Oncogene (2001) 20, 5865 ± 5877 ã 2001 Nature Publishing Group All rights reserved 0950 ± 9232/01 $15.00 www.nature.com/onc Sensitization for death receptor- or drug-induced apoptosis by re-expression of caspase-8 through demethylation or gene transfer Simone Fulda1, Martin U KuÈ fer1, Eric Meyer1, Frans van Valen2, Barbara Dockhorn-Dworniczak3 and Klaus-Michael Debatin*,1 1University Children's Hospital, Prittwitzstr. 43, D-89075 Ulm, Germany; 2Laboratory for Experimental Orthopedic Research, WestfaÈlische-Wilhelms University, D-48149 MuÈnster, Germany; 3Department of Pathology, WestfaÈlische-Wilhelms University, D-48149 MuÈnster, Germany Resistance of tumors to treatment with cytotoxic drugs, Introduction irradiation or immunotherapy may be due to disrupted apoptosis programs. Here, we report in a variety of Cell death by apoptosis plays a pivotal role in the dierent tumor cells including Ewing tumor, neuroblas- regulation of various physiological or pathological toma, malignant brain tumors and melanoma that conditions and has also been implied to mediate caspase-8 expression acts as a key determinant of therapy-induced cytotoxicity, e.g. in response to sensitivity for apoptosis induced by death-inducing cytotoxic drug treatment or g-irradiation (Peter and ligands or cytotoxic drugs. In tumor cell lines resistant Krammer, 1998; Grith and Lynch, 1998; Kaufmann to TRAIL, anti-CD95 or TNFa, caspase-8 protein and and Earnshaw, 2000; Debatin, 1997; Friesen et al., mRNA expression was decreased or absent without 1996; Fulda et al., 1997, 1998a,b). Apoptosis pathways caspase-8 gene loss. Methylation-speci®c PCR revealed may be initiated through dierent entry sites, such as hypermethylation of caspase-8 regulatory sequences in death receptors (receptor pathway) or mitochondria cells with impaired caspase-8 expression. Treatment with (mitochondrial pathway) resulting in activation of the demethylation agent 5-Aza-2'-deoxycytidine (5- eector caspases (Peter and Krammer, 1998; Grith dAzaC) reversed hypermethylation of caspase-8 resulting and Lynch, 1998; Kaufmann and Earnshaw, 2000; in restoration of caspase-8 expression and recruitment Debatin, 1997). Stimulation of death receptors of the and activation of caspase-8 at the CD95 DISC upon tumor necrosis factor (TNF) receptor superfamily such receptor cross-linking thereby sensitizing for death as CD95 (APO-1/Fas) results in receptor aggregation receptor-, and importantly, also for drug-induced and recruitment of the adaptor molecule Fas-associated apoptosis. Inhibition of caspase-8 activity also inhibited death domain (FADD) and caspase-8 (Trauth et al., apoptosis sensitization by 5-dAzaC. Similar to demethy- 1989; Oehm et al., 1992; Kischkel et al., 1995; lation, introduction of caspase-8 by gene transfer Chinnaiyan et al., 1996; Muzio et al., 1996; Medema sensitized for apoptosis induction. Hypermethylation of et al., 1997; Scadi et al., 1998). Upon recruitment caspase-8 was linked to reduced caspase-8 expression in caspase-8 becomes activated and initiates apoptosis by dierent tumor cell lines in vitro and, most importantly, direct cleavage of downstream eector caspases also in primary tumor samples. Thus, these ®ndings (Trauth et al., 1989; Oehm et al., 1992; Kischkel et indicate that re-expression of caspase-8, e.g. by al., 1995; Chinnaiyan et al., 1996; Muzio et al., 1996; demethylation or caspase-8 gene transfer, might be an Medema et al., 1997; Scadi et al., 1998; Thornberry eective strategy to restore sensitivity for chemotherapy- and Lazebnik, 1998). A second pathway is initiated at or death receptor-induced apoptosis in various tumors in the mitochondrial level (Kroemer and Reed, 2000). vivo. Oncogene (2001) 20, 5865 ± 5877. Apoptogenic factors such as cytochrome c, apoptosis inducing factor (AIF), caspase-2 or caspase-9 are Keywords: caspase-8; apoptosis; drugs; methylation; released from mitochondria into the cytosol triggering gene transfer caspase-3 activation through formation of the cyto- chrome c/Apaf-1/caspase-9-containing apoptosome complex (Kroemer and Reed, 2000; Susin et al., 1999a,b; Zou et al., 1999). In these cells, caspase-8 is activated downstream of mitochondria promoting further cleavage of eector caspases. Signals originat- ing from the CD95 receptor may be linked to mitochondria by Bid, a BH3 domain containing protein of the Bcl-2 family which assumes cyto- *Correspondence: K-M Debatin; chrome-c-releasing activity upon cleavage by caspase- E-mail: [email protected] Received 11 April 2001; revised 21 June 2001; accepted 26 June 8 thereby initiating a mitochondrial ampli®cation loop 2001 (Kroemer and Reed, 2000; Li et al., 1998; Yin et al., Sensitization for apoptosis by caspase-8 demethylation or gene transfer S Fulda et al 5866 A Oncogene Sensitization for apoptosis by caspase-8 demethylation or gene transfer S Fulda et al 5867 1999). Studies from caspase-8 knock-out mice indicate may act as a tumor suppressor in some tumors (Teitz that caspase-8 plays a necessary and non-redundant et al., 2000; Hopkins-Donaldson et al., 2000; Grotzer role in various forms of cell death (Varfolomeev et al., et al., 2000). 1998). Interestingly, mutations in the caspase-8 gene Despite aggressive therapies, resistance of many have only infrequently been found in cell lines or tumors, e.g. neuroblastoma, malignant brain tumors primary tumor samples (Teitz et al., 2000; Mandruzza- or soft tissue sarcoma, to current treatment protocols to et al., 1997). still remains a major concern in cancer therapy (De In addition to genetic alterations, epigenetic mod- Vita, 1990). Deregulated apoptosis programs may i®cations such as DNA methylation have recently contribute to tumor progression and treatment been implied in loss of gene function and tumorigen- resistance. Defects in apoptosis pathways may occur esis (Tycko, 2000; Herman and Baylin, 2000). In at dierent levels, at the receptor level, during signal particular, hypermethylation of normally unmethy- transduction or during the eector phase (Debatin, lated CpG islands located in the promotor region of 1997; Friesen et al., 1997; Tschopp et al., 1998; many tumor suppressor genes or other tumor-related Kroemer, 1997; Deveraux and Reed, 1999; Los et al., genes, including Rb, p16, p15, VHL, E-cadherin or 1997). We previously reported that increased recruit- p73, is associated with loss of expression in cancer cell ment and activation of caspase-8 through down- lines and primary tumors (Tycko, 2000; Herman and regulation of FLIP by metabolic inhibitors may Baylin, 2000). Recently, caspase-8 was reported to be sensitize for CD95-induced apoptosis (Fulda et al., silenced by hypermethylation in neuroblastoma with 2000). Recently, we found that TNF-related apoptosis- MycN ampli®cation and in peripheral neuroectoder- inducing ligand (TRAIL) eciently triggered apopto- mal tumor (PNET) cell lines implying that caspase-8 sis in the majority of Ewing tumor cell lines including B Figure 1 Death receptor-induced apoptosis in sensitive and resistant Ewing tumor cell lines. (a) Death receptor-induced apoptosis. Cells were treated for 24 h with 0.3 ± 100 ng/ml TRAIL, 0.1 ± 1 mg/ml anti-APO-1 monoclonal antibody or 10 ± 100 ng/ml TNFa in the absence (&) or presence (&)of1mg/ml CHX. Apoptosis was determined by FACS analysis of propidium iodide stained DNA content. (b) Activation of apoptosis pathways. Cells were treated for indicated times with 100 ng/ml TRAIL. Cell lysates (50 mg protein per lane) were separated by 12% SDS ± PAGE and analysed for protein expression by Western blot. Expression of b-actin was used to control for equal gel loading. Migration positions of cleavage products are indicated by arrows Oncogene Sensitization for apoptosis by caspase-8 demethylation or gene transfer S Fulda et al 5868 primary tumor cells, however, a subset of Ewing expression of caspase-8 was found in resistant Ewing tumor cells remained resistant even in the presence of tumor cell lines (CADO, GG-62, RDES, SKES, 6647, protein synthesis inhibitors (van Valen et al., 2000). ES-3 and ET2.1) compared to sensitive or intermediate Therefore, we further investigated molecular mechan- sensitive Ewing tumor cell lines (VH-64, TC-71, WE- isms regulating sensitivity or resistance in the present 68, ES-2), whereas expression levels of caspase-10, -9, study. -7 or -3 protein did not correlate with sensitivity or resistance (Figure 2c). Results Impaired caspase-8 expression without loss of the caspase-8 gene We previously found that TRAIL eciently triggered apoptosis in the majority of Ewing tumor cell lines To investigate whether caspase-8 protein expression (78%, 32/41) and also in primary tumor cells ex vivo was downregulated at the transcriptional level, we (100%, 3/3) (van Valen et al., 2000). However, a subset analysed caspase-8 mRNA expression by RT ± PCR. of Ewing tumor cell lines remained resistant to Caspase-8 mRNA expression correlated with protein TRAIL-induced apoptosis (22%, 9/41) even in the expression with undetectable levels in cell lines with presence of protein synthesis inhibitors (Fulda et al., absent caspase-8 protein expression (CADO, 6647), 2000; van Valen et al., 2000). Therefore, we further reduced levels in cell lines with low caspase-8 protein investigated the molecular mechanism(s) regulating expression (GG-62, RDES, SKES, ET2.1) and high sensitivity in these cells. expression in cells with high caspase-8 protein expres- sion (VH-64, ES-2) (Figure 3a,b). However, reduced caspase-8 protein expression was not uniformly