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Grant Dewson1,3, Roger T Snowden1,3, Jason B Almond1, Martin JS Dyer1,2 ... University of Leicester, PO Box 138, Lancaster Road, Leicester LE1 9HN, UK;.
Oncogene (2003) 22, 2643–2654

& 2003 Nature Publishing Group All rights reserved 0950-9232/03 $25.00 www.nature.com/onc

Conformational change and mitochondrial translocation of Bax accompany proteasome inhibitor-induced apoptosis of chronic lymphocytic leukemic cells Grant Dewson1,3, Roger T Snowden1,3, Jason B Almond1, Martin JS Dyer1,2 and Gerald M Cohen*,1 1

MRC Toxicology Unit, Hodgkin Building, University of Leicester, PO Box 138, Lancaster Road, Leicester LE1 9HN, UK; Department of Hematology, Robert Kilpatrick Clinical Sciences Building, PO Box 65, University of Leicester, Leicester Royal Infirmary, Leicester LE2 7LX, UK 2

Chemotherapy resistance remains a major clinical problem in patients with B-cell chronic lymphocytic leukemia (B-CLL). Proteasome inhibitors are able to induce apoptosis in chemotherapy-resistant B-CLL cells in vitro. Exposure of B-CLL cells to the proteasome inhibitors, MG132 and lactacystin, resulted in inhibition of proteasomal activity within 30 min of treatment and was accompanied by an increase in the level of ubiquitinated proteins. Proteasome inhibitors did not alter the levels of expression of the proapoptotic Bcl-2 family proteins, Bax and Bid, prior to the onset of apoptosis. Instead, proteasome inhibitors induced a caspase-independent conformational change in Bax (as shown by a conformation-specific Bax antibody) and its translocation to mitochondria, resulting in mitochondrial perturbation, as evidenced by loss of the mitochondrial membrane potential and cytochrome c release. Similar conformational change and subcellular localization of Bax were observed during apoptosis induced with fludarabine, chlorambucil and prednisolone. These data suggest that alteration of Bax conformation and its redistribution to mitochondria are common and early features of B-CLL apoptosis in response to proteasome inhibitors and other chemotherapeutic agents. Oncogene (2003) 22, 2643–2654. doi:10.1038/sj.onc.1206326 Keywords: Bax; chronic lymphocytic leukemia; proteasome inhibitors

Introduction B-cell chronic lymphocytic leukemia (B-CLL) is the most common adult leukemia in the Western world. It is identified by the gradual accumulation of a monoclonal population of CD5+/CD19+ B-lymphocytes, which arise primarily from a failure in apoptosis (Reed, 1998; Bannerji and Byrd, 2000). The disease is currently *Correspondence: GM Cohen; E-mail: [email protected] 3 Authors contributed equally to the work Received 16 September 2002; revised 11 December 2002; accepted 12 December 2002

incurable and drug resistance presents a major clinical problem; patients refractory to fludarabine have a median survival of less than 1 year (Kalil and Cheson, 1999). Proteasome inhibitors, which potently induce apoptosis in drug-resistant B-CLL cells (Chandra et al., 1998; Delic et al., 1998; Almond et al., 2001), may be novel chemotherapeutic agents for this and other diseases (Dou and Li, 1999; Orlowski, 1999; Murray and Norbury, 2000; Almond and Cohen, 2002). One novel proteasome inhibitor, PS-341, has entered Phase II clinical trials in various malignancies including CLL and chronic myelogenous leukemia, and Phase III trials in multiple myeloma (Adams and Elliott, 2000). Proteasome inhibitors inhibit the ubiquitin–proteasome system, which is the major nonlysosomal pathway for protein degradation in eucaryotic cells, thereby interfering with its ability to both selectively degrade damaged or misfolded proteins and also to regulate the cell cycle (Ciechanover, 1994; Baumeister et al., 1998). Induction of apoptosis by proteasome inhibitors is of major importance for their antitumor activity (Dou and Li, 1999; Orlowski, 1999; Murray and Norbury, 2000; Almond and Cohen, 2002). Apoptosis induced by chemotherapeutic agents, growth factor withdrawal and stress all result in perturbation of mitochondria and release of cytochrome c from the mitochondrial intermembrane space into the cytosol (Martinou and Green, 2001). Cytochrome c then binds to Apaf-1 so allowing binding of ATP/dATP and oligomerization of Apaf-1 to form the apoptosome, which binds and activates caspase-9, and in turn recruits and activates effector caspase-3 and -7 (Li et al., 1997; Cain et al., 1999; Bratton et al., 2001). These effector caspases cleave many of the substrates responsible for the biochemical and morphological changes associated with apoptosis (Cohen, 1997; Earnshaw et al., 1999). Recent studies have shown that, in addition to cytochrome c, other proapoptotic molecules, including AIF, endonuclease G, Smac/DIABLO and Omi/HtrA2 are also released from mitochondria during the induction of apoptosis (Van Loo et al., 2002). Evidence suggests that the proapoptotic Bcl-2 homologs, Bax and Bak, are pivotal regulators of the release of apoptogenic

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factors from the mitochondrial intermembrane space (Eskes et al., 1998; Jurgensmeier et al., 1998; Rosse´ et al., 1998). Cells deficient in Bax and Bak are resistant to a wide range of different apoptotic stimuli (Wei et al., 2001). Although the precise mechanism remains unclear, Bax undergoes translocation to the mitochondria during apoptosis in response to numerous stimuli, and induces cytochrome c release in intact cells (Rosse´ et al., 1998) and from isolated mitochondria (Jurgensmeier et al., 1998) possibly involving interaction with integral mitochondrial pore proteins or generation of multimeric Bax channels (Marzo et al., 1998; Shimizu et al., 1999; Antonsson et al., 2001). Various mechanisms have been proposed to explain the ability of proteasome inhibitors to induce apoptosis, including the accumulation of molecules such as p53 (Shinohara et al., 1996; Lopes et al., 1997), the cyclindependent kinase inhibitors p21 and p27 (Drexler, 1997; An et al., 1998; Naujokat et al., 2000), activation of stress-activated protein kinases (Meriin et al., 1998; MacFarlane et al., 2000) or a shift in the balance of Bcl2 family proteins in favor of apoptosis (Chang et al., 1998; Breitschopf et al., 2000; Li and Dou, 2000; Marshansky et al., 2001). Few of these studies have addressed the mechanism of proteasome inhibitorinduced apoptosis in primary cells. Previously, we have shown that proteasome inhibitors induce apoptosis in B-CLL cells by formation of an B700 kDa Apaf-1containing apoptosome complex and subsequent activation of caspases (Almond et al., 2001). We now demonstrate that proteasome inhibitors induce disruption of mitochondria by a mechanism involving caspase-independent translocation of Bax, resulting in the release of mitochondrial cytochrome c. We also provide evidence that the Bax conformational change and its subcellular redistribution are common features of B-CLL cell apoptosis in response to diverse stimuli.

Results Rapid inhibition of proteasomal activity and accumulation of ubiquitinated proteins in B-CLL cells treated with proteasome inhibitors Previously, we have shown that proteasome inhibitors induced apoptosis in B-CLL cells within 4–6 h (Almond et al., 2001). To investigate the mechanism of apoptosis, we wished to ascertain at what time proteasomal activity was inhibited. Purified B-CLL cells possessed the three major protease activities of the proteasome. The chymotrypsin-like activity was clearly predominant with much lower amounts of trypsin-like and PGPH activities (Figure 1a). Inhibition of the chymotrypsin-like activity was therefore used as a measure for proteasomal inhibition. Treatment of B-CLL cells with lactacystin (10 mm) resulted in almost complete inhibition of chymotrypsin-like activity within 30 min (Figure 1b), which was maintained for at least 6 h when the cells were beginning to undergo apoptosis. Oncogene

Figure 1 Lactacystin rapidly inhibits proteasome activity and induces accumulation of ubiquitinated proteins. (a) The three major proteasomal activities of B-CLL were assessed as described in Materials and methods. (b) B-CLL cells were incubated in the absence or presence of lactacystin (10 mm) for 30 min and the chymotrypsin-like proteasomal activity determined. (c) B-CLL cells were incubated for 0–4 h in the absence or presence of lactacystin (10 mm) or MG132 (1 mm) and immunoblotting was performed with an anti-ubiquitin antibody. Results presented from one patient that were representative of independent experiments performed on two patients

The inhibition of proteasomal activity by either lactacystin (10 mm) or the reversible, peptide aldehyde proteasome inhibitor MG132 (1 mm) was accompanied by a time-dependent increase in the level of ubiquitinated proteins, first observed 30 min after treatment (Figure 1c). In untreated B-CLL cells from some patients, significant levels of ubiquitinated proteins were observed, which were rapidly degraded on culture (Figure 1c, lanes 1–5). These ubiquitinated proteins present in control cells were most probably due to the stress caused by the isolation procedure. Thus, in BCLL cells proteasome inhibitors caused a rapid inhibition of proteasomal activity followed by a subsequent increase in ubiquitinated proteins, which clearly preceded the onset of apoptosis.

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Proteasome inhibitors do not cause an accumulation of proapoptotic Bax and Bid in B-CLL cells Inhibition of the degradation of proapoptotic Bcl-2 family members such as Bax, the truncated form of Bid (tBid) and Bik by proteasome inhibitors has been implicated in their induction of apoptosis in different cell lines (Breitschopf et al., 2000; Li and Dou, 2000; Marshansky et al., 2001). In addition, the Bax/Bcl-2 ratio may be an important determinant of the sensitivity of B-CLL cells to apoptosis (McConkey et al., 1996; Pepper et al., 1998). We therefore investigated whether proteasome inhibitors induced apoptosis in B-CLL cells by alteration of these proteins. Control B-CLL cells contained the 26, 21 and 22 kDa full-length forms of Bcl-2, Bax and Bid, respectively (Figure 2a–c). Little alteration was detected in the levels of Bcl-2, Bax or Bid in untreated cells over 18 h, except for increased levels of Bax detected at 8 and 18 h, which corresponded with the onset of spontaneous apoptosis (Figure 2a–c, lanes 1–6). Lactacystin induced marked apoptosis by 4 h, which was almost complete at 18 h (Figure 2, lanes 7–11). However at 4 h, lactacystin did not cause any detectable changes in the cellular levels of Bcl-2, Bax or Bid compared with untreated cells (Figure 2a–c, compare lanes 7–8 with lanes 1–3). Prolonged treatment with lactacystin for 8 and 24 h caused a marked reduction in the cellular levels of Bax and Bid (Figure 2b, c, lanes 10–11). However, considerable apoptosis as evidenced by

phosphatidylserine externalization was observed at these times (84% at 8 h, 98% at 24 h). The loss of full-length Bid was accompanied by the appearance of small amounts of a lower molecular weight immunoreactive band, which may represent tBid (Figure 2c, lane 11). Loss of these molecules was most probably due to either cleavage by caspases, which were activated during BCLL cell apoptosis as evidenced by processing of the initiator caspase-9 and the effector caspase-3 (Figure 2d, e), or secondary necrosis caused by the extensive cell death observed (Almond et al., 2001). Processing of caspase-9 and -3 to their catalytically active large subunits was first observed after 4 h of treatment with lactacystin (Figure 2d, e, lane 8), coincident with the onset of apoptosis as detected by phosphatidylserine externalization. The detection of tBid, proposed to cooperate with Bax and Bak in inducing cytochrome c release, in the latter stages of proteasome inhibitorinduced apoptosis may represent an amplification pathway downstream of initial caspase activation. Bik expression was undetectable either in untreated or lactacystin-treated B-CLL cells, whereas it was clearly detectable in Jurkat cells (data not shown), where it has been implicated in proteasome inhibitor-induced apoptosis (Marshansky et al., 2001). Similarly, the proapoptotic molecule, Bad, could not be detected in B-CLL cells (data not shown), in agreement with a previous report (Kitada et al., 1998). Therefore, the induction of apoptosis in B-CLL cells by proteasome inhibitors was due to neither an increase in the proapoptotic proteins Bax, tBid, Bik or Bad nor an increase in the Bax/Bcl-2 ratio. Proteasome inhibitors induce a caspase-independent perturbation of mitochondria in B-CLL cells

Figure 2 Proteasome inhibitors induce caspase activation but do not cause accumulation of proapoptotic Bcl-2 homologs. B-CLL cells were incubated in the absence or presence of lactacystin (10 mm) for the indicated times and immunoblotted for Bcl-2 (a), Bax (b), Bid (c), caspase-9 (d), caspase-3 (e) or actin (f) as a control. The percentage of apoptotic cells was assessed by externalization of phosphatidylserine. Results presented from one patient that were representative of independent experiments performed on two patients

We wished to determine whether proteasome inhibitorinduced apoptosis involved an early perturbation of mitochondria in B-CLL cells. B-CLL cells exposed to MG132 showed a time-dependent loss of mitochondrial membrane potential (DCm), as assessed by TMRE, with loss of membrane potential first being observed at 3 h (Figure 3b). This loss in mitochondrial membrane potential accompanied apoptosis as a similar timedependent increase in phosphatidylserine externalization was also observed (Figure 3a). Many of the morphological and biochemical features of apoptosis are associated with activation of caspases. These may include direct or indirect effects of effector caspases on mitochondria resulting in further perturbation of mitochondria following a postmitochondrial feedforward amplification loop. In order to assess the role of caspases in the mitochondrial perturbation induced by MG132, cells were pretreated with the cell permeable polycaspase inhibitor Z-VAD.fmk. Interestingly, Z-VAD.fmk prevented phosphatidylserine externalization without affecting perturbation of mitochondrial membrane potential (Figure 3a, b). These results suggested that proteasome inhibitors initially induce a caspase-independent perturbation of mitochondria. Oncogene

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Figure 4 Caspase-independent cytochrome c release and Bax redistribution during the apoptosis of B-CLL cells. B-CLL cells were preincubated with Z-VAD.fmk (200 mm) for 30 min where indicated prior to treatment with MG132 (1 mm) for the indicated times. Cytosolic and membrane fractions were generated as described in Materials and methods, resolved by SDS–PAGE and immunoblotted for cytochrome c (a) and Bax (b). Apoptosis was assessed by Annexin V binding. Results presented from one patient that were representative of independent experiments performed on three patients

Figure 3 Proteasome inhibitor-induced phosphatidylserine externalization, but not loss of DCm is caspase-dependent. B-CLL cells were incubated either alone (’-’) or in the presence of ZVAD.fmk (200 mm, &-&). B-CLL cells were also incubated with MG132 (1 mm) either alone (m-m) or following a 30 min prior incubation with Z-VAD.fmk (200 mm, n-n). Phosphatidylserine (PS) externalization and DCm were assessed as described in Materials and methods by Annexin V (a) and TMRE (b) staining, respectively. Results presented from one patient that were representative of independent experiments performed on three patients

Proteasome inhibitors induce a caspase-independent subcellular redistribution of cytochrome c and Bax Although proteasome inhibitors have been shown to induce the release of mitochondrial cytochrome c in BCLL cells (Chandra et al., 1998; Almond et al., 2001), this was only demonstrated at late times when one could not discern its importance in the subsequent induction of apoptosis. We therefore wished to determine whether the loss in mitochondrial membrane potential was accompanied by changes in mitochondrial cytochrome c. MG132 caused a time-dependent loss of mitochondrial cytochrome c, which was accompanied by a concomitant increase in cytosolic cytochrome c (Figure 4a, lanes 8–12). Pretreatment with Z-VAD.fmk did not prevent release of mitochondrial cytochrome c (Figure 4a, lane 13). The mechanism by which cytochrome c is released from the mitochondrial intermembrane space remains unclear, although the proapoptotic Bcl-2 protein, Bax, is Oncogene

implicated. In a number of model systems, Bax undergoes translocation to the mitochondrial membrane during apoptosis (Wolter et al., 1997) and facilitates the release of apoptogenic factors (Deng et al., 2002; Rosse´ et al., 1998). In order to investigate if similar changes occurred in B-CLL cells, we studied the subcellular distribution of Bax during proteasome inhibitor-induced apoptosis. In untreated B-CLL cells, no consistent time-dependent change in Bax was observed in either the cytosolic or membrane fractions (Figure 4b, lanes 1–6). In contrast, MG132 induced a time-dependent loss of Bax from the cytosol, accompanied by its recruitment to the membrane fraction (Figure 4b, lanes 8–12), detectable after 3 h when apoptosis by phosphatidylserine externalization was 35% (Figure 4b, lane 10). As with cytochrome c release, Bax redistribution was unaffected by pretreatment with Z-VAD.fmk (Figure 4b, lane 13). Taken together, these data suggested that both the translocation of Bax and the release of mitochondrial cytochrome c were caspaseindependent. Bax conformational change in B-CLL cell apoptosis In nonapoptotic cells, Bax is a soluble monomeric protein, diffusely distributed in the cytoplasm (Hsu and Youle, 1998). The hydrophobic C terminus of Bax is proposed to mediate intracellular localization and dimerization via interactions with the BH3 domain and N terminus (Goping et al., 1998; Nechushtan et al., 1999; Suzuki et al., 2000). Diverse apoptotic stimuli induce a conformational change in Bax involving dissociation of its termini, thereby exposing previously occluded epitopes, and facilitating translocation to the outer mitochondrial membrane and oligomerization (Hsu et al., 1997; Goping et al., 1998; Gross et al., 1998; Suzuki et al., 2000; Makin et al., 2001). Using a number of different Bax antibodies to investigate the

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conformational state of Bax during B-CLL cell apoptosis, we noted that anti-Bax Clone 3, a monoclonal antibody directed against amino acids 55–178 (spanning BH domains 1–3) of Bax, appeared to be conformationspecific (Figure 5). The quaternary structure of monomeric Bax is detergent sensitive with nonionic detergents, such as Triton X-100, capable of inducing its open

configuration, thereby mimicking the intramolecular change of Bax that occurs during apoptosis (Hsu and Youle, 1997, 1998). Detergent-induced conformational change facilitates oligomerization and immunoprecipitation with conformation-specific anti-Bax antibodies, such as 6A7 (Gross et al., 1998; Hsu and Youle, 1998; Nechushtan et al., 1999). In contrast, zwitterionic

Figure 5 Detection of Bax activation using a conformation-specific anti-Bax antibody during apoptosis of B-CLL cells. (a) Immunoprecipitation analysis with conformation-specific Bax antibodies. B-CLL cells were incubated in the absence or presence of MG132 (1 mm) for 20 h and lysates were generated in the presence of CHAPS or Triton X-100. Bax was immunoprecipitated with monoclonal antibodies 6A7 and Clone 3 as described in Materials and methods. Control was lysate incubated with Protein G beads alone. (b) B-CLL cells were incubated in the absence or presence of MG132 (1 mm) for 4 and 8 h and intracellular flow cytometry performed using anti-Bax monoclonal antibody Clone 3 (solid line) or secondary antibody alone as negative control (dashed line). Figures in parentheses indicate percentage of cells with Baxhi expression. (c) Forward and side scatter profiles of Baxhi/lo and Annexin V+ve/ ve B-CLL cells treated with MG132 for 8 h. Results presented from one patient that were representative of independent experiments performed on four patients Oncogene

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detergents, such as CHAPS, do not promote inappropriate Bax conformational change, thereby maintaining Bax in its native conformation (Hsu and Youle, 1997, 1998). B-CLL cell apoptosis was induced with MG132 for 20 h (71% apoptosis versus 9% apoptosis in control cells as assessed by Annexin V binding). The cells were solubilized in CHAPS or Triton X-100, and Bax was immunoprecipitated with monoclonal anti-Bax antibodies 6A7 and Clone 3. In the presence of CHAPS, both monoclonal antibodies immunoprecipitated Bax in apoptotic but not in control cells (Figure 5a, compare lanes 2 and 4 with lanes 1 and 3). However, in the presence of Triton X-100, both antibodies immunoprecipitated Bax in both apoptotic and nonapoptotic cells (Figure 5a, lanes 1–4). These results demonstrated that anti-Bax Clone 3 behaved as a conformation-specific antibody that recognized an epitope that is normally concealed and is only exposed during apoptosis. Intracellular flow cytometric analysis using the Clone 3 antibody on B-CLL cells exposed to MG132 resulted in the formation of two distinct populations of Baxexpressing cells, termed Baxlo and Baxhi (Figure 5b). A time-dependent increase was observed in the Baxhi population of cells, with 31 and 61% of cells being Baxhi after 4 and 8 h treatment, respectively (Figure 5b). In contrast, in the untreated cells no evidence of a Baxhi population was observed even after 8 h (Figure 5b). No change in fluorescence was observed in the secondary antibody control indicating that the observed increase in fluorescence was not because of altered autofluorescence of apoptotic cells or increased nonspecific binding as a result of apoptosis-associated membrane permeability (Figure 5b). Back gating of the Baxhi and Baxlo cells revealed the two populations to exhibit distinct size and granularity profiles as indicated by forward and side scatter, respectively (Figure 5c). Independent gating of Annexin V positive and negative cells similarly revealed populations with distinct forward and side scatter profiles (Figure 5c). The Baxlo and Baxhi cells exhibited almost identical forward and side scatter profiles to the Annexin V negative and positive cells, respectively (Figure 5c). Taken together, these results strongly suggest that Baxhi cells represent a population of apoptotic cells or cells destined to undergo apoptosis. Anti-Bax Clone 3 was used subsequently as, in our hands, it provided better discrimination of Bax conformation change by flow cytometry than the previously described 6A7 monoclonal antibody (Hsu and Youle, 1998). Proteasome inhibitors and chemotherapeutic agents induce a conformational change of Bax in B-CLL cells Although MG132 is generally used to inhibit the proteasome, under some circumstances it has also been shown to inhibit calpain (Tsubuki et al., 1996). We therefore investigated whether similar changes were induced by lactacystin, a more specific proteasome inhibitor. Both lactacystin and MG132 caused a timedependent increase in Baxhi expressing cells, indicative of Oncogene

Figure 6 Proteasome inhibitors and chemotherapeutic agents induce a conformational change of Bax. (a) B-CLL cells were pretreated with Z-VAD.fmk (200 mm) for 30 min where indicated and incubated for the indicated times in the absence or presence of MG132 (1 mm) or lactacystin (10 mm). (b) B-CLL cells were incubated for the indicated times in the absence or presence of fludarabine (20 mm), prednisolone (200 mm) or chlorambucil (20 mm). Results presented from one patient that were representative of independent experiments performed on three patients. (c) B-CLL cells derived from a single patient with fludarabine-refractory disease were incubated in the absence or presence of MG132 (1 mm), lactacystin (10 mm) or fludarabine (20 mm). Apoptosis was assessed by Annexin V staining and the percentage of cells with Baxhi expression was determined by intracellular flow cytometry. Apoptosis and Baxhi expression for vehicle controls were essentially identical to untreated cells (not shown)

an increase in cells expressing the open conformation of Bax, which was concomitant with or preceded externalization of phosphatidylserine (Figure 6a). Pretreatment with Z-VAD.fmk completely inhibited externalization of phosphatidylserine but had no effect on the conformational change of Bax, indicating that Bax activation occurred upstream of the activation of caspases (Figure 6a). To assess whether the alteration in the conformation of Bax was a common event during apoptosis of B-CLL

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cells and also its possible clinical relevance, cells were treated with chemotherapeutic agents commonly used in the treatment of B-CLL, namely fludarabine, chlorambucil and prednisolone. All three agents induced a timedependent increase in Baxhi-expressing cells albeit to a lesser extent than the proteasome inhibitors with a detectable conformational change occurring later between 8 and 24 h (Figure 6b). Interestingly, B-CLL cells derived from one patient with fludarabine-refractory disease exhibited little detectable evidence of apoptosis as determined by phosphatidylserine externalization or Bax conformational change following incubation with fludarabine (Figure 6c). Cells from this patient were still sensitive to the proteasome inhibitors, MG132 and lactacystin, which both induced Bax conformational change and phosphatidylserine exposure (Figure 6c). Taken together, these data suggest that both the proteasome inhibitors and the conventional chemotherapeutic agents induce a conformational change in Bax, which correlated very well with the induction of apoptosis and possibly with susceptibility to chemotherapy. Colocalization of conformationally altered Bax with mitochondria is a common event during apoptosis of B-CLL cells In its open configuration, the hydrophobic C terminus of Bax is unmasked and proposed to target the proapoptotic protein to intracellular membranes, principally the outer mitochondrial membrane (Nechushtan et al., 1999). As Bax was translocated to the membrane fraction during proteasome inhibitor-induced apoptosis (Figure 4b), we assessed its precise subcellular distribution using confocal microscopy. There was no detectable staining of Bax with the conformation-specific Clone 3 antibody in freshly isolated B-CLL cells and after 8 h in culture (Figure 7a, row 1 and data not shown). Bax staining was readily detectable following treatment for 8 h with both proteasome inhibitors MG132 and lactacystin (Figure 7a, rows 2 and 3), consistent with the data obtained using flow cytometric analysis (Figures 5 and 6). The conformationally altered form of Bax detected by the Clone 3 antibody exhibited an aggregated distribution and most commonly colocalized with mitochondria (Figure 7a, rows 2 and 3). Similarly, treatment with fludarabine, chlorambucil and prednisolone for 24 h induced aggregation of Bax (data not shown). In order to exclude the possibility that the colocalization of Bax with mitochondria was not simply an artefact because of the low cytoplasmic volume of B-CLL cells, we used another monoclonal Bax antibody, 4F11, that recognizes native cytoplasmic Bax (Penault-Llorca et al., 1998). In untreated, control B-CLL cells 4F11 detected a diffuse distribution throughout the entire cell with no colocalization with the mitochondria (Figure 7b). In B-CLL cells induced to undergo apoptosis with MG132, 4F11 detected a similar aggregated Bax distribution to Clone 3 (data not shown).

To further verify the subcellular localization of Bax as detected with antibodies Clone 3 and 4F11, its distribution was examined in MCF-7 cells that possess a relatively large cytoplasmic volume. Similar to B-CLL cells, Bax exhibited a diffuse, cytoplasmic distribution in control cells (Figure 7c, left panel). Upon induction of apoptosis with tumor necrosis factor-related apoptosisinducing ligand (TRAIL) Bax underwent aggregation and colocalized with mitochondria (Figure 7c, right panel), and conformationally altered Bax was detected by Clone 3 (Figure 7d, right panel). Taken together, the evidence supports a conformational change of Bax and its redistribution to the mitochondria during B-CLL cell apoptosis. Discussion Currently, proteasome inhibitors are being tested in a variety of hematologic malignancies including multiple myeloma and B-CLL, as well as various solid tumors (Adams, 2002). The selective proteasome inhibitor, PS341, exhibits activity against a variety of malignancies in vitro and in vivo, and is well tolerated in Phase II clinical studies of refractory multiple myeloma and CLL (Hideshima et al., 2001; Adams, 2002). In light of their clinical potential, it is important to elucidate the mechanism whereby proteasome inhibitors induce apoptosis. Cytochrome c release is critical in the activation of caspases in response to numerous apoptotic stimuli (Kluck et al., 1997). The precise mechanism by which cytochrome c is released from the mitochondrial intermembrane space remains unclear. However, it has been shown that the multidomain, proapoptotic Bcl-2 homologs Bax and Bak fulfil partially redundant, but essential roles in mitochondrial dysfunction in response to apoptotic stimuli (Lindsten et al., 2000; Wei et al., 2001). Bax is pivotal in mediating the integrity of the outer mitchondrial membrane and its permeability to apoptogenic factors in a number of systems. In a nonapoptotic cell, Bax is a monomeric protein exhibiting a diffuse cytosolic distribution. The three-dimensional structure of monomeric Bax predicts the hydrophobic C terminus to interact with and mask the BH3 domain, cooperatively inhibiting dimerization and membrane targeting (Suzuki et al., 2000). In response to apoptotic stimuli, Bax undergoes a conformational change to expose functionally vital domains and also reveal previously masked epitopes, thus allowing recognition of the active form by conformationspecific antibodies. We have demonstrated that proteasome inhibitor-induced apoptosis of B-CLL cells involves caspase-independent conformational change of Bax and its translocation to mitochondria (Figures 4–7), which correlated well with perturbation of the mitochondria and the release of cytochrome c (Figures 3 and 4). Bax expression has also been implicated as a determinant in the susceptibility of CLL cells to commonly used chemotherapeutic agents, with cheOncogene

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moresistance correlating with reduced Bax levels (Pepper et al., 1999; Bosanquet et al., 2002). We have demonstrated that apoptosis induced by chlorambucil, fludarabine and prednisolone also resulted in an alteration of Bax conformation. In each case apoptosis,

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as determined by externalization of phosphatidylserine, correlated well with aggregation and redistribution of Bax to the mitochondria. We have shown that the potential role of Bax in the apoptotic programme may be independent of changes in its expression, but

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dependent instead on its conformation state and subcellular localization. Therefore, the induction of Bax expression or the ratio of Bcl-2 family proteins may not necessarily be the determining factor in the sensitivity of B-CLL cells to an apoptotic stimulus. Inter- and intrapatient variation was observed in the apoptotic response to the agents tested, in agreement with that previously described (Bosanquet et al., 2002). In one patient with fludarabine-refractory disease, only very small increases in Baxhi cells were observed together with very little increase in externalization of phosphatidylserine in response to fludarabine, while the same cells remained responsive to proteasome inhibitors (Figure 6c). Such differential responses may be indicative of previous treatment and the generation of resistant clones. Moreover, it indicates the potential therapeutic relevance of proteasome inhibitors for the treatment of B-CLL when cells are resistant to conventional chemotherapeutic agents. These results also raise the possibility that in vitro screening assays based upon the activation of Bax in response to drug treatment may be a more accurate prognostic indicator of drug sensitivity or resistance than levels of Bax expression alone. The initiating stimulus leading to the change in Bax conformation is as yet undetermined. Although reports suggest that the configuration of Bax is effected by a kinase activity (Ghatan et al., 2000; Gilmore et al., 2000), there is no direct evidence to suggest that Bax conformation is directly phosphorylation dependent (Suzuki et al., 2000). Survival factors such as IL-5 in eosinophils (Dewson et al., 2001), NGF in sympathetic neurons (Putcha et al., 1999), granulocyte colonystimulating factor in neutrophils (Maianski et al., 2002) and IL-3 in an IL-3-dependent hematopoietic cell line (Yamaguchi and Wang, 2001) suppress Bax activation, thereby providing a mechanism for trophic factor-mediated inhibition of apoptosis. The conformational change and translocation of Bax may also be regulated by the phosphatidylinositol 3-kinase (PI3-K)/Akt pathway, with active PI3-K capable of maintaining a cytosolic Bax distribution (Yamaguchi and Wang, 2001; Tsuruta et al., 2002). Interestingly, inhibition of PI3-K-induced apoptosis and also potentiated fludarabine-induced apoptosis of B-CLL cells (Barragan et al., 2002). It remains to be seen whether chemotherapeutic agents induce the apoptosis of B-CLL cells by interfering with the homeostatic mechanisms involved in maintaining Bax in its soluble form. In this study, we have demonstrated that alteration of Bax conformation and its translocation to the mito-

chondria are common events in the apoptosis of B-CLL cells in response to various cytotoxic stimuli. Determining whether different drugs affect Bax configuration and redistribution by divergent or common pathways and the elucidation of such pathways, may have important therapeutic implications.

Materials and methods Reagents Proteasome inhibitors, MG132 (carbobenzoxy-l-leucyl-l-leucyl-l-leucinal) and lactacystin, were from Affiniti Research Products Ltd (Exeter, UK) and Calbiochem (Nottingham, UK), respectively. The cell permeable caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethyl ketone (Z-VAD.fmk) was from Enzyme Systems (Dublin, CA, USA). Anti-Bax Clone 3 and 4F11 monoclonal antibodies were from Transduction Laboratories (Lexington, KY, USA) and (Beckman Coulter Inc., Fullerton, CA, USA), respectively. An Nterminal polyclonal antibody against Bax was from Upstate Biotechnology (Lake Placid, NY, USA). Polyclonal antibodies directed against Bid, Bad and Bik were from Biosource (Camarillo, CA, USA), New England Biolabs (Beverley, MA, USA), and Pharmingen (Oxford, UK), respectively. Rabbit polyclonal antibodies directed against caspase-9 and -3 were used as previously described (Sun et al., 1999). A monoclonal antibody against Bcl-2 was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The antiubiquitin monoclonal antibody was from Zymed Laboratories Inc. (San Francisco, CA, USA). Species-specific Alexa 488t secondary antibodies for immunocytochemistry, tetramethylrhodamine ethyl ester (TMRE), and Mitotracker Red CMXRos were from Molecular Probes (Eugene, OR, USA). FITC-conjugated Annexin V was from Bender Medsystems (Vienna, Austria). Anti-mouse and anti-rabbit HRP conjugates were from Sigma (Poole, UK) and DAKO (Ely, UK), respectively. Chlorambucil and prednisolone were from Sigma (Poole, UK), and fludarabine phosphate from Schering (Berlin, Germany). Recombinant TRAIL was generated as previously described (MacFarlane et al., 1997). B-cell purification and culture B-CLL patients who had not received chemotherapy within the previous 6 weeks were studied. Peripheral blood samples, obtained after informed consent and with local ethical committee approval, were purified as previously described (MacFarlane et al., 2002). B-CLL cell purity was assessed by staining with anti-CD19/FITC and anti-CD5/RPE antibodies (Dako, Cambridge, UK) and analysed by flow cytometry. This method produced an B95% pure population of cells expressing both CD19 and CD5. Purified B-CLL cells were resuspended in RPMI 1640 medium, supplemented with 10% fetal calf serum and 1% glutamax at a density of 1–4  106 ml 1 and incubated at 371C in an atmosphere of 5% CO2 with the

b—————————————————————————————————————————————————— Figure 7 Bax aggregation and colocalization with mitochondria during B-CLL apoptosis. (a) B-CLL cells were incubated in the absence or presence of MG132 (1 mm) or lactacystin (10 mm) for 8 h. Immunocytochemistry and confocal analysis was performed with anti-Bax Clone 3 antibody (green), Mitotracker CMXRos (red) and Hoechst 33258 (blue). (b) Untreated control B-CLL cells were stained with anti-Bax 4F11. Results presented in panels a and b are from one patient that are representatives of independent experiments performed on three patients. Independent and merged images of independently captured single sections are shown. (c and d) MCF-7 cells were incubated in the absence or presence of TRAIL (500 ng/ml) for 4 h. Immunocytochemistry and confocal analysis was performed with anti-Bax 4F11 (c, green) or Clone 3 (d, green), Mitotracker Red CMXRos (red) and Hoechst 33258 (blue). Merged images of independently captured single sections are shown Oncogene

Proteasome inhibitors induce CLL cell apoptosis G Dewson et al

2652 proteasome inhibitors, lactacystin (10 mm) or MG132 (1 mm) or with the chemotherapeutic agents, fludarabine phosphate (20 mm), chlorambucil (20 mM), or prednisolone (200 mm). Pretreatment with Z-VAD.fmk (200 mm) was for 30 min at 371C where indicated. Cell line MCF-7 breast carcinoma cell line was maintained in RPMI 1640 supplemented with 10% fetal calf serum and 1% glutamax and cultured at 371C in an atmosphere of 5% CO2. Apoptosis was induced with TRAIL (500 ng/ml) at 371C for 4 h. Assays of proteasomal activity Chymotrypsin-like, trypsin-like, and peptidylglutamylpeptide (PGPH) activities were determined using the fluorogenic substrates succinyl-Leu-Leu-Val-Tyr-4-methyl-coumaryl-7amide, butyloxycarbonyl-Leu-Ser-Thr-Arg-4-methyl-coumaryl-7-amide, and benzyloxycarbonyl-Leu-Leu-Glu-4-methylcoumaryl-7-amide (Peptide Institute Inc., Osaka, Japan) respectively. After treatment, approximately 15  106 purified B-CLL cells were harvested by brief centrifugation at 400 g for 5 min, washed three times in ice-cold PBS and resuspended in lysis buffer (20 mm Tris pH 7.5, 0.1 mm EDTA, 1 mm 2-mercaptoethanol, 20% glycerol, 5 mm ATP, 0.05% Nonidet-P40). Samples were frozen in liquid nitrogen and thawed three times, and protein concentrations were determined by the Bradford assay (Bio Rad Laboratories, Hercules, CA, USA). Samples were incubated at 371C with fluorogenic substrate in assay buffer (50 mm HEPES pH 7.5, 5 mm EGTA), and fluorescence measured continually using excitation/emission wavelength of 380/460 nm in a luminescence fluorimeter (Perkin-Elmer Corp., Boston, MA, USA). Subcellular fractionation After treatment, cells were pelleted, washed with ice-cold PBS and incubated on ice in buffer (250 mm sucrose, 20 mm HEPES pH 7.4, 5 mm MgCl2, 10 mm KCl, 1 mm EDTA, 1 mm EGTA) supplemented with a cocktail of protease inhibitors (Roche, Lewes, UK) containing 0.05% digitonin for 20 min. Cytosolic (supernatant) and membrane (pellet) fractions were separated by centrifugation at 13 000 g for 10 min. Protein concentrations were determined by the Bradford assay and resolved by SDS–PAGE and immunoblotted for cytochrome c (Pharmingen, San Diego, CA, USA) and Bax (Nterminal, Upstate Biotechnology, Lake Placid, NY, USA). This method routinely generated pure cytosolic and membrane fractions as verified by immunoblotting with a monoclonal antibody against the inner mitochondrial membrane protein cytochrome c oxidase subunit II (Molecular Probes, OR, USA).

4 mg/ml anti-Bax Clone 3 or 6A7 monoclonal antibodies, or in the absence of antibody as a control, overnight with constant agitation at 41C. Lysates were incubated with Protein G : Sepharose for 1 h at 41C and unbound proteins were removed by washing the beads in lysis buffer containing the appropriate detergent. Bound protein was eluted off the beads by boiling in Laemmli sample buffer plus 5% 2mercaptoethanol and resolved by SDS–polyacrylamide gel electrophoresis. Western blot analysis After treatment, cells were washed once in ice-cold PBS and cell pellets were snap frozen in liquid nitrogen. Cell samples were prepared by resuspension in Laemmli sample buffer plus 5% 2-mercaptoethanol and boiled for 5 min. Proteins were resolved on 10–15% SDS–polyacrylamide gels and blotted onto nitrocellulose membranes (Hybond C-extra, Amersham, Bucks, UK). Nonspecific binding was blocked by incubation (1 h at room temperature) with 5% nonfat milk in TBS plus 0.1% Tween-20 (TBS-T). Membranes were incubated with specific primary antibody diluted to 0.1 mg/ml in blocking buffer (2 h at room temperature). The membrane was then incubated for 1 h at room temperature with species-specific HRP-conjugated secondary antibody, diluted 1 : 2000 in TBST. Incubations were performed with constant agitation, followed by washing with TBS-T. Membranes were developed by enhanced chemiluminescence according to the manufacturer’s instructions (Amersham Life Science Ltd, Bucks., UK) and exposed to photographic film. Assessment of mitochondrial membrane potential (DCm) Cells were loaded with 50 nm TMRE for 10 min at 371C. Alterations of DCm were determined from the fluorescence intensity of TMRE as the transmembrane distribution of this lipophilic, cationic rhodamine derivative is dependent on membrane potential (Scaduto and Grotyohann, 1999). The cells were run immediately for FACScans analysis (Becton Dickinson, Oxford, UK), using excitation at 488 nm and detection between 560 and 606 nm. Intracellular flow cytometric analysis B-CLL cells were washed in PBS, prior to fixation in 2% formaldehyde for 10 min at room temperature. Cells were washed in PBS and incubated in the presence of 1 mg/ml of anti-Bax Clone 3 monoclonal antibody diluted in permeabilization buffer (0.5% BSA, 0.1% saponin in PBS) for 45 min on ice. As a negative control cells were incubated in the absence of primary antibody. Cells were washed in permeabilization buffer and incubated with species-specific Alexa 488t conjugated secondary antibody, diluted 1 : 100 in permeabilization buffer for 30 min on ice in the dark. Cells were washed in permeabilization buffer, resuspended in PBS and analysed using the FACScans.

Immunoprecipitation After treatment, B-CLL cells were washed with PBS and solubilized in lysis buffer (10 mm HEPES, pH 7.4, 150 mm NaCl) in the presence of 1% Triton X-100 or 1% CHAPS and a cocktail of proteolytic inhibitors at a cell density of 3  108 ml 1 for 30 min on ice. The lysate was spun at 13 000 g for 15 min at 41C to pellet unsolubilized debris and precleared with Protein G : Sepharose beads in a 1 : 1 slurry with lysis buffer containing the appropriate detergent for 2 h at 41C. Protein concentrations were determined by the Bradford assay and 500 mg of precleared lysate were incubated with Oncogene

Immunocytochemistry and confocal analysis B-CLL cells were incubated with 100 nm Mitotracker Red CMXRos, for 30 min at 371C, washed in PBS, and cytospins were performed at 300 g for 6 min on slides at 1  106 cells/ml. MCF-7 cells were cultured and treated in chamber microscope slides (Nalge Nunc, IL) prior to incubation with Mitotracker Red CMXRos as above. Cells were fixed in 3.8% formaldehyde for 15 min at room temperature and washed five times in PBS. Cells were permeabilized for 5 min at room temperature in blocking buffer (3% BSA in PBS) including

Proteasome inhibitors induce CLL cell apoptosis G Dewson et al

2653 0.1% saponin followed by blocking of nonspecific binding in blocking buffer for 1 h at room temperature. Cells were incubated overnight at 41C with 2.5 mg/ml anti-Bax Clone 3 monoclonal antibody diluted in blocking buffer. Cells were then incubated with species-specific Alexa 488t conjugated secondary antibody diluted 1 : 300 in blocking buffer for 50 min at room temperature in the dark. The nuclei were stained with membrane permeable DNA-binding dye, Hoechst 33258 (250 ng/ml) for 10 min at room temperature in the dark prior to mounting with fluoromount (Vectashield, Vector Laboratories, CA, USA). Images were independently captured by confocal laser microscopy (model TCS 4D, Leica, Heidelberg, Germany) with the 488-

and 568-nm lines of the krypton/argon laser used for the excitation of Alexa 488t and Mitotracker Red CMXRos, respectively. Excitation of Hoechst 33258 was by ultraviolet laser. Acknowledgements We thank Dr Marion MacFarlane, Dr Shawn Bratton, and Prof. Pierluigi Nicotera for their advice and critique of the manuscript. We thank Kul Sikand for his invaluable assistance with the confocal analysis. This work was supported in part by the Medical Research Council and a grant from the European Union (Grant # QLG1-1999-00739).

References Adams J. (2002). Curr. Opin. Chem. Biol., 6, 493–500. Adams J and Elliott PJ. (2000). Oncogene, 19, 6687–6692. Almond JB and Cohen GM. (2002). Leukemia, 16, 433–443. Almond JB, Snowden RT, Hunter A, Dinsdale D, Cain K and Cohen GM. (2001). Leukemia, 15, 1388–1397. An B, Goldfarb RH, Siman R and Dou QP. (1998). Cell Death Differ., 5, 1062–1075. Antonsson B, Montessuit S, Sanchez B and Martinou JC. (2001). J. Biol. Chem., 276, 11615–11623. Bannerji R and Byrd JC. (2000). Curr. Opin. Oncol., 12, 22–29. Barragan M, Bellosillo B, Campas C, Colomer D, Pons G and Gil J. (2002). Blood, 99, 2969–2976. Baumeister W, Walz J, Zuhl F and Seemuller E. (1998). Cell, 92, 367–380. Bosanquet AG, Sturm I, Wieder T, Essmann F, Bosanquet MI, Head DJ, Dorken B and Daniel PT. (2002). Leukemia, 16, 1035–1044. Bratton SB, Walker G, Srinivasula SM, Sun XM, Butterworth M, Alnemri ES and Cohen GM. (2001). EMBO J., 20, 998– 1009. Breitschopf K, Zeiher AM and Dimmeler S. (2000). J. Biol. Chem., 275, 21648–21652. Cain K, Brown DG, Langlais C and Cohen GM. (1999). J. Biol. Chem., 274, 22686–22692. Chandra J, Niemer I, Gilbreath J, Kliche KO, Andreeff M, Freireich EJ, Keating M and McConkey DJ. (1998). Blood, 92, 4220–4229. Chang YC, Lee YS, Tejima T, Tanaka K, Omura S, Heintz NH, Mitsui Y and Magae J. (1998). Cell Growth Differ., 9, 79–84. Ciechanover A. (1994). Cell, 79, 13–21. Cohen GM. (1997). Biochem. J., 326, 1–16. Delic J, Masdehors P, Omura S, Cosset JM, Dumont J, Binet JL and Magdelenat H. (1998). Br. J. Cancer, 77, 1103–1107. Deng Y, Lin Y and Wu X. (2002). Genes Dev., 16, 33–45. Dewson G, Cohen GM and Wardlaw AJ. (2001). Blood, 98, 2239–2247. Dou QP and Li B. (1999). Drug Resist. Update, 2, 215–223. Drexler HC. (1997). Proc. Natl. Acad. Sci. USA, 94, 855–860. Earnshaw WC, Martins LM and Kaufmann SH. (1999). Annu. Rev. Biochem., 68, 383–424. Eskes R, Antonsson B, Osen-Sand A, Montessuit S, Richter C, Sadoul R, Mazzei G, Nichols A and Martinou JC. (1998). J. Cell Biol., 143, 217–224. Ghatan S, Larner S, Kinoshita Y, Hetman M, Patel L, Xia Z, Youle RJ and Morrison RS. (2000). J. Cell Biol., 150, 335–347.

Gilmore AP, Metcalfe AD, Romer LH and Streuli CH. (2000). J. Cell Biol., 149, 431–446. Goping IS, Gross A, Lavoie JN, Nguyen M, Jemmerson R, Roth K, Korsmeyer SJ and Shore GC. (1998). J. Cell Biol., 143, 207–215. Gross A, Jockel J, Wei MC and Korsmeyer SJ. (1998). EMBO J., 17, 3878–3885. Hideshima T, Richardson P, Chauhan D, Palombella VJ, Elliott PJ, Adams J and Anderson KC. (2001). Cancer Res., 61, 3071–3076. Hsu YT, Wolter KG and Youle RJ. (1997). Proc. Natl. Acad. Sci. USA, 94, 3668–3672. Hsu YT and Youle RJ. (1997). J. Biol. Chem., 272, 13829– 13834. Hsu YT and Youle RJ. (1998). J. Biol. Chem., 273, 10777– 10783. Jurgensmeier JM, Xie Z, Deveraux Q, Ellerby L, Bredesen D and Reed JC. (1998). Proc. Natl. Acad. Sci. USA, 95, 4997–5002. Kalil N and Cheson BD. (1999). Oncologist, 4, 352–369. Kitada S, Krajewska M, Zhang X, Scudiero D, Zapata JM, Wang HG, Shabaik A, Tudor G, Krajewski S, Myers TG, Johnson GS, Sausville EA and Reed JC. (1998). Am. J. Pathol., 152, 51–61. Kluck RM, Bossy-Wetzel E, Green DR and Newmeyer DD. (1997). Science, 275, 1132–1136. Li B and Dou QP. (2000). Proc. Natl. Acad. Sci. USA, 97, 3850–3855. Li P, Nijhawan D, Budihardjo I, Srinivasula SM, Ahmad M, Alnemri ES and Wang X. (1997). Cell, 91, 479–489. Lindsten T, Ross AJ, King A, Zong WX, Rathmell JC, Shiels HA, Ulrich E, Waymire KG, Mahar P, Frauwirth K, Chen Y, Wei M, Eng VM, Adelman DM, Simon MC, Ma A, Golden JA, Evan G, Korsmeyer SJ, MacGregor GR and Thompson CB. (2000). Mol. Cell, 6, 1389–1399. Lopes UG, Erhardt P, Yao R and Cooper GM. (1997). J. Biol. Chem., 272, 12893–12896. MacFarlane M, Ahmad M, Srinivasula SM, FernandesAlnemri T, Cohen GM and Alnemri ES. (1997). J. Biol. Chem., 272, 25417–25420. MacFarlane M, Cohen GM and Dickens M. (2000). Biochem. J., 348, 93–101. MacFarlane M, Harper N, Snowden RT, Dyer MJ, Barnett GA, Pringle JH and Cohen GM. (2002). Oncogene, 21, 6809–6818. Maianski NA, Mul FP, van Buul JD, Roos D and Kuijpers TW. (2002). Blood, 99, 672–679. Makin GW, Corfe BM, Griffiths GJ, Thistlethwaite A, Hickman JA and Dive C. (2001). EMBO J., 20, 6306–6315. Oncogene

Proteasome inhibitors induce CLL cell apoptosis G Dewson et al

2654 Marshansky V, Wang X, Bertrand R, Luo H, Duguid W, Chinnadurai G, Kanaan N, Vu MD and Wu J. (2001). J. Immunol., 166, 3130–3142. Martinou JC and Green DR. (2001). Nat. Rev. Mol. Cell Biol., 2, 63–67. Marzo I, Brenner C, Zamzami N, Jurgensmeier JM, Susin SA, Vieira HL, Prevost MC, Xie Z, Matsuyama S, Reed JC and Kroemer G. (1998). Science, 281, 2027–2031. McConkey DJ, Chandra J, Wright S, Plunkett W, McDonnell TJ, Reed JC and Keating M. (1996). J. Immunol., 156, 2624–2630. Meriin AB, Gabai VL, Yaglom J, Shifrin VI and Sherman MY. (1998). J. Biol. Chem., 273, 6373–6379. Murray RZ and Norbury C. (2000). Anticancer Drugs, 11, 407–417. Naujokat C, Sezer O, Zinke H, Leclere A, Hauptmann S and Possinger K. (2000). Eur. J. Haematol., 65, 221–236. Nechushtan A, Smith CL, Hsu YT and Youle RJ. (1999). EMBO J., 18, 2330–2341. Orlowski RZ. (1999) Cell Death Differ., 6, 303–313. Penault-Llorca F, Bouabdallah R, Devilard E, Charton-Bain MC, Hassoun J, Birg F and Xerri L. (1998). Pathol. Res. Pract., 194, 457–464. Pepper C, Hoy T and Bentley P. (1998). Leukemia Lymphoma, 28, 355–361. Pepper C, Thomas A, Hoy T and Bentley P. (1999). Br. J. Haematol., 104, 581–588. Putcha GV, Deshmukh M and Johnson Jr EM. (1999). J. Neurosci., 19, 7476–7485.

Oncogene

Reed JC. (1998). Semin. Hematol., 35, 3–13. Rosse´ T, Olivier R, Monney L, Rager M, Conus S, Fellay I, Jansen B and Borner C. (1998). Nature, 391, 496–499. Scaduto Jr RC and Grotyohann LW. (1999). Biophys. J., 76, 469–477. Shimizu S, Narita M and Tsujimoto Y. (1999). Nature, 399, 483–487. Shinohara K, Tomioka M, Nakano H, Tone S, Ito H and Kawashima S. (1996). Biochem. J., 317, 385–388. Sun XM, MacFarlane M, Zhuang J, Wolf BB, Green DR and Cohen GM. (1999). J. Biol. Chem., 274, 5053–5060. Suzuki M, Youle RJ and Tjandra N. (2000). Cell, 103, 645– 654. Tsubuki S, Saito Y, Tomioka M, Ito H and Kawashima S. (1996) J. Biochem. (Tokyo), 119, 572–576. Tsuruta F, Masuyama N and Gotoh Y. (2002). J. Biol. Chem., 277, 14040–14047. Van Loo G, Saelens X, Van Gurp M, MacFarlane M, Martin SJ and Vandenabeele P. (2002). Cell Death Differ., 9, 1031–1042. Wei MC, Zong WX, Cheng EH, Lindsten T, Panoutsakopoulou V, Ross AJ, Roth KA, MacGregor GR, Thompson CB and Korsmeyer SJ. (2001). Science, 292, 727–730. Wolter KG, Hsu YT, Smith CL, Nechushtan A, Xi XG and Youle RJ. (1997). J. Cell Biol., 139, 1281–1292. Yamaguchi H and Wang HG. (2001). Oncogene, 20, 7779–7786.