PK11195, a Ligand of the Mitochondrial Benzodiazepine Receptor, Facilitates the Induction of Apoptosis and Reverses Bcl-2-Mediated Cytoprotection

EXPERIMENTAL CELL RESEARCH ARTICLE NO.

241, 426 – 434 (1998)

EX984084

PK11195, a Ligand of the Mitochondrial Benzodiazepine Receptor, Facilitates the Induction of Apoptosis and Reverses Bcl-2-Mediated Cytoprotection Tamara Hirsch,1 Didier Decaudin,1 Santos A. Susin, Philippe Marchetti, Nathanael Larochette, Miche`le Resche-Rigon,* and Guido Kroemer2 Centre National de la Recherche Scientifique, Unite´ Propre de Recherche 420, 19 rue Guy Moˆquet, F-94801 Villejuif, France; and *Hoechst-Marion Roussel, 102 rue de Noisy, F-93230 Romainville, France

One critical step of the apoptotic process is the opening of the mitochondrial permeability transition (PT) pore leading to the disruption of mitochondrial membrane integrity and to the dissipation of the inner transmembrane proton gradient (DCm). The mitochondrial PT pore is a polyprotein structure which is inhibited by the apoptosis-inhibitory oncoprotein Bcl-2 and which is closely associated with the mitochondrial benzodiazepine receptor (mBzR). Here we show that PK11195, a prototypic ligand of the 18-kDa mBzR, facilitates the induction of DCm disruption and subsequent apoptosis by a number of different agents, including agonists of the glucocorticoid receptor, chemotherapeutic agents (etoposide, doxorubicin), gamma irradiation, and the proapoptotic second messenger ceramide. Whereas PK11195 itself has no cytotoxic effect, it enhances apoptosis induction by these agents. This effect is not observed for benzodiazepine diazepam, whose binding site in the mBzR differs from PK11195. PK11195 partially reverses Bcl-2 mediated inhibition of apoptosis in two different cell lines. Thus, transfection-enforced Bcl-2 overexpression confers protection against glucocorticoids and chemotherapeutic agents, and this protection is largely reversed by the addition of PK11195. This effect is observed at the level of DCm dissipation as well as at the level of nuclear apoptosis. To gain insights into the site of action of PK11195, we performed experiments on isolated organelles. PK11195 reverses the Bcl-2-mediated mitochondrial retention of apoptogenic factors which cause isolated nuclei to undergo apoptosis in a cellfree system. Mitochondria from control cells, but not mitochondria from Bcl-2-overexpressing cells, readily release such apoptogenic factors in response to atractyloside, a ligand of the adenine nucleotide translocator. However, control and Bcl-2-overexpressing mitochondria respond equally well to a combination of 1

T.H. and D.D. contributed equally to this paper. To whom correspondence and reprint requests should be addressed at 19 rue Guy Moˆquet, B.P. 8, F-94801 Villejuif, France. Fax: 33-1-49 58 35 09. E-mail: kroemer @infobiogen.fr. 2

0014-4827/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.

atractyloside and PK11195. Altogether, these findings indicate that PK11195 abolishes apoptosis inhibition by Bcl-2 via a direct effect on mitochondria. Moreover, they suggest a novel strategy for enhancing the susceptibility of cells to apoptosis induction and, concomitantly, for reversing Bcl-2-mediated cytoprotection. © 1998 Academic Press

Key Words: mitochondrial transmembrane potential; permeability transition; programmed cell death.

INTRODUCTION

The mitochondrial permeability transition (PT)3 pore, also called megachannel or multiple conductance channel, participates in the regulation of matrix Ca21, pH, DCm, and volume and functions as a Ca21-, voltage-, pH-, and redox-gated channel with several levels of conductance and little if any ion selectivity [1– 4]. Recent evidence suggests that opening of the PT pore, which is regulated by Bcl-2, is a critical event in the process leading to apoptosis [5, 6]. Opening of the PT pore can cause the dissipation of the inner mitochondrial transmembrane potential (DCm) and culminates in the disruption of outer membrane integrity leading to the liberation of intermembrane proteins from the mitochondrion [7–10]. Indeed, the liberation of intermembrane proteins such as cytochrome c and/or the dissipation of the DCm are common events of early apoptosis [5, 6, 11–14]. Depending on the experimental system and the cell type, an increase in the matrix volume causing physical disruption of the outer mitochondrial membrane precedes the dissipation of the DCm [12, 13, 15] or both the disruption of the outer membrane and the dissipation of the inner membrane 3 Abbreviations used: DCm, mitochondrial transmembrane potential; DiOC6(3), 3,39-dihexyloxacarbocyanine iodide; Eth, ethidium; HE, hydroethidine; mBzR, mitochondral benzodiazepin receptor; PK11195, 1-(2chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinoline-carboxamide; PS, phosphatidylserine; PT, permeability transition; ROS, reactive oxygen species.

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DCm occur in a near-to-simultaneous fashion [7, 8]. On theoretical grounds [1– 4], both the increase in matrix volume preceding DCm reduction and the dissipation of the DCm may be mediated by opening of the PT pore, which can operate at a reversible low conductance level (which would cause a net inflow of ions and water into the mitochondrial matrix) and at an irreversible high conductance level (which would lead to DCm disruption). The PT pore is a multiprotein complex formed at the contact site between the mitochondrial inner and outer membranes, exactly at the same localization at which the oncoprotein Bcl-2 is particularly abundant [16]. Its pharmacological inhibition prevents apoptosis in a number of different models [5, 6, 17], whereas opening of the PT pore induces apoptosis [18 –21]. The exact molecular composition of the pore is not known, although proteins from the cytosol (hexokinase), the outer membrane [mitochondrial benzodiazepine receptor (mBzR); mitochondrial porin, also called voltagedependent anion channel], the intermembrane space (creatine kinase), the inner membrane (adenine nucleotide translocator, ANT), and the matrix (cyclophilin D) have been implicated in PT pore formation and/or regulation [1, 22–27]. In accord with its complex composite architecture, the PT pore is regulated by multiple endogenous effectors including local ion and pH gradients, ADP/ATP, NAD(P)H, and proapoptotic signal transduction molecules such as Ca21 or reactive oxygen species [1– 4, 26, 27]. Moreover, several of the components in the PT pore may constitute pharmacological targets for apoptosis modulation [5, 6, 17]. Based on these considerations, we decided to investigate the apoptosis-modulatory effect of 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide (PK11195), a prototypic ligand of the 18-kDa mBzR, which is one of the molecules associating with the PT pore complex [23, 24]. Here we show that PK11195, which itself is not toxic, facilitates the induction of apoptosis by a number of different stimuli. More importantly, we report that PK11195 can reverse apoptosis inhibition by Bcl-2. MATERIALS AND METHODS Cells and culture conditions. Thymocytes from 4- to 6-week-old Balb/c mice, 2B4.11 T cell hybridoma cell lines stably transfected with a SFFV.neo vector containing the human bcl-2 gene or the neomycin resistance gene (Neo) only [28] (kindly provided by J. Ashwell, NIH, Bethesda, MA), B cell leukemia WEHI231 transfected with the human bcl-2 gene or a control Neo vector [29] (gift from C. Martı´nez-A., National Center of Biotechnology, Madrid, Spain), or human CEM-C7.H2 T lymphoblastic leukemia cells [30] (gift from R. Kofler, University of Innsbruck, Austria) were cultured in RPMI 1640 supplemented with 10% FCS, antibiotics, and Lglutamine. Induction of apoptosis. Cells (5–10 3 105/ml) were cultured in the presence of the indicated amount of PK11195, diazepam (Sigma),

dexamethazone (1 mM, Sigma), RU24858 (1 mM, synthesized by Roussel Uclaf) [31], the glucocorticoid receptor antagonist RU38486 (1 mM, Roussel Uclaf), doxorubicin (1 mg/ml; Pharmacia), etoposide (10 mM/ml; Sigma), cyclosporin A (10 mM; Sandoz), or C8 ceramide (25 mM; Biomol, Plymouth Meeting, PA) or treated by gamma irradiation (10 Gy). After the indicated interval, cells were recovered and tested for apoptosis-associated features. Cytofluorometric quantitation of apoptosis-associated parameters in intact cells. Following published protocols [18, 32, 33], the following fluorochromes were employed to determine different apoptosis-associated changes: 3,39-dihexyloxacarbocyanine iodide (DiOC6(3), 20 nM) for DCm determination; hydroethidine (HE, 4 mM) for the determination of superoxide anion generation; or annexin V-FITC conjugates (1 mg/ml; Nexins Research, Hoeven, The Netherlands) for determination of phosphatidylserine (PS) exposure on the outer plasma membrane. The frequency of hypoploid cells was determined by propidium iodide (PI) staining of ethanol-permeabilized cells. Cell-free system of apoptosis. Mitochondria from 2B4.11 T cell hybridoma cell lines were purified on a Percoll (Pharmacia, Uppsala, Sweden) gradient [34] and were resuspended in CFS buffer: 220 mM mannitol; 68 mM sucrose, 2 mM NaCl, 2.5 mM PO4H2K, 0.5 mM EGTA, 2 mM MgCl2, 5 mM pyruvate, 0.1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM dithiotreitol, 10 mM Hepes–NaOH, pH 7.4, as described [7, 8]. Mitochondria from 5 3 106 cells were incubated in a total volume of 15 mm CFS buffer in the presence or absence of atractyloside (5 mM; Sigma), tert-butylhydroperoxide (t-BHP; 300 mM; Sigma), PK11195 (1 mM), cyclosporin A (10 mM), and/or bongkrekic acid (BA; 25 mM; kindly provided by Dr. Hans J. Duine, Delft University, Delft, The Netherlands) for 30 min at 4°C, followed by spinning down of mitochondria (5 3 103 g; 20 min) and recovery of the supernatant and ultracentrifugation (1.5 3 105 g, 1 h, 4°C). HeLa cell nuclei (103 nuclei/ml) purified on a sucrose gradient [35] were incubated for 90 min in the presence of mitochondrial supernatant, stained with PI (10 mg/ml; Sigma; minimum 5 min at room temperature), and analyzed in an Epics Profile II cytofluorometer (Coulter, Hialeah, FL) to assess the frequency of hypoploid nuclei, as described [36]. Experimental design and statistical analysis. All experiments were performed at least three times yielding similar results, and typical results are shown. Statistical significance was calculated using the paired Student’s t test.

RESULTS AND DISCUSSION

PK11195 facilitates the induction of apoptosis by a variety of stimuli. PK11195 is the prototypic ligand of the 18-kDa subunit of the mBzR [23, 37–39]. Up to doses of 50 to 100 mM, PK11195 had no toxic effects on a variety of cell types including thymocytes (Fig. 1), acute T cell leukemia CEM-C7 cells (Fig. 2), 2B4.11 T cell hybridoma cell (Fig. 3), and WEHI231 B cell leukemia cells (Fig. 4). As shown in Fig. 1, a dose of cell-permeable C8 ceramide which itself did not induce thymocyte apoptosis (25 mM) did become apoptogenic in the presence of the isoquinoline carboxamide PK11195. In contrast, the benzodiazepine diazepam, a molecule which binds to another subunit of the mBzR [23] or to another binding site within the same molecules [40], had no major coapoptogenic effect (Figs. 1, 3, and 4). Among the different mBzR ligands which we have tested, PK11195 was the most efficient coinducer of apoptosis (relative potency: PK11195 . 49-chlordiazepam $ diazepam . Ro-5-4864; not shown), corre-

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FIG. 1. Synergism between ceramide and PK11195 in the induction of thymocyte apoptosis. Thymocytes were cultured for 4 h in the presence of C8 ceramide (25 mM), diazepam (100 mM), and/or PK11195 (100 mM), followed by determination of several apoptosis-associated parameters, namely, disruption of the DCm (determined by means of DiOC6(3)), generation of superoxide anion (determined with HE) (A), phosphatidylserine exposure on the plasma membrane surface (measured with FITC-annexin V), and cell viability (EthBr exclusion) (B). Numbers refer to the percentage of cells found in each quadrant.

lating with its antagonist potential on neuromuscular transmission [41, 42]. Synergic effects with PK11195 extended to a variety of different apoptosis inducers including the topoisomerase II inhibitor etoposide (Fig. 2), gamma irradiation (Fig. 4), the intercalating agent doxorubicin (Fig. 4), and, in the case of WEHI231 cells, cyclosporin A (Fig. 4). The apoptosis-inducing effect of cyclosporin A is probably linked to its immunosuppressive function, because a nonimmunosuppressive cyclo-

sporine A derivative (which still binds and inhibits mitochondrial cyclophilin D) does not induce apoptosis, neither alone, nor in combination with PK11195 (Ref. [43] and data not shown). In addition, PK11195 (but not diazepam) facilitated the induction of apoptosis by glucocorticoid receptor agonists including dexamethasone (Fig. 2) and the prototypic ‘‘dissociated’’ glucocorticoid receptor agonist RU24858 (Fig. 2), an agent that has lost its transactivating function yet preserves its

FIG. 2. PK11195 facilitates apoptosis induction in CEM-C7 acute T cell leukemia cells. Cells were cultured with 10 mM etoposide, 1 mM dexamethasone (DEX), RU24858, RU38486, and/or 75 mM PK11195 during the indicated period, followed by determination of the frequency of DiOC63)low (HE3 Eth)low, DiOC6(3)low (HE3 Eth)high, and hypoploid cells as described under Materials and Methods. Asterisks indicate a significant (P ,0.01) enhancement of apoptosis induction by PK11195 as compared to control cultures kept in the absence of PK11195.

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FIG. 3. PK11195 reverses Bcl-2-mediated cytoprotection in T cell hybridoma cells. 2B4.11 T cell hybridoma cell lines stably transfected with a SFFV.neo vector containing the human bcl-2 gene (B) or the neomycin resistance gene (Neo) only (A) were cultured for 12 h in the presence of dexamethasone (1 mM), PK11195 (50 mM), and/or diazepam (50 mM), followed by determination of the indicated apoptosisassociated parameters. Numbers in black circles indicate the frequency of subdiploid cells. The synergistic effect of PK111951DEX was highly significative (P,0.01) compared to controls treated with DEX or PK11195 alone. Similar results are obtained when etoposide instead of dexamethasone is used as apoptosis inducer.

transrepressive gene-regulatory activity [31]. As to be expected, no effect of the glucocorticoid receptor antagonist RU38486 was revealed by PK11195 (Fig. 2). In conclusion, PK11195 facilitates the induction of apoptosis in response to a wide array of agents and in very different cell types, including primary cells (Fig. 1) and transformed cells lines of human and murine origin (Figs. 2– 4). PK11195 facilitates the induction of both mitochondrial and postmitochondrial changes associated with apoptosis. The synergism between PK11195 and several proapoptotic agents extended to all hallmarks of apoptosis, namely, the early loss of the mitochondrial transmembrane potential (measured by means of the potential-sensitive dye DiOC6(3)), the increase in the generation of reactive oxygen species (measured by the superoxide anion-driven conversion of hydroethidine into ethidium; Figs. 1– 4), the aberrant exposure of phosphatidylserine residues on the plasma membrane surface (measured by means of FITC–Annexin V conjugate; Fig. 1), and nuclear DNA fragmentation (hypoploidy determined by propidium iodine staining of ethanol-fixed cells; Figs. 2– 4). These findings are compatible with the idea that PK11195 acts on a mitochondrial target to facilitate apoptosis induction [19] and confirm the strong association between mitochondrial, redox, plasma membrane, and nuclear features of apoptosis.

PK11195 partially reverses Bcl-2-mediated apoptosis inhibition in several different cell lines. Bcl-2 has cytoprotective effects with a wide spectrum of activities [6, 43]. Thus, transfection enforced Bcl-2 overexpression largely prevented the DCm disruption, superoxide anion production, and nuclear apoptosis induced by dexamethasone in T cell hybridoma cells (Fig. 3). Again, the simultaneous treatment with PK11195 had a hyperadditive apoptosis-facilitating effect, even in the presence of Bcl-2. Thus, PK11195 restored apoptosis induction in Bcl-2-overexpressing cells at least partially. This effect has been observed in two different cell types, namely, 2B4.11 T cell hybridoma cells (Fig. 3B) and WEHI231 B cell leukemia cells (Fig. 4B). PK11195 largely reversed the Bcl-2-conferred protection against glucocorticoids (Fig. 3B), gamma irradiation, doxorubicin, cyclosporin A (Fig. 4B), and etoposide (not shown). This effect is again observed at the levels of the mitochondrion, cellular redox potentials, and the nucleus (Figs. 3 and 4). PK11195 reverses the Bcl-2-mediated block of apoptosis induction in a cell-free system. Bcl-2 stabilizes the mitochondrial membrane barrier function via a direct local effect [7, 8, 12–15]. Thus, as previously described [7, 8], isolated mitochondria from Bcl-2-overexpressing cells failed to release apoptogenic factors when they were incubated for 10 min in the presence of atractyloside, a ligand of the adenine nucleotide translocator [7]

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FIG. 4. PK11195 enhances apoptosis susceptibility in WEHI 231 B cell leukemia cells overexpressing Bcl-2. WEHI 231 cells transfected with a neomycin control vector (Neo, A) or the human bcl-2 gene (B) were treated with gamma irradiation, doxorubicine (doxo), or cyclosporin A (CsA), alone or in combination with PK11195 (40 mM) or diazepam (40 mM), followed by cytofluorometric determination of the indicated parameters. Asterisks indicate significant (P,0.001) effects of PK11195.

(Fig. 5A). Control mitochondria treated with atractyloside readily released apoptogenic factors, as quantified by an in vitro assay in which isolated nulei are induced to undergo DNA fragmentation and to become hypoploid (Fig. 5A). PK11195 alone had no effect on isolated mitochondria (Fig. 5) nor did if affect the structure of isolated nuclei (not shown). PK11195 did not enhance the effect of atractyloside in control mitochondria (which is probably already optimal, Ref. [7]) from Neotransfected cells. In strict contrast to the data obtained with mitochondria from Neo-transfected cells, the combination of PK11195 and atractyloside but neither of these agents alone induced the release of apoptogenic factors from mitochondria purified from Bcl-2-transfected cells. Together, PK11195 and atractyloside were equally efficient in releasing apoptogenic factors from Bcl-2-over-

expressing and control mitochondria (Fig. 5A). Thus, PK11195 can overcome the resistance of Bcl-2-overexpressing mitochondria to the release of apoptogenic factors induced by atractyloside (Fig. 5A) or the prooxidant tert-butylhydroperoxide (Fig. 5B). Atractyloside and tert-butylhydroperoxide act on mitochondria to open the PT pore [1], and this effect is thought to account for the release of apoptogenic factors [7]. To understand the mechanism of the PK11195-facilitated liberation of apoptogenic factors, we therefore determined the effect of two inhibitors of the PT pore, cyclosporin A and bongkrekic acid (Fig. 5b). Either of these two agents abolishes the effect of PK111951atractyloside or that of PK111951t-BHP on mitochondria from Bcl-2-overexpressing cells (Fig. 5B). Altogether, these results indicate that (i) PK11195 neutralizes the capacity of

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FIG. 5. PK11195 overcomes the Bcl-2-mediated mitochondrial retention of apoptogenic factors. (A) Effect of PK11195 and Atr on mitochondria from 2B4.11 T cell hybridoma cells expressing the human bcl-2 gene or the control vector (Neo). Isolated mitochondria were incubated for 30 min in the presence of atractyloside (5 mM) and/or PK11195 (1 mM). The supernatant of mitochondria was then added to purified HeLa nuclei. After a culture period of 90 min the percentage of hypoloid nuclei was determined by PI staining and cytofluorometric analysis. The background value of isolated nuclei cultured in the absence of mitochondrial supernatants was 22 6 2%. (B) Effect of different agents on mitochondria purified from Bcl-2-overexpressing cells. Isolated mitochondria were incubated with the indicated combination of atractyloside (Atr), tert-butylhydroperoxide (t-BHP), PK11195, cyclosporin A (CsA), and/or bongkrekic acid (BA). Inhibitors (CsA, BA) were added 5 min before inducers (Atr, t-BHP, PK11195). The apoptogenic activity contained in the supernatants of mitochondria was then tested on purified HeLa nuclei as in A. Background values were subtracted from experimental values.

Bcl-2 to retain apoptogenic factors in isolated mitochondria and (ii) it acts on PT pores to facilitate apoptosis induction. Concluding remarks. In this work we describe a novel strategy for enhancing the susceptibility of cells to the induction of apoptosis. PK11195, a specific ligand of the 18-kDa mBzR, facilitated the induction of DCm disruption by a variety of different apoptosis triggers, including DNA damage (gamma irradiation, etoposide, doxorubicin), ligation of the glucocorticoid receptor (dexamethasone, RU24858), and the proapoptotic second messenger ceramide. Concomitantly, it enhanced the induction of classical signs of apoptosis such as phosphatidylserine exposure on the cell surface and nuclear DNA fragmentation. The mBzR has been previously shown to interact with a number of proteins involved in

the formation and/or regulation of the PT pore complex [23, 24]. Moreover, the PK11195 has been shown to facilitate PT pore opening induced by tumor necrosis factor (TNF-a) in L929 cells [44]. In that model, TNF induces necrosis [45], and necrosis induction is enhanced by PK11195 [44]. In contrast, we show here that PK11195 can facilitate the induction of apoptosis, correlating with the induction of PT pore-mediated DCm dissipation. These findings add to the notion that PT pore opening may be a rate-limiting step of any type of cell death (apoptosis and necrosis) [5] and that postmitochondrial events culminating in the activation of catabolic enzymes (caspases, noncaspase proteases, nucleases) and/or the availability of extramitochondrial ATP sources dictate the modality of cell death [21, 46, 47]. Bcl-2 is the prototypic representative of an apopto-

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sis-inhibitory oncogene family which contributes both to the genesis of cancer and to the difficulties in treating it. Most of the cytoprotective effect of Bcl-2 may be attributed to its capacity to protect the integrity of mitochondrial membranes [6 – 8, 12, 13, 43, 48, 49]. We have shown that Bcl-2 stabilizes mitochondrial membranes in a variety of different models of apoptosis [32, 43]. However, it appears that Bcl-2 fails to protect isolated mitochondria against thiol-crosslinking by diamide [7, 27] and the proteolytic attack mediated by caspases [14], correlating with the fact that Bcl-2 does not inhibit apoptosis induced by diamide [7, 27] and direct caspase activation [14, 50 –52]. The Bcl-2 effect is also overcome by treatment with paclitaxel (Taxol), an agent which causes hyperphosphorylation of Bcl-2 [53, 54] and favors opening of the PT pore [55]. Here we demonstrate another possibility to overcome Bcl-2-mediated chemo- or radioresistance. Treatment of cells with PK11195, a ligand of the mBzR, largely reversed cytoprotection mediated by Bcl-2. A quasi-stoichiometric relationship between the expression of Bcl-2 and that of the mBzR, at least in lymphoid cell lines, has been reported [56]. Moreover, Bcl-2 protects isolated mitochondria against opening of the PT pore induced by low doses of protoporphyrin IX, a ligand of the MBzR, and this inhibition is overcome by high doses of protoporphyrin IX [19], suggesting a functional interaction between Bcl-2 and the mBzR. In contrast, the Bcl-2-mediated protection against PT pore opening is not overcome by increasing the doses of other PT poretargeted agents such as atractyloside, a ligand of the adenine nucleotide translocator [7, 8]. In contrast to protoporphyrin IX, PK11195 itself has no PT-inducing effect and fails to release apoptogenic factors from control or Bcl-2-overexpressing mitochondria. However, it does act in synergy with atractyloside or the prooxidant t-BHP to overcome the Bcl-2-mediated retention of apoptogenic factors in isolated mitochondria, and this effect is blocked by two inhibitors of the PT pore, bongkrekic acid and cyclosporin A (Fig. 5). Since ligation of the mBzR does not cause a downregulation of Bcl-2 expression (Ref. [56] and data not shown), it thus appears probable that specific PK11195-driven conformational changes in the complex composed by the mBzR and the PT pore [1, 22–25] indirectly affect the mitochondrial membrane-stabilizing and antiapoptic function of Bcl-2. In this context, it appears intriguing that the purified PT pore is under the direct regulatory influence of proteins of the Bcl-2 family [26, 27]. Thus, the components of the PT pore, the mBzR, and the Bcl-2/Bax complex could form higher order molecular complexes which regulate the permeability of the inner and/or outer mitochondrial membranes. This scenario would explain how Bcl-2 can inhibit the atractyloside-induced release of apoptogenic factors and how PK11195 can neutralize this

inhibitory effect. Future studies addressing the putative physical interaction between the mBzR, the proteins composing the PT pore, and Bcl-2-related proteins should elucidate the mechanism via which mBzR and Bcl-2 exert antagonistic effects on mitochondrial membrane integrity. In conclusion, our present data suggest a novel strategy for enhancing the susceptibility of cells to apoptosis induction, namely, ligation of the mBzR. Ligation of the mBzR with PK11195 may be a valuable strategy for reversing chemoresistance mediated by Bcl-2. This work has been supported by grants from ANRS, ARC, CNRS, FRM, INSERM, LFC, and Hoechst-Marion-Roussel (to G.K.). We thank Dr. Pierre Carayon (Sanofi-Elf, Montpellier, France) for helpful suggestions.

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