Inhibition of TRAIL-Induced Apoptosis by Bcl-2 Overexpression

Oncogene (2002) 21, 2283 ± 2294 ã 2002 Nature Publishing Group All rights reserved 0950 ± 9232/02 $25.00 www.nature.com/onc ORIGINAL PAPERS Inhibition of TRAIL-induced apoptosis by Bcl-2 overexpression

Simone Fulda1, Eric Meyer1 and Klaus-Michael Debatin*,1

1University Children's Hospital, Prittwitzstr. 43, D-89075 Ulm, Germany

Primary or acquired resistance to current treatment toxic drug treatment, g-irradiation or cytotoxic cytokines protocols remains a major concern in clinical oncology (Peter and Krammer, 1998; Schulze-Ostho et al., 1998; and may be caused by defects in apoptosis programs. Kaufmann and Earnshaw, 2000; Debatin, 1997; Fulda et Since recent data suggest that TRAIL can bypass al., 1997, 1998b). Apoptosis pathways may be initiated apoptosis resistance caused by Bcl-2, we further through dierent entry sites, such as death receptors investigated the role of Bcl-2 in TRAIL-induced (receptor pathway) or mitochondria (mitochondrial apoptosis. Here we report that overexpression of Bcl-2 pathway) resulting in activation of eector caspases conferred protection against TRAIL in neuroblastoma, (Peter and Krammer, 1998). Stimulation of death glioblastoma or breast carcinoma cell lines. Bcl-2 receptors of the tumor necrosis factor (TNF) receptor overexpression reduced TRAIL-induced cleavage of superfamily such as CD95 (APO-1/Fas) results in caspase-8 and Bid indicating that caspase-8 was receptor aggregation and recruitment of the adaptor activated upstream and also downstream of mitochondria molecule Fas-associated death domain (FADD) and in a feedback ampli®cation loop. Importantly, Bcl-2 caspase-8 (Medema et al., 1997). Upon recruitment blocked cleavage of caspases-9, -7 and -3 into active caspase-8 becomes activated and initiates apoptosis by subunits and cleavage of the caspase substrates DFF45 direct cleavage of downstream eector caspases (Peter or PARP. Also, Bcl-2 blocked cleavage of XIAP and and Krammer, 1998). A second pathway is initiated at overexpression of XIAP conferred resistance against the mitochondrial level (Kroemer and Reed, 2000). TRAIL indicating that apoptosis was also ampli®ed Apoptogenic factors such as cytochrome c, apoptosis through a feedforward loop between caspases and XIAP. inducing factor (AIF), caspase-2 or caspase-9 are In contrast, in SKW lymphoblastoid cells, TRAIL- released from mitochondria into the cytosol triggering induced activation of caspase-8 directly translated into caspase-3 activation through formation of the cyto- full activation of caspases, cleavage of XIAP, DFF45 or chrome c/Apaf-1/caspase-9-containing apoptosome PARP and apoptosis independent of Bcl-2 overexpres- complex (Kroemer and Reed, 2000; Susin et al., 1999; sion, although Bcl-2 similarly inhibited loss of mitochon- Hengartner, 2000). In these cells, caspase-8 is activated drial membrane potential and the release of cytochrome downstream of mitochondria promoting further clea- c, AIF and Smac from mitochondria in all cell types. By vage of eector caspases. Signals originating from the demonstrating a cell type dependent regulation of the CD95 receptor may be linked to mitochondria by Bid, a TRAIL signaling pathway at dierent level, e.g. by Bcl-2 BH3 domain containing protein of the Bcl-2 family and by XIAP, these ®ndings may have important clinical which assumes cytochrome-c-releasing activity upon implication. Thus, strategies targeting the molecular cleavage by caspase-8 thereby initiating a mitochondrial basis of resistance towards TRAIL may be necessary ampli®cation loop (Kroemer and Reed, 2000). in some tumors for cancer therapy with TRAIL. In CD95-mediated apoptosis we recently identi®ed Oncogene (2002) 21, 2283 ± 2294. DOI: 10.1038/sj/ two dierent apoptosis signaling cell types (Scadi et al., onc/1205258 1998). In type I cells, caspase-8 is activated at the CD95 DISC in large quantities resulting in direct processing of Keywords: TRAIL; apoptosis; caspases; IAP caspase-3. This step is independent of mitochondrial activation and cannot be blocked by overexpression of Bcl-2. In contrast, in type II cells, the amount of active Introduction caspase-8 generated at the DISC is limited. In type II cells, apoptosis proceeds through mitochondrial pertur- Cell death by apoptosis plays a pivotal role in the bations and generation of active caspase-3 and -8 regulation of various physiological or pathological downstream of mitochondria and Bcl-2 overexpression conditions and has also been implied to mediate blocked activation of both caspase-3 and -8. therapy-induced cytotoxicity, e.g. in response to cyto- Besides CD95 ligand and TNFa, TRAIL is another member of the TNF/NGF superfamily of death inducing ligands (Walczak and Krammer, 2000). TRAIL can interact with ®ve distinct receptors, two *Correspondence: K-M Debatin; of which contain a cytoplasmic death domain and signal E-mail: [email protected] Received 23 August 2001; revised 10 December 2001; accepted 14 apoptosis. Analysis of the native TRAIL death-inducing December 2001 signaling complex (DISC) revealed ligand-dependent Regulation of TRAIL-induced apoptosis S Fulda et al 2284 ab

3 ng/ml; 10 ng/ml; 30 ng/ml; 100 ng/ml

Figure 1 Eect of Bcl-2 overexpression on TRAIL-induced apoptosis. (a) Eect of Bcl-2 overexpression on TRAIL-induced DNA fragmentation in neuroblastoma cells. SHEP neuroblastoma cells transfected with vector control (Neo) or Bcl-2 were treated for indicated times with 3 ± 100 ng/ml TRAIL and/or 1 mg/ml CHX. Apoptosis was determined by FACS analysis of propidium iodide stained DNA content. Percentage of speci®c apoptosis was calculated as follows: 1006[experimental apoptosis (%) 7 spontaneous apoptosis (%) / 100% 7 spontaneous apoptosis (%)]. Mean and s.d. of triplicates are shown, similar results were obtained in three independent experiments. (b) Eect of Bcl-2 overexpression on TRAIL-induced phosphatidylserine exposure in neuroblastoma cells. SHEP neuroblastoma cells transfected with vector control (Neo) or Bcl-2 were treated for 24 h with 100 ng/ml TRAIL and 1 mg/ml CHX. Apoptosis was determined by FACS analysis of FITC-annexin V stained cells. Fluorescence intensity (abscissa) is plotted against cell counts (ordinate). (c) Eect of Bcl-2 overexpression on TRAIL-induced long term survival in neuroblastoma cells. SHEP neuroblastoma cells transfected with vector control (Neo) or Bcl-2 were left untreated or were treated for 7 days with 100 ng/ml TRAIL. Clonogenic survival was determined by staining colonies with crystal violet and visualized by digital camera. (d) Eect of Bcl-2 overexpression on TRAIL-induced DNA fragmentation in SKW lymphoblastoid cells. SKW cells transfected with vector control (Neo) or Bcl-2 were treated for indicated times with 3 ± 100 ng/ml TRAIL and/or 1 mg/ml CHX. Apoptosis was determined by FACS analysis of propidium iodide stained DNA content as described above. (e) TRAIL DISC analysis in SHEP and SKW cells. SHEP or SKW cells were left untreated or were treated with 1000 ng/ml TRAIL before lysis. TRAIL was added to lysates of untreated cells for precipitation of unstimulated TRAIL receptors. DISC formation was analysed by immunoprecipitation and Western blot analysis as described in Materials and methods

Oncogene Regulation of TRAIL-induced apoptosis S Fulda et al 2285 recruitment of FADD and caspase-8 demonstrating that completely inhibited apoptosis in response to treatment TRAIL utilizes a pathway similar to the one used by the with TRAIL (Figure 1a). Combined treatment with CD95 receptor (Sprick et al., 2000). TRAIL has been TRAIL together with the protein synthesis inhibitor shown to induce apoptosis in various tumor cell lines in CHX further sensitized vector control cells for vitro and also suppresses the growth of TRAIL sensitive apoptosis induction, whereas Bcl-2 overexpression tumors in SCID mice in vivo (Walczak and Krammer, protected SHEP cells even against treatment with 2000; Walczak et al., 1999). Unlike the other death TRAIL together with CHX (Figure 1a). Apoptosis ligands CD95 or TNFa, systemic administration of was also determined by annexin V staining. Following soluble human TRAIL did not cause systemic toxicity treatment with TRAIL and CHX the majority of vector in mice or nonhuman primates, although further studies control cells were positive for annexin V, whereas only a may be necessary to evaluate the possible cytoxicity of small percentage of Bcl-2 transfected cells were positive TRAIL on human normal tisues, e.g. hepatocytes for annexin V (Figure 1b). To test whether Bcl-2 (Walczak and Krammer, 2000; Walczak et al., 1999; overexpression only causes a delay in the apoptosis Lawrence et al., 2001; Ichikawa et al., 2001; Nagane et response or has an eect on long-term survival, we al., 2001). Thus, TRAIL may be a promising novel performed clonogenic assays. Treatment of vector therapeutic agent for human cancer. control cells with TRAIL almost completely abrogated Resistance of many tumors such as neuroblastoma to colony formation compared to untreated vector control current treatment protocols still remains a major cells, whereas the eect of TRAIL treatment on colony concern in cancer therapy (Berthold et al., 1990). formation was blocked by Bcl-2 overexpression (Figure Deregulated apoptosis programs may contribute to 1c). This demonstrates that Bcl-2 overexpression not tumor progression and treatment resistance (Debatin, only delayed cell death, but also had a protective eect 1997; Kaufmann and Earnshaw, 2000). Thus, the on long term survival of tumor cells in response to identi®cation of molecular defect(s) in apoptosis TRAIL treatment. We also investigated the eect of signaling is crucial to develop novel strategies to target Bcl-2 overexpression on TRAIL-induced apoptosis in resistance. Defects in apoptosis pathways may occur at SKW lymphoblastoid cells, since TRAIL has previously dierent levels, e.g. at the mitochondrial level by Bcl-2 been reported to signal apoptosis independent of or during the eector phase by inhibitor of apoptosis mitochondria. In SKW cells, Bcl-2 overexpression did proteins (IAP's) (Kroemer, 1997; Deveraux and Reed, not prevent apoptosis following treatment with TRAIL 1999). Bcl-2 is highly expressed in many cell lines and or the combination of TRAIL and CHX (Figure 1d). In primary tumors including neuroblastoma or malignant addition, DISC formation following treatment with brain tumors, and its expression has been associated TRAIL was reduced in SHEP cells compared to SKW with drug resistance (Kroemer, 1997; Hoehner et al., cells (Figure 1e). These ®ndings suggest that Bcl-2 1995; Fels et al., 2000). Thus, novel strategies are overexpression conferred protection against TRAIL- necessary to target resistance of tumors with Bcl-2 induced apoptosis in a cell type speci®c manner. overexpression. We previously found that overexpression of Bcl-2 protected SHEP neuroblastoma cells Bcl-2 overexpression inhibits TRAIL-induced against apoptosis induced by agonistic anti-CD95 mitochondrial alterations monoclonal antibody or cytotoxic drugs (Fulda et al., 1998a, 2000). Recent data suggest that TRAIL induced To exclude that the dierential protection of Bcl-2 in apoptosis independent of mitochondria and could be SHEP and SKW cells against TRAIL-induced apoptosis used in Bcl-2 overexpressing tumor cells which were was the result of dierent expression levels of Bcl-2, we resistant to chemotherapy (Walczak et al., 2000; Keogh analysed Bcl-2 protein expression. High Bcl-2 expression et al., 2000; Kim et al., 2001; Gazitt et al., 1999; Suliman was detected in the cytosolic and mitochondrial fraction et al., 2001). In search for strategies to overcome of both SHEP and SKW cells (Figure 2a). This indicates resistance, we therefore investigated the role of Bcl-2 in that the dierent protective eect of Bcl-2 against TRAIL-induced apoptosis in the present study. TRAIL-induced apoptosis could not be attributed to low Bcl-2 protein levels in SKW cells. In addition, overexpression of Bcl-2 similarly blocked loss of the mitochondrial membrane potential following treatment Results with TRAIL and/or CHX in both SHEP and SKW cells (Figure 2b). Also, the release of cytochrome c, AIF and Bcl-2 overexpression inhibits TRAIL-induced apoptosis in Smac from mitochondria was inhibited in SHEP and neuroblastoma cells SKW cells (Figure 2c). These ®ndings indicate that We previously found that overexpression of Bcl-2 overexpression of Bcl-2 similarly blocked mitochondrial protected SHEP neuroblastoma cells against apoptosis perturbations upon treatment with TRAIL in both induced by agonistic anti-CD95 monoclonal antibody SHEP and SKW cells. or cytotoxic drugs. To determine the role of Bcl-2 in To see whether caspases were activated upstream of TRAIL-induced apoptosis, we compared the pattern of mitochondria we analysed the eect of caspase apoptosis following treatment with TRAIL in SHEP inhibitors on the mitochondrial membrane potential neuroblastoma cells transfected with Bcl-2 expression in response to treatment with TRAIL. Loss of the vector or control vector. Overexpression of Bcl-2 almost mitochondrial membrane potential upon TRAIL

Oncogene Regulation of TRAIL-induced apoptosis S Fulda et al 2286 treatment was markedly inhibited in the presence of the caspases including caspase-3, caspase-7 and caspase-9. broad range caspase inhibitor zVAD.fmk or the In addition, XIAP has recently been reported to act as a relatively speci®c caspase-8 inhibitor zIETD.fmk in ubiquitin-ligase for activated caspase-3 or -7 thereby both SHEP and SKW cells (Figure 2d). This indicates promoting the degradation of activated caspases. that caspases such as caspase-8 were activated prior to Therefore, we analysed XIAP expression by Western mitochondrial dysfunction at the receptor level follow- blot analysis. Upon TRAIL treatment XIAP was ing ligation of TRAIL receptors. cleaved in SHEP vector control cells, whereas cleavage of XIAP was completely inhibited in Bcl-2 transfected SHEP cells (Figure 4a). The slight reduction of full- Bcl-2 overexpression reduces TRAIL-induced activation length XIAP expression without appearance of cleavage of caspases in neuroblastoma cells products observed in Bcl-2 transfected SHEP cells To gain insight into the dierential regulation of (Figure 4a) may be due to inhibition of protein synthesis TRAIL-induced apoptosis, we then analysed activation by CHX consistent with previous results (Fulda et al., of caspases by Western blot. In SHEP cells, over- 2000). In contrast, XIAP was similarly cleaved in vector expression of Bcl-2 reduced cleavage of the initiator control or Bcl-2 transfected SKW cells (Figure 4a). To caspase-8, Bid and caspase-9, and inhibited cleavage of further investigate whether apoptosis was blocked at a caspase-3 and caspase-7 into active fragments (Figure level downstream of mitochondria by absent cleavage of 3a). Interestingly, in SHEP vector control cells cleavage XIAP in SHEP cells, we analysed the eect of XIAP of caspase-3 resulted in generation of the p24 inter- overexpression. As shown in Figure 4b stable trans- mediate fragment and the p17/10 active subunits, fected clones expressed high levels of XIAP protein. whereas in Bcl-2 overexpressing SHEP cells, only the Apoptosis in response to treatment with TRAIL or p24 intermediate fragment, but no active p17/10 TRAIL together with CHX was strongly inhibited by subunits were detected (Figure 3a). This suggests that XIAP (Figure 4c) demonstrating that TRAIL-induced Bcl-2 did not prevent the initial cleavage of caspase-3 apoptosis was also regulated by inhibitor of apoptosis between the large and small subunit to generate the p24 proteins such as XIAP in SHEP cells. intermediate fragment, but blocked further autoproces- sing of caspase-3 into the p17/10 active subunits. In Bcl-2 overexpression inhibits TRAIL-induced apoptosis in contrast, in SKW cells Bcl-2 overexpression did not glioblastoma and breast carcinoma cells inhibit cleavage of caspase-8, caspase-7, caspase-3 and cleavage of Bid, while cleavage of caspase-9 was reduced To exclude that our ®ndings were restricted to SHEP in Bcl-2 overexpressing SKW cells (Figure 3b). neuroblastoma cells, we then investigated TRAIL- Also, we tested for activation of eector caspases by induced apoptosis in LN-229 glioblastoma and MCF- analysing cleavage of cellular substrates such as DFF45, 7 breast carcinoma cells. High expression levels of Bcl- a prototype substrate of caspase-3, and PARP, a 2 in transfected cells was controlled by Western blot prototype substrate of caspase-7 (Germain et al., 1999; analysis (Figure 5a). While treatment with TRAIL Wolf et al., 1999; Slee et al., 2001). Importantly, alone was only marginally eective to induce apopto- cleavage of DFF45 and PARP was completely blocked sis, treatment with TRAIL together with CHX induced by Bcl-2 in SHEP cells compared to vector control cells apoptosis in LN-229 or MCF-7 cells in a dose and (Figure 3c). Also by ¯ow cytometry, Bcl-2 overexpres- time-dependent manner (Figure 5b, c). In contrast, sion was found to inhibit activation of caspase-3 in overexpression of Bcl-2 protected LN-229 or MCF-7 SHEP cells (data not shown). In contrast, in SKW cells cells against apoptosis triggered by TRAIL or the Bcl-2 overexpression did not inhibit cleavage of DFF45 combination of TRAIL and CHX (Figure 5b, c). or PARP (Figure 3d). Together, these ®ndings indicate Western blot analysis revealed that Bcl-2 delayed that in SKW cells, TRAIL-induced activation of cleavage of caspase-8 and Bid and strongly inhibited caspase-8 upstream of mitochondria directly translated cleavage of caspase-9, caspase-7 or caspase-3 (Figure into activation of executioner caspases such as caspase-3 5d, e). Importantly, Bcl-2 completely blocked cleavage and cleavage of cellular substrates independent of of XIAP and PARP (Figure 5d, e). These ®ndings mitochondria. In contrast, in SHEP neuroblastoma indicate that a mitochondrial ampli®cation step was cells, the generation of active caspase-3 and -7 also required in LN-229 glioblastoma and MCF-7 fragments and cleavage of caspase substrates was breast carcinoma cells for full activation of caspases, completely blocked by Bcl-2 indicating that a mitochon- cleavage of antiapoptotic proteins such as XIAP and drial ampli®cation step was required for complete apoptosis in response to treatment with TRAIL. activation of eector caspases and apoptosis.

Discussion Bcl-2 overexpression inhibits TRAIL-induced cleavage of XIAP in neuroblastoma cells and XIAP overexpression Despite aggressive therapies, resistance to current confers protection against TRAIL treatment protocols has been a major obstacle in IAP's such as XIAP exert their antiapoptotic function, clinical oncology. Most anticancer agents act through at least in part, downstream of the mitochondrial induction of apoptosis in target cells and defects in checkpoint by inhibiting proteolytically processed apoptosis programs may confer resistance (Debatin,

Oncogene Regulation of TRAIL-induced apoptosis S Fulda et al 2287 1997; Kaufmann and Earnshaw, 2000). Apoptosis is of XIAP conferred protection against TRAIL. Since mediated through both the death receptor and the XIAP binds to and inhibits processed caspase-9, -7 and - mitochondrial pathway (Hengartner, 2000). At least in 3 and can also promote caspase degradation through its some cellular systems, mitochondria also seem to be ubiquitin-protein ligase activity, downregulation of necessary for death receptor-induced apoptosis (Kroe- XIAP will amplify the apoptotic process (Holcik et al., mer and Reed, 2000). The mitochondrial pathway of 2001; Suzuki et al., 2001). This points to an additional apoptosis can be blocked by the antiapoptotic protein control level of the TRAIL signaling pathway down- Bcl-2 which has been localized to cellular membranes, stream of mitochondria by XIAP that is linked to the e.g. the mitochondrial membrane (Kroemer, 1997). Bcl- mitochondrial checkpoint, since Bcl-2 can regulate 2 is highly expressed in many cell lines and primary inactivation of XIAP by caspase cleavage and by Smac tumors including neuroblastoma or glioblastoma release from mitochondria (Holcik et al., 2001; Du et al., (Kroemer, 1997; Hoehner et al., 1995; Fels et al., 2000). XIAP is inactivated by at least two mechanisms, 2000). High Bcl-2 expression has been correlated with e.g. by proteolytic cleavage and by XIAP-interacting poor prognosis in some instances and also likely plays a proteins such as Smac (Holcik et al., 2001). Of the known role in chemoresistance (Kroemer, 1997; Hoehner et al., human IAP related proteins, XIAP is the most potent 1995; Fels et al., 2000). Thus, novel strategies are inhibitor and a direct role of XIAP in the resistance of necessary to target resistance, e.g. due to Bcl-2 over- cancer cells to radiation and chemotherapy was recently expression. One potential strategy is a direct induction demonstrated using a XIAP antisense approach (Holcik of cell death by death receptors. Unlike the other death et al., 2001; Sasaki et al., 2000). Thus, the TRAIL ligands CD95 ligand or TNFa, systemic administration signaling pathway may be regulated in a cell type of TRAIL does not cause toxicity in mice or non- dependent manner at dierent levels, e.g. at the human primates (Walczak and Krammer, 2000; Walc- mitochondrial level by Bcl-2 and at a level downstream zak et al., 1999; Lawrence et al., 2001; Ichikawa et al., of mitochondria by XIAP. 2001; Nagane et al., 2001). Thus, TRAIL may be a Initial studies on the biochemical pathways of promising novel therapeutic agent for human cancer. TRAIL-induced apoptosis in lymphoid cell lines Recent data suggest that TRAIL can induce apoptosis demonstrated that overexpression of Bcl-2 did not independent of mitochondria and can also be used in confer protection, whereas apoptosis following antic- Bcl-2 overexpressing tumor cells which were resistant to ancer drug treatment was blocked (Walczak et al., 2000; chemotherapy (Walczak et al., 2000; Keogh et al., 2000; Keogh et al., 2000; Kim et al., 2001; Gazitt et al., 1999; Kim et al., 2001; Gazitt et al., 1999; Suliman et al., Suliman et al., 2001). These data suggested that TRAIL 2001). We previously found that Bcl-2 overexpression induced apoptosis via a caspase cascade independent of blocked apoptosis induced by CD95 ligation or mitochondria and could be used in Bcl-2 overexpressing cytotoxic drugs in neuroblastoma cells (Fulda et al., tumor cells which were resistant to chemotherapy. In 1998a, 2000). In search for strategies to overcome contrast, recent reports and our ®ndings indicate that resistance, we therefore investigated the role of Bcl-2 in Bcl-2 also conferred protection against TRAIL in some TRAIL-induced apoptosis in the present study. tumors (Hinz et al., 2000; Sun et al., 2001; Rokhlin et Here we report that overexpression of Bcl-2 al., 2001). The dierences between the studies may be conferred protection against TRAIL-induced apoptosis due to dierent recombinant TRAIL preparations in neuroblastoma, glioblastoma or breast carcinoma which may vary in their cytotoxic potential (Lawrence cell lines. Ampli®cation of the TRAIL-induced death et al., 2001; Ichikawa et al., 2001). Dierent expression signal by mitochondria was required for full activation levels of Bcl-2 may also play a role. However, we found of the caspase cascade to inactivate XIAP in these cells. no association between Bcl-2 protein expression levels This conclusion is based on several pieces of evidence. and protection against TRAIL in the present study, Bcl-2 inhibited cleavage of the eector caspases since SKW lymphoblastoid cells were not protected by caspase-3 or caspase-7 into active subunits and cleavage Bcl-2 against TRAIL despite high levels of Bcl-2 of cellular caspase substrates, e.g. cleavage of DFF45, a protein. Alternatively, there may be intrinsic dierences prototype substrate of caspase-3, and cleavage of PARP, between cell types, since a lack of protection by Bcl-2 a primary target of caspase-7 (Germain et al., 1999; Wolf against TRAIL has predominantly been reported for et al., 1999; Slee et al., 2001). Cleavage of caspase-8 and lymphoid cell lines (Walczak et al., 2000; Keogh et al., Bid was partially blocked by Bcl-2 suggesting that 2000; Kim et al., 2001; Gazitt et al., 1999; Suliman et al., caspase-8 was activated upstream and downstream of 2001). To this end, we found protection by Bcl-2 against mitochondria. These results support recent data demon- TRAIL treatment in solid tumor cell lines derived from strating that executioner caspases, e.g. caspase-3, can neuroblastoma, glioblastoma or breast carcinoma cell serve as a feedback loop by cleaving caspase-8 to amplify lines, whereas Bcl-2 did not confer protection in the the apoptotic process (Slee et al., 1999, 2001). In lymphoblastoid cell line SKW indicating for the ®rst addition, our ®ndings indicate that executioner caspases time a cell type dependent (type I and type II) regulation can serve as a feedforward loop by cleaving XIAP to of the TRAIL signaling pathway. allow the caspase cascade to progress to completion, For CD95, we previously identi®ed two dierent since full activation of the caspase cascade through a apoptosis signaling cell types (Scadi et al., 1998). In mitochondrial ampli®cation step was necessary for type I cells, e.g. SKW lymphoblastoid cells, activation cleavage and inactivation of XIAP. Also, overexpression of caspase-8 following CD95 crosslinking directly

Oncogene Regulation of TRAIL-induced apoptosis S Fulda et al 2288 ab

Oncogene Regulation of TRAIL-induced apoptosis S Fulda et al 2289 proceeded to activation of executioner caspases such as Bcl-2 is highly expressed in many cell lines and caspase-3 independent of mitochondria. In these cells, primary tumors including neuroblastoma or malignant overexpression of Bcl-2 did not confer protection brain tumors, and its expression has been associated against CD95-induced apoptosis. In contrast, in type with drug resistance (Kroemer, 1997; Hoehner et al., II cells apoptosis following CD95 crosslinking de- 1995; Fels et al., 2000). Current attempts to improve pended on mitochondrial activity which was blocked cancer survival largely depend on strategies to target by Bcl-2 overexpression. SHEP neuroblastoma cells tumor cell resistance and on the development of novel display characteristics of both type I and type II cells. treatment modalities, e.g. immunotherapy, gene ther- In SHEP cells, loss of the mitochondrial membrane apy or cytotoxic cytokines such as TRAIL. TRAIL is a potential depended on caspase activity indicating that promising candidate for clinical investigation, since it activation of caspases, e.g. caspase-8 following TRAIL induces apoptosis in a broad spectrum of human tumor receptor ligation occurred prior to loss of the cell lines and exhibits potent antitumor activity without mitochondrial membrane potential which is character- normal tissue toxicity in various human cancer istic of type I cells. In addition, activation of caspases xenograft models (Nagane et al., 2001). However, our partially depended on mitochondria and was inhibited ®ndings indicate that TRAIL may not be eective in by Bcl-2 overexpression, since activation of caspase-8 some tumors, e.g. in tumors with enhanced Bcl-2 or did not directly translate into full activation of caspase- XIAP expression. Thus, our ®ndings may have 3, a feature characteristic of type II cells. Our data on important implications for cancer therapy. Because Bcl-2 inhibition of TRAIL-induced apoptosis suggest a the potential success of novel treatment approaches similar cell type dependent (type I and type II) including TRAIL largely depend on intact apoptosis organization of the TRAIL signaling pathway. pathways in tumor cells, strategies that target the

Figure 2 Eect of Bcl-2 overexpression on TRAIL-induced mitochondrial alterations. (a) Analysis of Bcl-2 expression in SHEP neuroblastoma and SKW lymphoblastoid cells. Cellular or mitochondrial lysates were separated by 10 ± 20% SDS ± PAGE and analysed for protein expression by Western blot. Expression of b-actin or COX4 was used to control for equal gel loading. (b) Eect of Bcl-2 overexpression on loss of Dcm. SHEP neuroblastoma or SKW lymphoblastoid cells transfected with vector control (Neo) or Bcl-2 were treated for 12 h with 100 ng/ml TRAIL and/or 1 mg/ml CHX. DCm was determined by staining cells with the potential-sensitive ¯uorochrome DiOC6(3) and analysed by ¯ow cytometry. Mean and s.d. of triplicates are shown, similar results were obtained in three independent experiments. (c) Eect of Bcl-2 overexpression on the release of cytochrome c, AIF and Smac. SHEP neuroblastoma or SKW lymphoblastoid cells transfected with vector control (Neo) or Bcl-2 were treated for 24 h with 100 ng/ml and 1 mg/ml CHX. Mitochondrial, cytosolic or nuclear extracts were prepared as described in Materials and methods. Expression of cytochrome c, AIF or Smac was determined by Western blot analysis as described above. (d) Eect of caspase inhibition on loss of DCm. Cells were treated for 12 h with 100 ng/ml TRAIL, 50 mM zVAD.fmk and/or 50 mM zIETD.fmk. DCm was determined as described above

Oncogene Regulation of TRAIL-induced apoptosis S Fulda et al 2290

c d

Figure 3 Eect of Bcl-2 overexpression on TRAIL-induced activation of caspases. (a±d). Eect of Bcl-2 overexpression on TRAIL-induced activation of caspases in SHEP neuroblastoma (a) or SKW lymphoblastoid (b) cells or cleavage of caspase substrates in SHEP (c) or SKW (d) cells. Cells transfected with vector control (Neo) or Bcl-2 were treated for indicated times with 100 ng/ml and 1 mg/ml CHX. Protein expression was determined by Western blot analysis as described above

Oncogene Regulation of TRAIL-induced apoptosis S Fulda et al 2291 a b

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Figure 4 Inhibition of TRAIL-induced apoptosis by XIAP. (a) Eect of Bcl-2 overexpression on TRAIL-induced cleavage of XIAP. SHEP neuroblastoma (upper panel) or SKW lymphoblastoid (lower panel) cells transfected with vector control (Neo) or Bcl- 2 were treated for indicated times with 3 ± 100 ng/ml and 1 mg/ml CHX. Protein expression was determined by Western blot analysis as described above. In addition to XIAP, a nonspeci®c XIAP immune-reactive molecule migrating slightly larger than XIAP was detected using the mouse monoclonal anti-XIAP antobody H62120 (Deveraux and Reed, 1999). (b) Analysis of XIAP expression. SHEP neuroblastoma cells were transfected with vector control (Neo) or XIAP cDNA. Protein expression of myc-tagged XIAP was determined by Western blot analysis as described above. (c) Eect of XIAP overexpression on TRAIL-induced apoptosis. SHEP neuroblastoma cells transfected with vector control (Neo) or XIAP were treated for indicated times with 3 ± 100 ng/ml TRAIL and/ or 1 mg/ml CHX. Apoptosis was determined as described above

molecular basis of resistance towards TRAIL may be medium (Life Technologies, Inc., Eggenstein, Germany) as necessary in certain tumors as a complementary tool previously described (Fulda et al., 1998a; Scadi et al., 1998; for cancer therapy with TRAIL. Rieger et al., 1998; Ruiz-Ruiz et al., 2000). Cells (0.56105) per ml were cultured in 24-well-plates for determination of apoptosis or in 75 cm2 ¯asks (Falcon, Heidelberg, Germany) for protein isolation. Materials and methods Determination of apoptosis Cell culture Cells were incubated with recombinant human TRAIL SHEP neuroblastoma, LN-229 glioblastoma, MCF-7 breast (PeproTech Inc., Rocky Hill, NJ, USA), cycloheximide carcinoma or SKW6.4 B lymphoblastoid transfected with (CHX, Sigma, Deisenhofen, Germany), benzyloxycarbonyl- vector control or Bcl-2 were maintained in RPMI 1640 Val-Ala-Asp-¯uoromethylketone (zVAD.fmk; Bachem, Hei-

Oncogene Regulation of TRAIL-induced apoptosis S Fulda et al 2292 a d

3 ng/ml; 10 ng/ml; 30 ng/ml; 100 ng/ml

Figure 5 Eect of Bcl-2 overexpression on TRAIL-induced apoptosis in glioblastoma or breast carcinoma cells. (a) Analysis of Bcl-2 expression. Bcl-2 expression of LN-229 glioblastoma or MCF-7 breast carcinoma cells transfected with vector control (Neo) or Bcl-2 was determined by Western blot analysis as described above. (b and c) Eect of Bcl-2 overexpression on TRAIL-induced apoptosis. LN-229 glioblastoma (b) or MCF-7 breast carcinoma (c) cells transfected with vector control (Neo) or Bcl-2 were treated for indicated times with 3 ± 100 ng/ml TRAIL and/or 1 ± 3 mg/ml CHX. Apoptosis was determined as described above. (d and e) Eect of Bcl-2 overexpression on TRAIL-induced activation of caspases. LN-229 glioblastoma (d) or MCF-7 breast carcinoma (e) cells transfected with vector control (Neo) or Bcl-2 were treated for indicated times with 100 ng/ml TRAIL and/or 1 ± 3 mg/ml CHX. Protein expression was determined by Western blot analysis as described above

Oncogene Regulation of TRAIL-induced apoptosis S Fulda et al 2293 delberg, Germany) or benzyloxycarbonyl-Ile-Glu(OMe)-Thr- Transfection experiments Asp(OMe)-¯uoromethylketone (zIETD.fmk; Bachem). Quan- ti®cation of DNA fragmentation was performed by ¯uores- SHEP neuroblastoma cells were transfected with expression cence-activated cell-sorting (FACS) analysis of propidium plasmid pcDNA3 vector containing myc-tagged XIAP cDNA iodide stained nuclei as previously described (Fulda et al., or empty vector using lipofectamine transfection reagent (Life 1998a; Nicoletti et al., 1991). Analysis of phosphatidylserine Technologies, Inc.) and cultured in 0.5 mg/ml G418 (Life exposure was performed by ¯ow cytometry of FITC-annexin Technologies, Inc.). V stained cells (Bender Med System, Munich, Germany) according to the manufacturer's instructions. Preparation of mitochondria, cytosolic or nuclear extracts Preparation of mitochondria or cytosolic extracts was Clonogenicity assay performed using the ApoAlert cell fractionation kit (Clontech Cells (103) were seeded in a six-well plate and treated for 7 Laboratories) according to the manufacturer's instructions. days with 100 ng/ml TRAIL. Clonogenic survival was determined by staining colonies using crystal violet and Assessment of mitochondrial transmembrane potential visualized by digital camera. The cationic lipophilic ¯uorochrome 3,3'-dihexyloxacarbo- cyanide iodide (DiOC (3)) (460 ng/ml, Molecular Probes, Western blot and DISC analysis 6 Eugene, OR, USA) was used to measure the mitochondrial Western blot analysis was performed as previously described transmembrane potential (DCm). Cells were incubated for (Fulda et al., 1997) using mouse anti-caspase-8 monoclonal 15 min at 378C in the presence of the ¯uorochrome, washed antibody C15 (1 : 10 dilution of hybridoma supernatant) in PBS/1% FCS and immediately analysed by ¯ow cytometry (Scadi et al., 1997), mouse anti-caspase-3 monoclonal (FACScan, Becton Dickinson). DiOC6(3) was recorded in antibody (1 : 1000, Transduction Laboratories, Lexington, ¯uorescence 1. KY, USA), mouse anti-caspase-7 monoclonal antibody (1 : 1000, Transduction Laboratories), rabbit anti-caspase-9 polyclonal antibody (1 : 1000, PharMingen, San Diego, CA, USA), rabbit anti-poly(ADP-ribose) polymerase (PARP) polyclonal antibody (1 : 5000, Boehringer Mannheim, Man- nheim, Germany), mouse anti-XIAP monoclonal antibody (1 : 1000, H62120, Transduction Laboratories), mouse anti- DFF45 monoclonal antibody (1 : 1000, Transduction Labora- Acknowledgments tories), mouse anti-Bcl-2 monoclonal antibody (1 : 1000, We thank Petra Miller-Rostek for expert technical assis- Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1 : 1000, tance, PH Krammer (DKFZ, Heidelberg, Germany) for PharMingen), rabbit anti-AIF polyclonal antibody (1 : 5000, anti-FLICE antibody, X Wang for anti-Smac antibody kindly provided by G Kroemer), rabbit anti-Smac polyclonal (University of Texas, Dallas, TX, USA), G Kroemer antibody (1 : 5000, kindly provided by X Wang), mouse anti- (CNRS, Villejuif, France) for anti-AIF antibody, JC Reed COX4 monoclonal antibody (1 : 1000, Clontech Laboratories, (Burnham Institute, San Diego, CA, USA) for XIAP Inc., Palo Alto, CA, USA), or mouse anti-b-actin monoclonal cDNA plasmid, A Lopez-Rivas (University of Granada, antibody (1 : 5000, Sigma) followed by goat anti-mouse IgG Spain) for MCF-7 breast carcinoma and M Weller or goat anti-rabbit IgG (1 : 5000, Santa Cruz Biotechnology). (University of Tuebingen, Germany) for LN-229 glioblas- Analysis of the TRAIL DISC was performed as previously toma cells. This work has been partially supported by described (Mitsiades et al., 2000). Enhanced chemilumines- grants from the Deutsche Forschungsgemeinschaft, the cence (ECL, Amersham Pharmacia, Freiburg, Germany) was Bundesministerium fuÈ r Forschung and Technologie, Bonn, used for detection. Expression of b-actin was used to control the Deutsche LeukaÈ mieforschungshilfe, the Wilhelm San- for equal gel loading. der-Stiftung and the European community.

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