SP6 promoter site within pcDNA3. The PCR product for p18 Bax was gel purified using Ultrapure-DA columns (Millipore, Bedford, MA), digested with EcoRI and subcloned into the EcoRI site in pcDNA3.1. The pcDNA3.1/Bax and pcDNA3.1/Bax-t plasmids were then used for transformation of INV␣F⬘ competent cells (Invitrogen). Plasmid DNA was isolated using the Qiagen (Valencia, CA) maxiprep kit. Proper orientation of the inserts was determined by restriction mapping and the authenticity of the inserts was confirmed by DNA sequence analysis. In 2 Abbreviations used: HEK, human embryonic kidney; Z-VADfmk, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone; CsA, cyclosporin A; PT, permeability transition; PTP, permeability transition pore; CK, creatine kinase; cyc D, cyclophilin D; HEX, hexokinase; PBR, peripheral benzodiazepine receptor; ⌬⌿ m, the mitochondrial inner membrane potential; ␤-gal, ␤-galactosidase; PAGE, polyacrylamide gel electrophoresis; AraA, aristolochic acid.

order to demonstrate that each construct was capable of encoding for its respective fusion protein, each purified plasmid was subjected to in vitro translation in the presence of [ 35S]methionine (NEN, Wilmington, DE) using a coupled transcription/translation reaction (TNT T7 Quick System, Promega, Madison, WI). From each translation reaction, 5 ␮l was diluted with 20 ␮l of 2⫻ loading buffer and boiled for 3 min with 5 ␮l of the mixture electrophoresed on 14% SDS–PAGE gels. Autoradiography was performed for 1– 4 h at ⫺70°C. Transient transfection of human embryonic kidney 293T cells and ␤-galactosidase activity assay. For transient transfection, 2 ⫻ 10 5 293T cells were plated on six-well plates 22–24 h prior to transfection. Immediately before transfection, cells were washed once with PBS and then cotransfected with 1 ␮g of the reporter construct pcDNA3.1/lacZ and 2 ␮g of either the empty pcDNA3.1 control plasmid or one of the Bax fusion constructs (pcDNA3.1/Bax or pcDNA3.1/Bax-t) using 10 ␮l of Superfect (Qiagen) in 2 ml of DMEM supplemented with serum and antibiotics for 2.5 h at 37°C. The transfection medium was then removed and cells were washed once with PBS followed by the addition of 2 ml of DMEM supplemented with serum and antibiotics to each well. To determine transfection efficiency, cells transfected with the empty pcDNA3.1 control plasmid and pcDNA3.1/lacZ reporter construct were fixed and stained with 5-bromo-4-chloro-3-indolyl-␤-D-galactopyranoside (X-gal) at 24 h after transfection (Invitrogen). To analyze for expression of the fusion proteins after transfection of the constructs, cells at 24 h after transfection were trypsinized, washed, and lysed to generate whole cell protein lysates as described [12]. Lysates were then electrophoresed on 14% SDS–PAGE gels and immunoblotted with a mouse monoclonal anti-Xpress antibody at 1:5000 (Invitrogen) that recognizes the Xpress epitope found in recombinant proteins expressed from the pcDNA3.1 plasmid. Immunoblotting with a mouse monoclonal anti-actin antibody at 1:4000 (Chemicon, Temecula, CA) was used to ensure equal protein loading of the gels. For the time course experiments, cells at 6, 12, and 24 h after transfection were lysed in 35 ␮l of 0.25 M Tris, pH 8.0, and 10 ␮l of the lysates were used in a ␤-galactosidase (␤-gal) activity assay according to the manufacturer’s protocol (Invitrogen). For the Z-VAD-fmk (Enzyme Systems Products, Livermore, CA) and CsA (Sigma, St. Louis, MO) experiments, Z-VAD-fmk (50 ␮M), CsA (1 ␮M), or Z-VAD-fmk plus CsA was added to the cell culture immediately following transfection. Cell lysates were made from cells collected at 24 h after transfection and subjected to the ␤-gal activity assay. Loss of cell viability was measured by the decrease in ␤-gal activity relative to the control. Percentage of ␤-gal activity was calculated using the following: (␤-gal activity of cells cotransfected with a pcDNA3.1/Bax or pcDNA3.1/ Bax-t vector and the pcDNA3.1/lacZ reporter construct/over the ␤-gal activity of control cells cotransfected with the empty pcDNA3.1 vector and the pcDNA3.1/lacZ reporter construct) ⫻ 100. Experiments were done independently three times and the data are expressed as means ⫾ SEM. DNA fragmentation assay. The following protocol was adapted from McGahon et al. [19]. Cells were transfected as described above and cultured in the absence or presence of 50 ␮M Z-VAD-fmk. At 24 h after transfection, cells (5 ⫻ 10 5) were harvested and centrifuged at 250g. Supernatants were removed and the pelleted cells were resuspended in 20 ␮l of lysis buffer (20 mM EDTA, 100 mM Tris, pH 8.0, and 0.8% sodium lauryl sarcosine; all from Sigma). After complete resuspension, 10 ␮l of a 1 mg/ml RNase/T1 cocktail mix (Ambion, Austin, TX) was added and the lysed cells were incubated in a 37°C water bath for 1.5 h. Upon completion of this incubation, 10 ␮l of 20 mg/ml proteinase K (Boehringer Mannheim, Indianapolis, IN) was added to each sample followed by incubation for at least 5 h at 50°C with constant rotation. The recovered DNA (10 ␮l per sample) was electrophoresed on 1.0% agarose gels containing 1 ␮g/ml ethidium bromide in TAE buffer (40 mM Tris–acetate, pH 8.0, and 2 mM EDTA) to visualize DNA fragmentation.

CLEAVAGE OF BAX ENHANCES CELL DEATH

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Bax Expression and Time Course of Induction of Cell Death in 293T Cells

FIG. 1. Bax constructs used to induce cell death in 293T cells (a, b) or in COS-7 and CHO cells (c [20]). The different Bcl-2 homology (BH) and transmembrane (TM) domains are boxed. (a) full-length p21 Bax, (b) truncated p18 Bax-t missing amino acids 1–38, (c) truncated Bax⌬RT missing amino acids 1–19, and (d) in vitro transcription/translation of Bax cDNAs. The Bax protein in lane 1 was translated from a Bax cDNA cloned into pcDNA3 which lacks the N-terminal Xpress tag and serves as a size comparison for the Bax fusion proteins derived from cDNAs subcloned into the pcDNA3.1 plasmid containing the Xpress tag.

RESULTS

Generation of Bax Expression Constructs Figure 1 shows the Bax constructs, Bax and Bax-t, used to induce cell death in 293T cells in this study. In order to test the ability of Bax expression constructs to induce apoptosis when transiently expressed in 293T cells, we first subcloned full-length p21 Bax and truncated p18 Bax (Bax-t) cDNAs into the vector pcDNA3.1 which produces recombinant proteins bearing an Xpress tag at the N-terminus. The truncated Bax cDNA is missing amino acids 1–38 (Bax-t) (Fig. 1b) representing the cleavage product of Bax detected both in vivo and in vitro [12, 13]. For comparison, another Bax expression construct missing amino acids 1–19 (Bax⌬RT) has been used by Goping et al. [20] to induce cell death in COS-7 and CHO cells (Fig. 1c). We first determined if the Bax fusion constructs produced the correct-sized proteins. Each construct was subjected to an in vitro coupled transcription/translation reaction in the presence of [ 35S]methionine and the resulting products were analyzed by SDS–PAGE and autoradiography. The Bax expression constructs produced proteins of the expected mass, with the N-terminal Xpress tag adding approximately 4 kDa to each protein product (Fig. 1d). Translation of the Bax-t protein was approximately 1.8-fold less compared with p21 Bax as determined by densitometric analysis.

To investigate the cell-death-promoting activities of the two different Bax expression constructs, we used a well-established transient transfection system in 293T cells that has been used previously to study Bax-induced apoptosis [21, 22]. Previous reports had demonstrated that transient expression of Bax in 293T cells induced cytochrome c release, caspase activation, and DNA fragmentation [21, 22]. The relative levels of expression of the Bax and Bax-t proteins were determined at 24 h after transfection by immunoblotting with an antibody against the Xpress epitope tag found at the N-terminus of the Bax fusion proteins (Fig. 2a). The blot was reprobed with an antibody against actin to control for protein loading of the gel. The expression levels of the Bax and Bax-t fusion proteins in vivo were similar to each other. In order to quantitate the levels of cell death induced by Bax and Bax-t, we cotransfected one of the Bax expression constructs or empty pcDNA3.1 vector along with a ␤-gal reporter plasmid (pcDNA3.1/lacZ) into 293T cells. At 6, 12, and 24 h after transfection, cell lysates were made and assayed for ␤-gal activity in vitro using the ␤-galactoside substrate ortho-nitrophenyl-␤-D-galactopyranoside. A decrease in ␤-gal reporter gene activity has been shown to parallel the loss of cell viability [23, 24]. At 24 h after transfection, at least 60% of control cells transfected with empty pcDNA3.1 and pcDNA3.1/lacZ expressed ␤-galactosidase as measured by X-gal staining (data not shown). As illustrated in Fig. 2b, transfection of Bax or Bax-t expression plasmids resulted in a marked reduction in ␤-gal activity at 12 and 24 h after transfection. At 24 h after transfection, expression of Bax resulted in greater than a 70% decrease in ␤-gal activity relative to the control. Interestingly, transfection with Bax-t resulted in greater than a 90% decrease in ␤-gal activity relative to the control at 24 h. This represented greater than a 2.5-fold increase in cytotoxic activity of p18 Bax compared to p21 Bax. Since the expression levels of the Bax constructs were similar at 24 h after transfection of 293T cells (Fig. 2a), the enhanced cytotoxicity of Bax-t cannot be attributed to higher levels of protein expression. Transfection and expression of Bax or Bax-t resulted in DNA fragmentation (Fig. 2c). In addition, both Bax constructs were able to induce cell shrinkage, plasma membrane blebbing, and detachment from the plate (Fig. 3). Thus both Bax proteins used in these experiments induced apoptosis in 293T cells as described previously for full-length Bax [21, 22]. However, the cleaved p18 form of Bax was characterized by enhanced cytotoxic properties.

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Bax-t was only partially inhibited by Z-VAD-fmk as ␤-gal activity was restored to less than half of the control activity (Fig. 4). Although DNA fragmentation induced by Bax-t was inhibited by Z-VAD-fmk treatment (Fig. 2c), the presence of the inhibitor did not prevent the shedding of Bax-t-transfected cells into the culture medium (data not shown). These results taken together underscore the enhanced cytotoxic activity of Bax-t relative to full-length Bax. The mechanism by which Bax induces release of cytochrome c from mitochondria is currently unknown. One hypothesis is that Bax regulates in some fashion the opening of a pore in the mitochondria called the PTP [25, 26]. Recent reports have demonstrated that Bax can interact with proteins within the PTP complex and that Bax regulation of the pore is required for the release of cytochrome c [27–31]. One component of the PTP is the matrix protein cyclophilin D [18]. Opening of the PTP can be inhibited by CsA that targets cyclophilin D [18]. Indeed, in some systems CsA treatment of cells is sufficient to block both PT and apoptosis [26, 28]. Since Z-VAD-fmk was only partially effective at inhibiting apoptosis induced by Bax-t, we wanted to determine if the enhanced cytotoxic activity of Bax-t might be due to direct effects on the PTP. If CsA can

FIG. 2. Bax expression and time course of induction of cell death in 293T cells. (a) The relative expression levels of the Bax fusion proteins were determined at 24 h after transfection by immunoblotting with an antibody against the Xpress epitope found in the Bax fusion proteins. Blots were reprobed with an antibody against actin to control for equal protein loading of the gel. (b) Cells were cotransfected with one of the Bax plasmids or empty pcDNA3.1 vector along with a ␤-galactosidase reporter plasmid. At the indicated time points, cell lysates were made and analyzed for ␤-gal activity. The percentage of ␤-gal activity was calculated relative to control cells transfected with empty pcDNA3.1 vector and the reporter plasmid. Experiments were done independently three times and the data are expressed as means ⫾ SEM. (c) DNA fragmentation was evaluated in cells transfected with Bax or Bax-t plasmids. Cells were then left untreated or treated with 50 ␮M Z-VAD-fmk immediately following transfection. At 24 h after transfection, DNA was extracted and electrophoresed on 1% agarose gels to visualize DNA fragmentation.

Bax-Mediated Apoptosis Can Be Inhibited by the Pancaspase Inhibitor Z-VAD-fmk but Not by the PTP Inhibitor Cyclosporin A Next we determined if the general caspase inhibitor Z-VAD-fmk could inhibit the cytotoxicity of either Bax construct. Cells transfected with Bax constructs and cultured in the presence of Z-VAD-fmk did not undergo morphological changes nor did they exhibit DNA fragmentation (Fig. 2c and data not shown). In addition, ␤-gal activity in these lysates was restored to control levels (Fig. 4). However, the cell death induced by

FIG. 3. Transfection of Bax constructs induces apoptosis. 293T cells were cotransfected with one of the Bax plasmids or empty pcDNA3.1 vector. At 24 h after transfection cell cultures were analyzed by light microscopy. Cells in the control culture retained a flat morphology and lacked condensed, round, highly refractive apoptotic cells. In contrast, numerous condensed, round, and highly refractive apoptotic cells were detectable in cultures transfected with the Bax or Bax-t constructs (⫻400).

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DISCUSSION

FIG. 4. Bax-mediated apoptosis can be inhibited by the pancaspase inhibitor Z-VAD-fmk but not by the PTP inhibitor CsA. 293T cells were cotransfected with one of the Bax plasmids or empty pcDNA3.1 vector along with a ␤-galactosidase reporter plasmid. Cells were then left untreated or treated with 50 ␮M Z-VAD-fmk or 1 ␮M CsA or a combination of both immediately following transfection. At 24 h after transfection cell lysates were made and analyzed for ␤-gal activity. The percentage of ␤-gal activity was calculated relative to control cells transfected with empty pcDNA3.1 vector and the reporter plasmid. Experiments were done independently three times and the data are expressed as means ⫾ SEM.

block the PTP, then CsA together with Z-VAD-fmk might restore ␤-gal activity to control levels. In order to test this, first we determined the concentration of CsA that was nontoxic to 293T cells transfected with the ␤-gal reporter gene by culturing cells for 24 h in different concentrations of CsA (1, 2.5, 5, and 10 ␮M). The highest concentration of CsA that could be used without inducing cell death was 1 ␮M (data not shown). Cells treated with Z-VAD-fmk, CsA, or a combination of Z-VAD-fmk plus CsA for 24 h showed ␤-gal activity that was similar to the control cells (Fig. 4). Expression of Bax or Bax-t in the absence of CsA resulted in a marked reduction in ␤-gal activity at 24 h as observed previously (Fig. 2b). Loss of ␤-gal activity induced by either Bax or Bax-t was unaffected by the presence of CsA indicating that this inhibitor of the PTP provided no protection against Bax-mediated apoptosis. Furthermore, the combination of Z-VAD-fmk plus CsA did not result in any increased protection against Bax-tmediated cell death over Z-VAD-fmk treatment alone. Taken together, these results suggest that Bax-mediated apoptosis in 293T cells occurs in a manner independent of the opening of the PTP.

Using a transient transfection system in 293T cells developed by others to study Bax-mediated apoptosis [21, 22], we have shown that two different Bax fusion proteins were able to induce apoptosis. Previous reports had demonstrated that transient expression of full-length Bax in 293T cells resulted in cytochrome c release, activation of caspases, DNA fragmentation, and changes in morphology consistent with apoptosis [21, 22]. We observed that apoptosis induced by fulllength Bax could be inhibited by the pancaspase inhibitor Z-VAD-fmk similar to previous observations [21, 22]. However, Bax-t, missing amino acids 1–38 at the N-terminus, exhibited enhanced cytotoxic activity that was only partially inhibited by Z-VAD-fmk treatment. The ability of the truncated p18 Bax to exhibit greater cytotoxicity relative to p21 Bax is consistent with a previous report that demonstrated that transient expression of Bax with deletion of amino acids 1–19 of Bax resulted in its enhanced cytotoxicity in both COS-7 and CHO cells (Bax⌬RT, see Fig. 1c [20]). Bax⌬RT arose during transcription–translation of Bax cDNA apparently from an internal initiation site using the methionine at codon 20, whereas the truncated p18 Bax construct used in these studies has been observed in vivo in a variety of different cell types undergoing apoptosis [11, 14 –17]. It appears that this portion of the N-terminus of Bax (amino acids 1–19) is required for retention of Bax in the cytosol under nonapoptotic conditions. Deletion of this region promotes targeting of Bax to mitochondria and insertion into the mitochondrial membrane [20]. Given these findings, one interpretation of our data is that p18 Bax with its extended deletion from amino acids 1–38 at the Nterminus of Bax may abolish the regulatory function of the N-terminal retention domain. However, cleavage of Bax at the N-terminus during 9-AC-induced apoptosis in HL-60 cells is a late event dependent upon caspasemediated activation of calpain [13]. These results suggest that cleavage of Bax is not involved in the targeting of Bax to mitochondria. Rather, we favor the interpretation that calpain-mediated cleavage of p21 Bax downstream of caspase activation in HL-60 cells serves to enhance the cytotoxic activity of p18 Bax at the mitochondria. Our data demonstrate that transient expression of the p18 Bax cleavage product in 293T cells induces higher levels of cell death compared to full-length Bax. The enhanced cytotoxicity of the p18 form of Bax is highlighted by the observation that the pancaspase inhibitor Z-VAD-fmk can only partially inhibit cell death caused by this construct compared to the complete inhibition of cell death induced by p21 Bax. Our findings in this study demonstrating increased cytotoxicity of p18 Bax would be consistent with work reported by others demonstrating caspase-

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FIG. 5. Model for Bax-mediated breakdown of mitochondrial membranes and disruption of ⌬⌿ m. Homodimers of Bax initially destabilize the outer mitochondrial membrane (OM) facilitating release of cytochrome c from the intermembrane space (IMS) independent of the opening of the mitochondrial permeability transition pore (PTP). The PTP complex contacts the inner and outer mitochondrial membranes and consists of several different proteins (VDAC, voltage-dependent anion channel; ANT, adenine nucleotide translocator; HEX, hexokinase; PBR, peripheral benzodiazepine receptor; CK, creatine kinase; cyc D, cyclophilin D). Once Bax has broken down the outer mitochondrial membrane it would have access to the intermembrane space where it would undergo proteolysis by calpain resulting in the p18 Bax cleavage fragment. The p18 Bax may then adopt a conformation that allows it to readily attack and disrupt the inner mitochondrial membrane (IM) resulting in collapse of the mitochondrial inner membrane potential (⌬⌿ m). Thus cleavage of p21 Bax during apoptosis to the p18 form may serve to increase the intrinsic cytotoxic properties of Bax and enhance its cell death function at the mitochondria.

dependent as well as caspase-independent Bax-mediated cell deaths [25]. In the case of the latter, cell death was due to direct mitochondrial damage resulting in compromised function of this organelle [25]. Thus, p18 Bax may have an enhanced ability to cause cell death by activating caspases more efficiently relative to p21 Bax and by directly inducing mitochondrial dysfunction. The ability of Bax to induce release of cytochrome c and apoptosis may be due to the induction of the mitochondrial PT. Previous reports had demonstrated that Bax expression in some cell lines induced cell death accompanied by the loss of mitochondrial membrane potential (⌬⌿ m) indicating the opening of the PTP [25, 28]. This collapse in mitochondrial ⌬⌿ m as well as the resulting cell death was inhibited by CsA, which is known to target the PTP component cyclophilin D [18, 26, 28]. In addition, use of in vitro systems with purified mitochondria and recombinant Bax has demonstrated that Bax-induced release of cytochrome c can be inhibited by a variety of

compounds that block the PTP, including CsA [27–29]. Finally, Bax has been shown to copurify with the adenosine nucleotide translocator and the voltage-dependent anion channel, both components of the PTP [27–30]. Taken together, these data suggest that Bax-induced release of cytochrome c is associated with the induction of the PT. However, using our model system in 293T cells, p21 and p18 Bax-mediated apoptosis was insensitive to CsA as well as CsA plus aristolochic acid (AraA) (data not shown) suggesting that Bax does not induce the PT. This combination has been shown previously to be an effective inhibitor of the PT [26]. This does not rule out Baxmediated effects through other CsA/AraA-insensitive pore proteins. Our results are in agreement with other reports that have demonstrated Bax induced cytochrome c release both in vitro and in vivo independent of the PT and loss of ⌬⌿ m [22, 31]. Indeed, cytochrome c release and caspase activation appear to lie upstream of the PT during Bax-induced apoptosis of 293T cells [22]. Cleavage of p21 Bax at the mitochondria during the

CLEAVAGE OF BAX ENHANCES CELL DEATH

latter stages of apoptosis could have ramifications for the ability of this molecule to affect continuous changes in this organelle during apoptosis (Fig. 5). For example, Bax is capable of decreasing outer mitochondrial membrane stability resulting in dissolution of the permeability barrier function of that membrane to molecules such as cytochrome c [32]. Once Bax has broken down the outer mitochondrial membrane independent of the opening of the mitochondrial PTP, it would have access to the inner mitochondrial membrane, which would presumably be a target for Bax-mediated disruption as well [32]. Interestingly, calpain activity has been targeted to the intermembrane space of mitochondria and has been shown to be involved in the mitochondrial PT during necrosis [33–35]. It is entirely possible that Bax initially forms pores in the outer mitochondrial membrane causing the release of cytochrome c and subsequently gains access to the mitochondrial intermembrane space. Once in the intermembrane space, p21 Bax may be cleaved by calpain resulting in enhanced cytotoxic activity of the p18 Bax cleavage fragment, which we have demonstrated, allowing it to attack the inner mitochondrial membrane. Indeed, at least one report has demonstrated movement of Bax from the outer to the inner mitochondrial membrane [28]. A potential consequence of Bax-mediated breakdown of the inner mitochondrial membrane would be the collapse of ⌬⌿ m, which has been observed very late in the apoptotic cascade in several reports of apoptosis using HL-60 cells as a model system [32, 36 –38]. The fact that cleavage of the proapoptotic molecule Bid during apoptosis, albeit by caspases, allows it to cause mitochondrial damage and cytochrome c release suggests that proteolysis of some of the proapoptotic members of the Bcl-2 family enhances their cell death function [7, 8]. This work was supported by a grant from the Elsa U. Pardee Foundation. The authors acknowledge Dr. L. Devi for kindly supplying primers and Heide Pleskin and Michelle Alonso for their assistance in the preparation of the figures.

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