CDDO induces apoptosis via the intrinsic pathway in ... - Nature

Leukemia (2004) 18, 948–952 & 2004 Nature Publishing Group All rights reserved 0887-6924/04 $25.00 www.nature.com/leu

CDDO induces apoptosis via the intrinsic pathway in lymphoid cells S Inoue1, RT Snowden1, MJS Dyer1,2 and GM Cohen1 1

MRC Toxicology Unit, University of Leicester, Leicester, UK; and 2Department of Hematology, University of Leicester, Leicester Royal Infirmary, Leicester, UK

The peroxisome-proliferator-activated receptor (PPAR) c agonist, CDDO, is under investigation for use in various malignancies. The mechanisms by which CDDO induces apoptosis are controversial. We have therefore sought to determine these mechanisms using primary chronic lymphocyte leukemic (CLL) cells and Jurkat cell lines with defined apoptotic abnormalities. In these cells, CDDO induced-apoptosis involved caspaseindependent loss in mitochondrial membrane potential followed by caspase processing. The pattern of CDDO-induced caspase processing, defined by use of a caspase inhibitor, strongly suggested that caspase-9 was the apical caspase. Moreover, CDDO induced apoptosis in caspase-8 and FADDdeficient but not in Bcl-xL overexpressing Jurkat cells. In CLL cells, CDDO induced an early release of mitochondrial cytochrome c and Smac that preceded apoptosis. Thus, in both cell types, CDDO induced apoptosis primarily by the intrinsic pathway with caspase-9 as the apical caspase. This has important implications in the design of novel agents for the treatment of CLL and other malignancies. Leukemia (2004) 18, 948–952. doi:10.1038/sj.leu.2403328 Published online 26 February 2004 Keywords: chronic lymphocytic leukemia (CLL); apoptosis; CDDO; intrinsic pathway

Introduction Apoptosis is induced by two distinct cell death pathways, involving either perturbation of mitochondria, resulting in release of proapoptotic mediators, such as cytochrome c, Smac/DIABLO and Omi/HtrA2, or stimulation of cell surface death receptors, often referred to as the intrinsic or extrinsic pathway, respectively.1,2 The release of cytochrome c results in the formation of the Apaf-1 apoptosome, which recruits and activates caspase-9 as the apical caspase, which then activates caspase-3 and -7.3–5 Stimulation of death receptors by CD95 (Fas) or TRAIL (TNF-related apoptosis inducing ligand) results in receptor aggregation and recruitment of FADD/MORT1 (Fasassociated death-domain) and activation of caspase-8 as the apical caspase.1,6 Peroxisome-proliferator-activated receptors (PPARs) (a, d and g) induce expression of genes critical to diabetes, obesity, inflammation and cancer.7,8 PPARg ligands, such as the triterpenoid, 2cyano-3,12-dioxoolean-1,9-dien-28-oic acid (CDDO), have been synthesized as potential anticancer agents partly due to their ability to inhibit cell proliferation and induce apoptosis in many cancer cell lines.8–10 CDDO is reported to induce apoptosis by the extrinsic pathway in human myeloid leukemic, osteosarcoma and CLL cells11–13 and by the intrinsic pathway in lung cancer cells.14 As CDDO and derivatives are being developed for the treatment of various cancers including hematological malignanCorrespondence: GM Cohen, MRC Toxicology Unit, Hodgkin Building, University of Leicester, PO Box 138, Lancaster Road, Leicester LE1 9HN, UK; Fax: þ 44 116 2525616; E-mail: [email protected] Received 11 December 2003; accepted 29 January 2004; Published online 26 February 2004

cies, it is essential to understand how they induce apoptosis. We demonstrate that CDDO induces apoptosis primarily by the intrinsic pathway in CLL and Jurkat cells.

Materials and methods Peripheral blood samples from CLL patients, obtained after informed consent and with local ethical committee approval, were purified as previously described15 except that T cells were removed using anti-CD3 Dynabeads (Dynal, Merseyside, UK). CLL cell purity was assessed by staining with anti-CD19/FITC and anti-CD5/RPE antibodies (Dako, Cambridge, UK) and analyzed by flow cytometry. This method produced an B95% pure population of cells expressing both CD19 and CD5. The purified CLL cells were resuspended in RPMI 1640 medium (4 106/ml) in six or 24-well plates at 371C in an atmosphere of 5% CO2 and incubated for the indicated times with CDDO (5 mM) or the proteasome inhibitor, MG132 (1 mM) as described.15,16 Wild-type, caspase-8-deficient, FADD-deficient and Bcl-xL stably transfected Jurkat cells were cultured as described.17,18 CLL or Jurkat cells were pre-treated for 1 h with benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethyl ketone (ZVAD.fmk) (Enzyme Systems, CA, USA), a caspase inhibitor. Apoptosis was quantified by phosphatidylserine (PS) externalization or loss of mitochondrial membrane potential (cm) in the presence of propidium iodide (PI) as previously described.19,20 CDDO was from Glaxo Smith Kline (Stevenage, UK). Cell samples were prepared and proteins detected by Western blotting.18 The caspase-9 antibody was from MBL (Nagoya, Japan). After treatment, cells were pelleted, washed with icecold 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 protease inhibitors (Roche, 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.20

Results and discussion

CDDO activates the intrinsic pathway in Jurkat cells To investigate CDDO-induced apoptosis in Jurkat cells, we compared it with etoposide, a DNA topoisomerase II inhibitor, and TRAIL, which induce apoptosis by the intrinsic and extrinsic pathways, respectively.19 CDDO, etoposide and TRAIL induced apoptosis, assessed by caspase processing, PS externalization and decreases in cm (Figure 1). Caspase-3 was processed to its active p19 and p17 forms and caspase-8 to its p43 and p41 forms (Figure 1, lanes 5–7, 11 and 13). Z-VAD.fmk inhibited CDDO- and etoposide-induced PS externalization but not their induction of a decrease in cm (Figure 1, lanes 8–10 and 12). These results demonstrated that both CDDO and etoposide induced an initial mitochondrial perturbation. Z-VAD.fmk

CDDO induces apoptosis via the intrinsic pathway S Inoue et al

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Figure 1 CDDO induces apoptosis in Jurkat cells. Wild-type Jurkat cells were cultured for up to 42 h either alone (lane 1) or in the presence of CDDO (1 mM) (lanes 5–7). Cells were also cultured for up to 42 h with Z-VAD.fmk (25 mM) either alone (lanes 2–4) or with CDDO (lanes 8–10) as indicated. Jurkat cells were also cultured for 6 h with either etoposide (50 mM) or TRAIL (1 mg/ml) in the presence or absence of Z-VAD.fmk (25 mM) as indicated. Cells were then analyzed by immunoblotting as described in Materials and methods for the processing of caspase-3 and -8. The pro-forms and processed forms of the caspases are indicated. Apoptosis was assessed by PS externalization or by the percentage of cells with low mitochondrial membrane potential (cm) in the presence of PI as described in Materials and methods. The values shown are the mean values from three different experiments.

completely inhibited both CDDO-, etoposide- and TRAILinduced caspase-8 processing and TRAIL-induced caspase-3 and -8 processing (Figure 1, lanes 8–10, 12 and 14). In TRAILinduced apoptosis, Z-VAD.fmk inhibits caspase-8 processing at the death inducing signaling complex (DISC),21 so inhibiting processing of all downstream caspases including caspase-3. In marked contrast, Z-VAD.fmk did not prevent CDDO- and etoposide-induced formation of the p20 form of caspase-3 (Figure 1, lanes 8–10 and 12), suggesting that CDDO initially activates caspase-9, which cleaves caspase-3 at Asp175 between the large and small subunits forming the p20 fragment. Normally, the autocatalytic activity of caspase-3 removes the Nterminal prodomain to form the p19/p17 fragments, but this is blocked by Z-VAD.fmk so the p20 fragment predominates. Thus with CDDO and etoposide, our data are compatible with active caspase-3, activating caspase-6, which then activates caspase83,18,22 and that the inhibition of caspase-3 by Z-VAD.fmk blocks processing of all downstream caspases including caspase-8. Further evidence that caspase-3 was activated prior to caspase-8 was provided by the earlier appearance of processed subunits of cleaved caspase-3 compared to caspase8 (Figure 1, compare lanes 5 and 6), although this difference may be partly due to differing sensitivity of antibodies. Taken together these results support the hypothesis that in Jurkat cells CDDO induces apoptosis primarily by the intrinsic pathway. If as proposed by others CDDO induces apoptosis by the extrinsic pathway, then it should not induce apoptosis in caspase-8-deficient Jurkat cells. Initially, we determined the sensitivity of caspase-8-deficient Jurkat cells to both etoposide and TRAIL. TRAIL, which induces apoptosis by activation of the extrinsic pathway with caspase-8 as the apical caspase, did not induce apoptosis assessed by processing of caspase-3, PS

Figure 2 CDDO induces apoptosis in caspase-8-deficient Jurkat cells. Caspase-8-deficient Jurkat cells were cultured for up to 42 h either alone (lane 1) or in the presence of CDDO (1 mM) (lanes 5–7) in the presence or absence of Z-VAD.fmk (25 mM) as indicated and described in the legend to Figure 1. Caspase-8-deficient Jurkat cells were also cultured for 6 h with either etoposide (50 mM) or TRAIL (1 mg/ ml). Cells were then analyzed by immunoblotting and apoptosis assessed as described in the legend to Figure 1. The values shown are the mean values from three different experiments.

externalization or an increase in the percentage of cells with low cm (Figure 2, lane 13), whereas etoposide induced apoptosis by all these criteria (Figure 2, lane 11). Thus, the caspase-8-deficient cells were susceptible to apoptosis induced by the intrinsic but not the extrinsic pathway. Exposure of the caspase-8-deficient Jurkat cells to CDDO resulted in the timedependent induction of apoptosis as assessed by processing of caspase-3, PS externalization and a decrease in cm (Figure 2, lanes 5–7). In the presence of CDDO alone, caspase-3 was processed primarily to its p19 and p17 forms, whereas in the presence of Z-VAD.fmk it was processed only to its p20 form as the autocatalytic removal of its prodomain was inhibited (Figure 2, lanes 8–10). Similarly etoposide-induced caspase-3 processing was inhibited at its p20 form in the presence of ZVAD.fmk (Figure 2, lane 12). These data demonstrate that caspase-8 is not required for CDDO-induced apoptosis and that CDDO may induce apoptosis by the intrinsic pathway. In order to further demonstrate the ability of CDDO to induce apoptosis by the intrinsic pathway, we used FADD-deficient Jurkat cells, FADD being required for the induction of apoptosis by the extrinsic but not the intrinsic pathway. TRAIL did not induce apoptosis in FADD-deficient Jurkat cells, as assessed by processing of caspase-8 or -3, PS externalization or a decrease in cm (Figure 3, lane 13), whereas etoposide induced apoptosis by all these criteria (Figure 3, lane 11). Thus, the FADD-deficient cells were susceptible to apoptosis induced by the intrinsic but not the extrinsic pathway. Exposure of the FADD-deficient Jurkat cells to CDDO resulted in the time-dependent induction of apoptosis as assessed by processing of caspase-3 and -8, PS externalization and a decrease in cm (Figure 3, lanes 5–7). However, less processing of caspase-8 was observed in the FADD-deficient cells compared to the wild-type Jurkat cells. The effects of Z-VAD.fmk were again very similar to those observed in wild-type Jurkat cells, in particular the inhibition of CDDOand etoposide-induced caspase-3 processing at its p20 form (Figure 3, lanes 9–10 and 12). These data further demonstrate that CDDO can induce apoptosis primarily by the intrinsic pathway but cannot totally exclude some contribution from the extrinsic pathway. Leukemia


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Figure 3 CDDO induces apoptosis in FADD-deficient Jurkat cells. FADD-deficient Jurkat cells were cultured for up to 42 h either alone (lane 1) or in the presence of CDDO (1 mM) (lanes 5–7) in the presence or absence of Z-VAD.fmk (25 mM) as indicated and described in the legend to Figure 1. FADD-deficient Jurkat cells were also cultured for 6 h with either etoposide (50 mM) or TRAIL (1 mg/ml). Cells were then analyzed by immunoblotting and apoptosis assessed as described in the legend to Figure 1. The values shown are the mean values from three different experiments.

If CDDO induces apoptosis solely by the intrinsic pathway, then antiapoptotic Bcl-2 family members should inhibit CDDOinduced apoptosis. Etoposide-induced apoptosis as assessed by PS externalization and a decrease in cm as well as processing of caspase-3 and -8 was completely inhibited in Jurkat cells overexpressing Bcl-xL (Figure 4, lane 11). Similarly, CDDOinduced apoptosis assessed by all these criteria was largely inhibited in Jurkat cells overexpressing Bcl-xL, although a small increase was observed in the percentage of cells with a decreased cm particularly after 42 h (Figure 4, lanes 5–7). This small increase in the percentage of cells with a low cm is compatible with CDDO inducing an initial perturbation of mitochondria, which would lead to the release of proapoptotic mediators from the mitochondria so resulting in a marked amplification of the apoptotic stimulus.2 However, as this release is blocked in cells overexpressing Bcl-xL, this results in an inhibition of CDDO-induced apoptosis. In marked contrast, TRAIL induced apoptosis in Jurkat cells overexpressing Bcl-xL as assessed by an increase in PS externalization, a decrease in cm and processing of caspase-3 and -8 (Figure 4, lane 13). With TRAIL, a distinctive p20 form of caspase-3 was observed, similar to that seen in death receptor-induced apoptosis in cells overexpressing Bcl-2 or Bcl-xL (Figure 4, lane 13).18 Some of this p20/p12 form is inactive because it is inhibited by endogenous X-linked inhibitor-of-apoptosis protein (XIAP).18 Z-VAD.fmk largely inhibited TRAIL-induced processing of caspase-3 and -8 (Figure 4, lane 14). Taken together, these results strongly support the hypothesis that CDDO induces apoptosis in Jurkat cells primarily by the intrinsic pathway.

CDDO activates the intrinsic pathway in CLL cells CDDO is of potential benefit in the treatment of CLL, as it induces apoptosis in refractory CLL cells and sensitizes some CLL cells to TRAIL.13 To determine whether the discrepancy with regard to activation of the intrinsic or extrinsic pathway was due to cell-type specificity, we further examined the Leukemia

Figure 4 CDDO induces apoptosis in Bcl-xL overexpressing Jurkat cells. Bcl-xL over-expressing Jurkat cells were cultured for up to 42 h either alone (lane 1) or in the presence of CDDO (1 mM) (lanes 5–7) in the presence or absence of Z-VAD.fmk (25 mM) as indicated and described in the legend to Figure 1. Bcl-xL over-expressing Jurkat cells were also cultured for 6 h with either etoposide (50 mM) or TRAIL (1 mg/ ml). Cells were then analyzed by immunoblotting and apoptosis assessed as described in the legend to Figure 1. The values shown are the mean values from three different experiments.

mechanism of CDDO-induced apoptosis in CLL cells and compared it with MG132, which activates the intrinsic pathway as evidenced by its ability to induce the translocation of Bax, release of cytochrome c, and formation of an B700 kDa Apaf-1 apoptosome.15,20 In agreement with our previous results, MG132 induced apoptosis in CLL cells as evidenced by the increase in PS externalization, the decrease in cm and the processing of caspase-3, -8 and -9 (Figure 5, lane 7). Pretreatment with Z-VAD.fmk largely inhibited the increase in PS externalization but not the decrease in cm (Figure 5, lane 8) supporting the suggestion that MG132 initially perturbs mitochondria. CDDO caused a time-dependent induction of apoptosis in CLL cells as evidenced by the increase in PS externalization, the decrease in cm and the processing of caspase-3, -8 and -9 (Figure 5, lanes 3–5). Z-VAD.fmk inhibited the CDDO- and MG132-induced processing of caspase-3 primarily at the p20 form (Figure 5, lanes 6 and 8) similar to that observed in Jurkat cells (Figure 1, lane 8–10 and 12). ZVAD.fmk completely inhibited the CDDO- and MG132induced processing of caspase-8 (Figure 5, lanes 6 and 8). CDDO also induced the processing of caspase-9 to its p35 and p37 fragments and an uncharacterized B25 kDa product, which were only partially inhibited by Z-VAD.fmk (Figure 5, lanes 3– 6), suggesting that caspase-9 was still active. Processing of caspase-9 at Asp 315 and Asp 330 yielding the p35 and p37 fragments is mediated by Apaf-1 and caspase-3, respectively, and result in an amplification loop.23,24 Inhibition of caspase-3 by Z-VAD.fmk inhibits this amplification loop and alters caspase-9 processing. Taken together these results strongly support the hypothesis that CDDO causes an initial perturbation of mitochondria in CLL cells, which results in the release of proapoptotic mediators from the mitochondria and the subsequent activation of caspases with caspase-9 as the apical caspase.1,3,5 In order to confirm this hypothesis, we investigated the release from CLL cells exposed to CDDO of two such proapoptotic


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Figure 5 CDDO activates the intrinsic pathway in CLL cells. Purified CLL cells were cultured for 7.5 h either alone (lane 1) or in the presence of Z-VAD.fmk (200 mM) (lane 2). Cells were also cultured for the indicated times with CDDO (5 mM) either alone (lanes 3–5) or in the presence of Z-VAD.fmk (200 mM) (lane 6). Cells were also cultured with MG132 (1 mM) in the presence or absence of Z-VAD.fmk (200 mM) (lanes 7–8). Cells were then analyzed by immunoblotting for the processing of caspase-3, -8 and -9 as described in Materials and methods. The pro-forms and processed forms of the caspases are indicated. An uncharacterized B25 kDa processed fragment of caspase-9 was detected. Equal protein loading was observed using tubulin as a loading control (data not shown). Apoptosis was assessed by measurement of PS externalization or loss in mitochondrial membrane potential (cm) in the presence of PI as described in Materials and methods. The values shown are the mean values from five different patients.

Figure 6 CDDO induces the mitochondrial release of cytochrome c and Smac in CLL cells. CLL cells were purified and treated as described in the legend to Figure 5. Cytosolic and mitochondrial membrane fractions were obtained as described in Materials and methods, resolved by SDS-PAGE and immunoblotted for cytochrome c or Smac.

molecules from mitochondria, namely cytochrome c and Smac. CDDO induced a time-dependent release of both cytochrome c and Smac from mitochondria, which preceded or accompanied the induction of apoptosis (Figure 6, lanes 2–4). Both the CDDO- and MG132-induced release of cytochrome c and Smac were not inhibited by Z-VAD.fmk (Figure 6, lanes 5 and 7). These results further support our suggestion that CDDO induces the activation of the intrinsic pathway in CLL cells.

It is unclear why our studies have demonstrated that CDDOinduced apoptosis by the intrinsic pathway, whereas others proposed that it used the extrinsic pathway.11–13 Although these differences may be partly due to cell-type specificity they may also result from the methods used. In some studies, caspase activity was assessed using fluorometric substrates, not necessarily specific for the intended caspase.11,12 The conclusion that CDDO activated the extrinsic pathway in CLL cells was dependent on data obtained by electroporation into CLL cells, which may have inadvertently introduced experimental artefacts.13 Although CDDO is a weak PPARg agonist,8,9,25 its ability to induce apoptosis is independent of this activity in both CLL and Jurkat cells.13,26 In CLL cells, troglitazone, a potent PPARg agonist failed to induce apoptosis, whereas potent PPARg agonists induce apoptosis in PPARg-negative Jurkat cells.13,26 Although our data that CDDO induces apoptosis by the intrinsic pathway in CLL and Jurkat cells is compelling, we cannot totally exclude the involvement of the extrinsic pathway in some cells under certain conditions. This new understanding of the mechanism of CDDO-induced apoptosis is important in the design of more potent analogs, which should be of value in the treatment of some hematological malignancies.

Acknowledgements We thank Dr J Blenis (Harvard Medical School, Boston, MA, USA) for supplying the FADD- and caspase-8-deficient cells. Dr Satoshi Inoue is partially supported by the Daiwa Anglo-Japanese Foundation.

References 1 Bratton SB, MacFarlane M, Cain K, Cohen GM. Protein complexes activate distinct caspase cascades in death receptor and stressinduced apoptosis. Exp Cell Res 2000; 256: 27–33. 2 van Loo G, Saelens X, van Gurp M, MacFarlane M, Martin SJ, Vandenabeele P. The role of mitochondrial factors in apoptosis: a Russian roulette with more than one bullet. Cell Death Differ 2002; 9: 1031–1042. 3 Li P, Nijhawan D, Budihardjo I, Srinivasula SM, Ahmad M, Alnemri ES et al. Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell 1997; 91: 479–489. 4 Cain K, Brown DG, Langlais C, Cohen GM. Caspase activation involves the formation of the aposome, a large (approximately 700 kDa) caspase-activating complex. J Biol Chem 1999; 274: 22686–22692. 5 Bratton SB, Walker G, Srinivasula SM, Sun XM, Butterworth M, Alnemri ES et al. Recruitment, activation and retention of caspases9 and -3 by Apaf-1 apoptosome and associated XIAP complexes. EMBO J 2001; 20: 998–1009. 6 Ashkenazi A, Dixit VM. Death receptors: signaling and modulation. Science 1998; 281: 1305–1308. 7 Kersten S, Desvergne B, Wahli W. Roles of PPARs in health and disease. Nature 2000; 405: 421–424. 8 Murphy GJ, Holder JC. PPAR-gamma agonists: therapeutic role in diabetes, inflammation and cancer. Trends Pharmacol Sci 2000; 21: 469–474. 9 Willson TM, Lambert MH, Kliewer SA. Peroxisome proliferatoractivated receptor gamma and metabolic disease. Annu Rev Biochem 2001; 70: 341–367. 10 Suh N, Wang Y, Honda T, Gribble GW, Dmitrovsky E, Hickey WF et al. A novel synthetic oleanane triterpenoid, 2-cyano-3,12dioxoolean-1,9-dien-28-oic acid, with potent differentiating, antiproliferative, and anti-inflammatory activity. Cancer Res 1999; 59: 336–341. 11 Ito Y, Pandey P, Place A, Sporn MB, Gribble GW, Honda T et al. The novel triterpenoid 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid induces apoptosis of human myeloid leukemia cells by a Leukemia


952 12

13 14

15

16

17

18

Leukemia

caspase-8-dependent mechanism. Cell Growth Differ 2000; 11: 261–267. Ito Y, Pandey P, Sporn MB, Datta R, Kharbanda S, Kufe D. The novel triterpenoid CDDO induces apoptosis and differentiation of human osteosarcoma cells by a caspase-8 dependent mechanism. Mol Pharmacol 2001; 59: 1094–1099. Pedersen IM, Kitada S, Schimmer A, Kim Y, Zapata JM, Charboneau L et al. The triterpenoid CDDO induces apoptosis in refractory CLL B cells. Blood 2002; 100: 2965–2972. Kim KB, Lotan R, Yue P, Sporn MB, Suh N, Gribble GW et al. Identification of a novel synthetic triterpenoid, methyl-2-cyano3,12-dioxooleana-1,9-dien-28-oate, that potently induces caspasemediated apoptosis in human lung cancer cells. Mol Cancer Ther 2002; 1: 177–184. Almond JB, Snowden RT, Hunter A, Dinsdale D, Cain K, Cohen GM. Proteasome inhibitor-induced apoptosis of B-chronic lymphocytic leukaemia cells involves cytochrome c release and caspase activation, accompanied by formation of an approximately 700 kDa Apaf-1 containing apoptosome complex. Leukemia 2001; 15: 1388–1397. MacFarlane M, Harper N, Snowden RT, Dyer MJ, Barnett GA, Pringle JH et al. Mechanisms of resistance to TRAIL-induced apoptosis in primary B cell chronic lymphocytic leukaemia. Oncogene 2002; 21: 6809–6818. Harper N, Hughes M, MacFarlane M, Cohen GM. Fas-associated death domain protein and caspase-8 are not recruited to the tumor necrosis factor receptor 1 signaling complex during tumor necrosis factor-induced apoptosis. J Biol Chem 2003; 278: 25534–25541. Sun XM, Bratton SB, Butterworth M, MacFarlane M, Cohen GM. Bcl-2 and Bcl-xL inhibit CD95-mediated apoptosis by preventing mitochondrial release of Smac/DIABLO and subsequent inactivation of X-linked inhibitor-of-apoptosis protein. J Biol Chem 2002; 277: 11345–11351.

19 Sun XM, MacFarlane M, Zhuang J, Wolf BB, Green DR, Cohen GM. Distinct caspase cascades are initiated in receptor-mediated and chemical-induced apoptosis. J Biol Chem 1999; 274: 5053–5060. 20 Dewson G, Snowden RT, Almond JB, Dyer MJ, Cohen GM. Conformational change and mitochondrial translocation of Bax accompany proteasome inhibitor-induced apoptosis of chronic lymphocytic leukemic cells. Oncogene 2003; 22: 2643–2654. 21 Medema JP, Scaffidi C, Kischkel FC, Shevchenko A, Mann M, Krammer PH et al. FLICE is activated by association with the CD95 death-inducing signaling complex (DISC). EMBO J 1997; 16: 2794–2804. 22 Slee EA, Harte MT, Kluck RM, Wolf BB, Casiano CA, Newmeyer DD et al. Ordering the cytochrome c-initiated caspase cascade: hierarchical activation of caspases-2, -3, -6, -7, -8, and -10 in a caspase-9-dependent manner. J Cell Biol 1999; 144: 281–292. 23 Bratton SB, Lewis J, Butterworth M, Duckett CS, Cohen GM. XIAP inhibition of caspase-3 preserves its association with the Apaf-1 apoptosome and prevents CD95- and Bax-induced apoptosis. Cell Death Differ 2002; 9: 881–892. 24 Srinivasula SM, Ahmad M, Fernandes-Alnemri T, Alnemri ES. Autoactivation of procaspase-9 by Apaf-1-mediated oligomerization. Mol Cell 1998; 1: 949–957. 25 Suh N, Wang Y, Williams CR, Risingsong R, Gilmer T, Willson TM et al. A new ligand for the peroxisome proliferator-activated receptor-gamma (PPAR-gamma), GW7845, inhibits rat mammary carcinogenesis. Cancer Res 1999; 59: 5671–5673. 26 Fehlberg S, Trautwein S, Goke A, Goke R. Bisphenol A diglycidyl ether induces apoptosis in tumour cells independently of peroxisome proliferator-activated receptor-gamma, in caspasedependent and -independent manners. Biochem J 2002; 362: 573–578.