Oncogene (2001) 20, 5789 ± 5798 ã 2001 Nature Publishing Group All rights reserved 0950 ± 9232/01 $15.00 www.nature.com/onc Analysis of FasL and TRAIL induced apoptosis pathways in glioma cells Melissa J Knight1,2,3,4, Christopher D Rikin1,2,3, Andrea M Muscat1,2,3, David M Ashley1,2,3 and Christine J Hawkins*,1,2,3 1Department of Haematology and Oncology, Royal Children's Hospital, Parkville, Australia; 2Murdoch Children's Research Institute, Royal Children's Hospital, Parkville, Australia; 3Department of Paediatrics, University of Melbourne, Parkville, Australia; 4Department of Surgery, Royal Melbourne Hospital, University of Melbourne, Parkville, Australia FasL and TNF-related apoptosis-inducing ligand therapies which induce apoptosis, such as radio- and (TRAIL) belong to a subgroup of the TNF superfamily chemotherapy. This study focuses on the death which induce apoptosis by binding to their death domain receptor mediated apoptotic pathways in glioma cells, containing receptors. In the present study we have induced by FasL (CD95L/Apo1L) and Tumor Necrosis utilized a panel of seven cell lines derived from human Factor-related apoptosis-inducing ligand (TRAIL/ malignant gliomas to characterize molecular pathways Apo2L). through which FasL and TRAIL induce apoptosis in FasL and TRAIL belong to a subgroup of the sensitive glioma cells and the mechanisms of resistance in Tumor Necrosis Factor (TNF) superfamily with the cell lines which survive the death stimuli. Our ®ndings unique ability to induce apoptosis. They do so via their indicate that FADD and Caspase-8 are essential for cognate death receptors Fas and DR4 (TRAIL-R1)/ FasL and TRAIL mediated apoptosis in glioma cells. DR5 (TRAIL-R2/Trick2) respectively, which are One sensitive cell line (D270) can be protected from characterized by a cytoplasmic death domain (reviewed FasL and TRAIL induced death by anti-apoptotic Bcl-2 by Reed, 2000). FasL and TRAIL can also bind to family members while another (D645) cannot, implying antagonist `decoy' receptors. DcR3/TR6/M68 is a that these lines may represent glioma examples of type II secreted soluble Fas decoy receptor which competes and type I cells respectively. For the ®rst time we with Fas for binding to FasL (Bai et al., 2000; Pitti et demonstrate resistance to FasL but not to TRAIL within al., 1998; Yu et al., 1999). Alternative splicing of the the one glioma cell line. Furthermore, we report distinct Fas gene can also give rise to mRNAs encoding mechanisms of resistance within dierent glioma lines, membrane bound proteins which lack the death including downregulation of Caspase-8 in U373MG. domain, such as the Fas Decoy Receptor (FDR) Cycloheximide sensitized four of the resistant cell lines (Jenkins et al., 2000). TRAIL can bind to the decoy suggesting the presence of labile inhibitors. None of the receptors DcR1 (TRAIL-R3/Trid/Lit) and DcR2 known apoptosis inhibitors examined accounted for the (TRAIL-R4/TRUNDD) which lack the intracellular observed resistance, suggesting novel inhibitors may exist death domain and are therefore incapable of inducing in glioma cells. Oncogene (2001) 20, 5789 ± 5798. the apoptotic pathway. TRAIL decoy receptors are expressed almost exclusively in normal cells (reviewed Keywords: apoptosis; glioma; resistance; TRAIL; Fas; by Reed 2000). Bcl-2 Studies in lymphoid cells suggest FasL and TRAIL engage similar apoptotic pathways in these cells. Aggregation of the death receptors by multimerized Introduction ligand induces the formation of a death-inducing signaling complex (DISC) (Kischkel et al., 1995; Sprick Malignant gliomas are the most frequent primary brain et al., 2000), involving the receptors' death domains tumors in adults and the prognosis of patients remains (DDs), the adaptor molecule FADD (Mort-1) and extremely poor (Hosli et al., 1998). Apoptosis is a Caspase-8 (FLICE, Mch5, MACH), which interact via highly evolutionarily conserved process fundamental to their death eector domains (DEDs). normal development and homeostasis. Defects in The subsequent activation of Caspase-8 at the Fas apoptosis regulation are implicated in a variety of DISC has been shown to propagate alternate apoptotic human diseases, including cancer (Reed, 1999). It is pathways (Scadi et al., 1998). In type I cells, active likely alterations in apoptotic pathways play a role in Caspase-8 directly cleaves downstream eector cas- the tumorigenesis of glioma and resistance to current pases, such as Caspase-3, which in turn cleave an array of substrates, leading to the systematic destruction of the cell (Wolf and Green, 1999). However in type II cells, DISC formation is less ecient and mitochon- *Correspondence: C Hawkins, Department of Haematology and drial ampli®cation of the death receptor induced signal Oncology, Royal Children's Hospital, Victoria Australia 3052; E-mail: [email protected] is required for apoptosis (Scadi et al., 1999b). Active Received 8 May 2001; revised 22 June 2001; accepted 5 July 2001 Caspase-8 cleaves the BH3-only Bcl-2 family member FasL and TRAIL induced apoptosis in glioma MJ Knight et al 5790 Bid and truncated Bid (tBID) translocates to the were sensitive to FasL, with the remainder being FasL mitochondria (Li et al., 1998). This results in the resistant (Figure 1a,b). D645 was also very sensitive to release of cytochrome-c, which binds to Apaf-1 and TRAIL induced apoptosis. U118MG, D270 and recruits Caspase-9 to form the `apoptosome' (Luo et U87MG displayed partial sensitivity to TRAIL, while al., 1998; Zou et al., 1999). This complex is involved in U251MG and U373MG were resistant (Figure 1d,e). the `intrinsic' apoptotic pathway, which is triggered by D54 was completely resistant to FasL, but sensitive to serum withdrawal (Zhang et al., 2000b) and DNA TRAIL. Death receptor mediated apoptosis has been damage (Coultas and Strasser, 2000). The Bcl-2 protein shown to converge downstream with the `intrinsic' family regulates apoptotic signals involving the apoptotic pathway which can be induced by che- mitochondria (reviewed by Gross et al., 1999). motherapy drugs. To examine sensitivity to such an Seven glioma cell lines were used in this study and apoptotic stimulus, cells were treated with the initially characterized for their sensitivity to FasL, chemotherapeutic agent cisplatin (Figure 1g,h). D270 TRAIL and cisplatin. The death receptor apoptotic was particularly sensitive to cisplatin with a second signal transduction pathways used by sensitive cells line, U118MG displaying intermediate sensitivity. and the mechanisms employed by resistant cells to D645, the most sensitive cell line to FasL and TRAIL, elude apoptosis were investigated. was resistant to cisplatin. U373MG and U251MG were resistant to all three death stimuli. Results Caspase-3 activity correlates with sensitivity Apoptosis triggered by all three agents studied has Glioma cell lines differ in their susceptibility to death been demonstrated in other cell types to involve stimuli activation of caspases. The tetrapeptide DEVD is A panel of seven glioma cell lines was characterized for cleaved by eector caspases such as Caspase-3. FasL and TRAIL sensitivity (refer to Table 1 and Cleavage of the ¯uorogenic substrate DEVD-AMC Discussion) using the sensitive lymphoid cell line was used to quantify activated Caspase-3 (and similar Jurkat as a control. Two distinct methods were used caspases) in each of the cell lines following treatment to assess cell viability with comparable results. The with FasL and TRAIL. Figure 1c,f demonstrate that glioma cell lines displayed dierential sensitivity to the amount of Caspase-3-like activity correlates with each of the death stimuli. D645, D270 and U118MG the susceptibility of the cells to apoptosis. Absolute Table 1 Summary of glioma cell line characteristics and apoptotic pathway analysis DISC Bcl-2 family Type I or Tumor components proteinsa Sensitivity Sensitized by Type IIb Proposed death receptor type Sex Age (Figure 3a) (Figure 3b) (Figure 1) CHX (Figure 5) (Figure 4) resistance mechanism U118 Anaplastic M 50 Intact F, High 3 May express low levels glioma T, Intermediate 3 of labile FasL and C, Intermediate x TRAIL inhibitor U251 Glioblastoma M 75 Intact Low Bcl-2 F, Resistant x Mutated component or multiforme T, Resistant x inhibitor with long C, Resistant x half-life U87 Glioblastoma F 44 Intact Low Bid F, Intermediate 3 May express low levels multiforme Low Bcl-xL T, Intermediate 3 of labile FasL and C, Low x TRAIL inhibitor U373 Glioblastoma M 50 Low levels F, Resistant x Low levels of multiforme FADD and T, Resistant x Caspase-8 Caspase-8 C, Resistant x D54 Mixed F 36 Intact High Bid F, Resistant 3 Fas specific labile anaplastic Low Bcl-2 T, High upstream inhibitor glioma C, Resistant x D270 Glioblastoma M 42 Intact High Bid F, High 3 Type II May express low levels multiforme High Bad T, Intermediate 3 of labile TRAIL Low Bcl-xL C, High x inhibitor D645 Glioblastoma F 10 Intact F, High Type I multiforme T, High C, Resistant x F=FasL, T=TRAIL, C=Cisplatin. aChange from the average only. bType I: inhibited by FADD DN and CrmA, Type II: inhibited by FADD DN, CrmA, Bcl-2 and Bcl-xL Oncogene FasL and TRAIL induced apoptosis in glioma MJ Knight et al 5791 Figure 1 Analysis of FasL (a,b,c), F-LZ-TRAIL (d,e,f) and cisplatin (g,h,i) induced apoptosis in the glioma line panel. Glioma cell lines were treated with TRAIL, FasL or control medium for 24 h (a,b,d,e)or6h(c,f) and 10 mg/ml cisplatin for 24 h (g,h)or12h (i). (a,d,g) Cell viability was assessed by colorimetric MTT assay. (b,e,h) Membrane integrity was measured by propidium iodide uptake. The data are expressed as percentage of viability relative to the cells exposed to the control media. Graphs represent the average and standard deviation of three individual experiments.