CCNU-Dependent Potentiation of TRAIL/Apo2l-Induced Apoptosis in Human Glioma Cells Is P53-Independent but May Involve Enhanced Cytochrome C Release

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CCNU-dependent potentiation of TRAIL/Apo2L-induced apoptosis in human glioma cells is p53-independent but may involve enhanced cytochrome c release

Till A RoÈ hn1, Bettina Wagenknecht1, Wilfried Roth1, Ulrike Naumann1, Erich Gulbins2, Peter H Krammer3, Henning Walczak4 and Michael Weller*,1

1Laboratory of Molecular Neuro-Oncology, Department of Neurology, University of TuÈbingen, Medical School, TuÈbingen, Germany; 2Institute of Physiology, University of TuÈbingen, Medical School, TuÈbingen, Germany; 3Department of Immunogenetics, German Cancer Research Center, Heidelberg, Germany; 4Department of Apoptosis Regulation, German Cancer Research Center, Heidelberg, Germany

Death ligands such as CD95 ligand (CD95L) or tumor apy may be an eective therapeutic strategy for these necrosis factor-related apoptosis-inducing ligand/Apo2 lethal neoplasms. Oncogene (2001) 20, 4128 ± 4137. ligand (TRAIL/Apo2L) induce apoptosis in radio- chemotherapy-resistant human malignant glioma cell Keywords: brain; apoptosis; neuroimmunology; cyto- lines. The death-signaling TRAIL receptors 2 kines; immunotherapy (TRAIL-R2/death receptor (DR) 5) and TRAIL-R1/ DR4 were expressed more abundantly than the non- death-inducing (decoy) receptors TRAIL-R3/DcR1 and Introduction TRAIL-R4/DcR2 in 12 human glioma cell lines. Four of the 12 cell lines were TRAIL/Apo2L-sensitive in the Death receptor targeting is an attractive approach of absence of a protein synthesis inhibitor, cycloheximide experimental treatment for solid tumors that are (CHX). Three of the 12 cell lines were still TRAIL/ resistant to radiotherapy and chemotherapy, including Apo2L-resistant in the presence of CHX. TRAIL-R2 malignant gliomas (Weller et al., 1998a). Among the expression predicted sensitivity to apoptosis. Coexposure family of cytokines referred to as death ligands, to TRAIL/Apo2L and cytotoxic drugs such as TRAIL/Apo2L has attracted special interest for topotecan, lomustine (1-(2-chloroethyl)-3-cyclohexyl-1- therapeutic purposes. This is because TRAIL/Apo2L nitrosourea, CCNU) or temozolomide resulted in seemed to be rather inactive against untransformed synergistic killing. Synergistic killing was more often cells but readily induced apoptosis in various cancer observed in cell lines retaining wild-type p53 activity cell lines (Wiley et al., 1995; Pitti et al., 1996), including (U87MG, LN-229) than in p53 mutant cell lines (LN- human malignant glioma cell lines (Rieger et al., 1998). 18, T98G, U373MG). Drug exposure resulted in Moreover, TRAIL/Apo2L inhibited tumor growth enhanced TRAIL-R2 expression, but decreased without causing systemic toxicity in xenograft models TRAIL-R4 expression in U87MG cells. Ectopic expres- of breast or colon cancer and glioma in nude mice sion of dominant-negative p53V135A abrogated the drug- (Ashkenazi et al., 1999; Roth et al., 1999; Walczak et induced changes in TRAIL-R2 and TRAIL-R4 expres- al., 1999; Nagane et al., 2000). This selectivity of action sion, but had no eect on synergy. Thus, neither wild- against neoplastic cells may be related to the complex type p53 function nor changes in TRAIL receptor patterns of TRAIL/Apo2L receptor expression. expression were required for synergy. In contrast, TRAIL/Apo2L interacts with at least four cell surface synergy resulted possibly from drug-induced cytochrome receptors, two of which transmit a death signal c release from mitochondria, serving as an ampli®er of (TRAIL-R1/DR4, TRAIL-R2/DR5) and two of which the TRAIL/Apo2L-mediated cascade of caspase activa- (TRAIL-R3/DcR1, TRAIL-R4/DcR2) do not (Ashke- tion. These data provide novel insights into the role of nazi and Dixit, 1998). Preferential expression of the TRAIL/Apo2L system in malignant gliomas and agonistic receptors might confer high susceptibility to illustrate that TRAIL/Apo2L-based immunochemother- apoptosis whereas expression of TRAIL-R3 and TRAIL-R4 might be protective. Whether receptor expression patterns truly underlie the resistance of untransformed cells to TRAIL/Apo2L-induced apoptosis, remains to be investigated. Here, we characterize *Correspondence: M Weller, Laboratory of Molecular Neuro- TRAIL-R expression and sensitivity to TRAIL/ Oncology, Department of Neurology, University of TuÈ bingen, Apo2L-induced apoptosis in a panel of human School of Medicine, Hoppe-Seyler-Strasse 3, 72076 TuÈ bingen, malignant glioma cell lines and study the molecular Germany; E-mail: [email protected] Received 19 January 2001; revised 22 February 2001; accepted 10 mechanisms underlying synergistic glioma cell killing April 2001 by TRAIL/Apo2L and cancer chemotherapeutic drugs. TRAIL/Apo2L-induced apoptosis of human glioma cells TA RoÈhn et al Results 4129

Expression of TRAIL-R in human malignant glioma cell lines The expression of TRAIL-R at the cell surface was assessed by ¯ow cytometry. Representative pro®les are shown in Figure 1a. Quantitative data for TRAIL-R1, -R2, -R3 and -R4 are summarized in Figure 1b. All glioma cell lines expressed TRAIL-R2. The speci®c ¯uorescence indexes (SFI) assumed values of more than 20, e.g., in A172 cells. In contrast, the SF1 for TRAIL-R1 did not exceed three. Only four cell lines (LN-18, LN-428, D247, LN-319) exhibited a distinct level of TRAIL-R1 expression. TRAIL-R3 reached a signi®cant level of expression in LN-428 only. TRAIL-R4 was more abundantly expressed, with four lines exhibiting a SFI of more than two. Note that the SFI values among the dierent receptors cannot be compared directly since the SFI is determined both by the level of expression and by the anity of the antibodies used. The TRAIL-R expression levels in the tumor cell lines were compared with those in the SV40- transformed non-neoplastic SV-FHAS astrocyte cell line. These cells expressed lower TRAIL-R2 levels than any of the neoplastic (glioma) cell lines, but detectable levels of both TRAIL-R3 and TRAIL-R4 (Figure 1b).

Sensitivity to TRAIL/Apo2L-induced apoptosis The sensitivity of the glioma cells to TRAIL/Apo2L- induced apoptosis was assessed in short-term (16 h) cytotoxicity assays. These experiments were performed in the absence or presence of CHX, an inhibitor of protein synthesis because inhibition of protein synthesis enhances glioma cell sensitivity to cytotoxic cytokines (Weller et al., 1994). In the absence of CHX, eight of 12 cell lines exhibited EC50 values above 2000 ng/ml and may thus be considered TRAIL/Apo2L-resistant (Table 1). CHX greatly enhanced TRAIL/Apo2L-induced apoptosis in eight of 12 cell lines. CHX was dispensable for apoptosis in the highly sensitive LN-18 cell line, and CHX failed to promote apoptosis in U138MG, U373MG and LN-308 cells, and in the SV-FHAS astrocyte cell line. The latter three glioma cell lines (U138MG, U373MG, LN-308) are characterized by low TRAIL-R2 and absent TRAIL-R1 expression. The other two cell lines with low TRAIL-R2 expression, D247MG and LN-319, express signi®cant levels of TRAIL-R1, which appear to confer sensitivity to apoptosis. Statistical analysis revealed strong correlation between TRAIL-R2 expression and apoptosis in response to TRAIL/Apo2L at 2000 ng/ml in the absence of CHX (r=0.72, P=0.008). This correlation failed to achieve statis- Figure 1 TRAIL receptor expression in human malignant tical signi®cance in the presence of CHX (r=0.56, glioma cell lines. (a) Representative ¯ow cytometry pro®les for P=0.06). No correlation between the expression of TRAIL-R1, -R2, -R3 and -R4 expression in U87MG and LN-428 cells. Solid lines represent speci®c signal, dotted lines correspond TRAIL-R1, TRAIL-R3 or TRAIL-R4 and sensitivity to the signal obtained with an isotype control antibody. (b) SFI to apoptosis became apparent. values were calculated for each receptor in each cell line

Oncogene TRAIL/Apo2L-induced apoptosis of human glioma cells TA RoÈhn et al 4130 Table 1 Sensitivity of human malignant glioma cell lines to TRAIL/ Apo2L

EC50 TRAIL/Apo2L EC50 TRAIL/Apo2L +CHX (ng/ml) (ng/ml)

LN-18 550 550 U138MG 42000 42000 U87MG 325 550 LN428 42000 60 D247MG 42000 550 T98G 360 550 LN-319 42000 140 LN-229 42000 300 A172 480 550 U251MG 42000 550 U373MG 42000 42000 LN-308 42000 42000 SV-FHAS 42000 42000

The cells were exposed to TRAIL/Apo2L for 16 h in the absence or presence of CHX (10 mg/ml). Survival was assessed by crystal violet staining. Data are expressed as mean EC50 values (n=3, s.e.m.510%)

Synergistic cytotoxic effects of TRAIL/Apo2L and cancer chemotherapeutic drugs The glioma cells were co-treated with various concentrations of TRAIL/Apo2L and the drugs for 24 h. These experiments were performed with LN-229, U87MG, U373MG, T98G and LN-18 cells. Topotecan, CCNU, cisplatin, teniposide, vincristine, temozolomide and doxorubicin were studied in combination with TRAIL/Apo2L. Synergistic eects determined by isobologram analysis (Berenbaum, 1981) were observed for topotecan and CCNU in LN-229 and U87MG cells, for cisplatin in U87MG cells, for teniposide in LN-229 cells, for vincristine and temozolomide in LN- 229, U87MG and T98G cells, and for doxorubicin in LN-229 and T98G cells. There was no synergy in LN- 18 cells which are highly TRAIL/Apo2L-sensitive constitutively. Moreover, U373MG cells which are resistant to TRAIL/Apo2L (Table 1) were not sensitized to TRAIL/Apo2L by any drug. Representa- tive isobologram analyses for TRAIL/Apo2L and topotecan, CCNU or temozolomide in U87MG cells are shown in Figure 2. Figure 2 Synergistic induction of cell death by TRAIL/Apo2L and cancer chemotherapy. U87MG cells were treated for 24 h with increasing concentrations of TRAIL/Apo2L and topotecan Wild-type p53 activity or p53-mediated changes in (a), CCNU (b) or temozolomide (c). Data are expressed as TRAIL-R expression are not required for synergy isobolograms (Berenbaum, 1981) Functional p53 activity in the 12 glioma cell lines has been previously studied (Weller et al., 1998b) and recently been con®rmed using a p53 reporter assay p53 mutant in human glioma cells (Naumann et al., (Schmidt et al., 2001). Coexposure to TRAIL/Apo2L 1998) (Figure 3a). The dominant-negative activity of and cytotoxic drugs resulted in more synergistic killing p53V135A in U87MG and LN-229 cells was ascertained in cell lines retaining wild-type p53 activity (U87MG, by the inhibition of camptothecin-induced p21 accu- LN-229) than in p53 mutant cell lines (LN-18, T98G, mulation in cells engineered to express p53V135A, U373MG). We therefore next determined whether compared with hygro control cells (data not shown). there is a causal relationship between p53 activity p53V135A-transfected U87MG and LN-229 cells exhib- and synergy. U87MG and LN-229 cells were engi- ited unaltered sensitivity to TRAIL/Apo2L. Trans- neered to express murine mutant p53V135A which has fected U87MG, but not LN-229, cells were more previously been characterized as a dominant-negative sensitive to CCNU than hygro control cells (Figure

Oncogene TRAIL/Apo2L-induced apoptosis of human glioma cells TA RoÈhn et al 4131

Figure 3 Synergy of TRAIL/Apo2L and CCNU: the role of p53. (a) U87MG or LN-229 cells were transfected with a p53V135A expression plasmid and analysed for transgene expression by immunoblot. b-actin immunodetection con®rmed equal protein loading (not shown). (b) Hygro control (®lled squares) or p53V135A-transfected (open squares) U87MG (left) or LN-229 (right) cells were treated with CCNU or TRAIL/Apo2L for 24 h. (c) Hygro control or p53V135A-transfected cells were treated with CCNU (U87MG EC15 concentration, LN-229 36 EC15 concentration, for a 24 h exposure, respectively) for 3 h and then analysed for TRAIL-R2 or TRAIL-R4 expression by ¯ow cytometry. Bold lines represent the speci®c signal of CCNU-treated cells, dashed lines correspond to untreated cells and the dotted lines on the left of all panels represent the isotype control antibody applied to untreated cells. (d) The cells were treated with CCNU or TRAIL/Apo2L alone or in combination for 24 h. Black bars indicate the eect of CCNU alone, open bars the eect of TRAIL/Apo2L alone, vertically striped bars the predicted eect of co-treatment according to the fractional product method, and the outer obliquely striped bar the actual cytotoxicity achieved with co-treatment. Survival in b and d was assessed by crystal violet staining. Data are expressed as mean percentages and s.e.m. (n=3) of survival in b and of cytotoxicity in d

Oncogene TRAIL/Apo2L-induced apoptosis of human glioma cells TA RoÈhn et al 4132 3b). We next assessed changes in TRAIL-R expression as a possible mechanism underlying synergy (Figure 3c). Exposure to CCNU enhanced TRAIL-R2 expression in both cell lines and decreased TRAIL-R4 expression in U87MG cells, consistent with a more apoptosis-sensitive phenotype. No such eect was seen in U373MG cells which are mutant for p53 (data not shown). The changes in TRAIL-R2 and TRAIL-R4 expression were p53-dependent since they were not observed in U87MG cells expressing dominant-negative p53V135A (Figure 3c). However, expression of p53V135A did not interfere with the synergistic killing by TRAIL/Apo2L and CCNU (Figure 3d). Similarly, p53V135A abrogated the CCNU-mediated increase in TRAIL-R2 expression, but did not aect synergistic killing mediated by CCNU and TRAIL, in LN-229 cells. Thus, neither functions of wild-type p53, that are blocked by dominant-negative p53V135A, nor drug- induced changes in TRAIL receptor expression are required for synergy.

Synergy of TRAIL/Apo2L and CCNU linked to mitochondrial cytochrome c release triggered by subtoxic concentrations of CCNU The molecular events during synergistic induction of apoptosis by TRAIL/Apo2L and cancer chemotherapeutic drugs were further characterized in U87MG cells co-treated with TRAIL/Apo2L and CCNU at approximate EC15 concentrations. A prominent release of cytochrome c from mitochondria was induced by CCNU alone, but not by TRAIL/Apo2L alone (Figure 4a). Co-exposure to TRAIL/Apo2L and CCNU resulted in enhanced cytochrome c release compared with CCNU treatment alone. In contrast, CCNU alone failed to induce the processing of caspases 3, 7 or 9, whereas caspase cleavage was readily induced by TRAIL/Apo2L alone (Figure 4b). The processing of caspases 9 and 3 was accelerated and more prominent in cells co-treated with TRAIL/Apo2L and CCNU than in cells treated with TRAIL/Apo2L alone. Note, for example, the reduction of procaspase levels at 4 h of co-treatment for all three caspases, or the more ecient processing to p35/caspase 9 or p17/caspase 3 in co-treated cells at 2 h. In contrast, no such eect was observed for caspase 7. Enhanced caspase activity triggered by co-treatment as opposed to single agent treatment became also apparent in an enzymatic ¯uorimetric assay for DEVD-amc-cleaving caspase activity (Figure 4c).

Caspase 8 processing is required for synergistic activity of Figure 4 Enhanced mitochondrial cytochrome c release during TRAIL/Apo2L and CCNU synergistic induction of apoptosis by TRAIL/Apo2L and CCNU. The role of TRAIL-R3 and TRAIL-R4 in TRAIL/ (a) Cytosolic proteins were isolated from U87MG cells treated for 2 h with CCNU (200 mM), TRAIL/Apo2L (125 ng/ml) or both, Apo2L signaling has remained obscure. We examined and were assessed for cytochrome c content by immunoblot. (b) whether synergy of TRAIL/Apo2L and CCNU strictly The cells were treated as in a for 2 or 4 h. Total cellular lysates depended on death signaling through TRAIL-R1 and were assessed for the processing of caspases 9, 7 and 3 by TRAIL-R2. To test this, we generated U87MG cells immunoblot. b-actin immunodetection con®rmed equal protein loading (not shown). (c) The cells were treated as in b. DEVD- expressing the viral preferential caspase 8 inhibitor, amc-cleaving activity was measured by ¯uorimetry (**P50.01, crm-A (Figure 5a). As predicted from our previous t-test)

Oncogene TRAIL/Apo2L-induced apoptosis of human glioma cells TA RoÈhn et al 4133 studies with crm-A-transfected LN-18 and LN-229 cells (Glaser et al., 1999), crm-A-transfected U87MG cells were protected from TRAIL/Apo2L-induced apoptosis but retained sensitivity to CCNU (Figure 5b,c). Ectopic expression of crm-A also prevented any

Figure 6 Bcl-2 gene transfer fails to prevent CCNU-induced cytochrome c release and does not interfere with the synergy of TRAIL/Apo2L and CCNU. (a) U87MG cells were transfected with a neo control plasmid or a bcl-2 expression plasmid and examined for bcl-2 transgene expression by immunoblot. b-actin immunodetection con®rmed equal protein loading (not shown). (b) U87MG puro control or bcl-2-transfected cells were exposed to CD95L (200 U/ml) or CCNU at EC15 concentration for 2 h and then assessed for cytochrome c release as in Figure 4a. (c) The cells were treated with approximate EC15 concentrations of CCNU or TRAIL/Apo2L alone or in combination for 24 h and analysed for synergy of cell death induction (for details and legend to the bars, see Figure 3d)

Figure 5 Crm-A gene transfer abrogates TRAIL/Apo2L-induced apoptosis and synergy with CCNU. (a) U87MG cells were transfected with a puro control plasmid or a crm-A expression TRAIL/Apo2L or CCNU for 24 h. (d) The cells were treated plasmid and examined for crm-A transgene expression by with approximate EC15 concentrations of CCNU or TRAIL/ immunoblot. b-actin immunodetection con®rmed equal protein Apo2L alone or in combination for 24 h and analysed for synergy loading (not shown). (b, c) U87MG puro control (®lled squares) of cell death induction (for details and legend to the bars, see or crm-A-transfected cells (open squares) were exposed to Figure 3d)

Oncogene TRAIL/Apo2L-induced apoptosis of human glioma cells TA RoÈhn et al 4134 augmentation of CCNU cytotoxicity by TRAIL/ not associated with resistance to TRAIL/Apo2L- Apo2L (Figure 5d), indicating that death signaling induced apoptosis. through caspase 8 is essential for the synergy of CCNU A comparison of the data reported here (Table 1) and TRAIL/Apo2L. and our previous data on the p53 status in the same cell lines revealed that cell lines retaining p53 function assessed by reporter assay or accumulation of p53 in Synergy of TRAIL/Apo2L and CCNU: effects of ectopic response to genotoxic stress (Van Meir et al., 1994; bcl-2 expression Weller et al., 1998b; Schmidt et al., 2001) did not dier We also examined how bcl-2 aected the synergistic in their TRAIL/Apo2L sensitivity from p53 mutant cell glioma cell killing induced by TRAIL/Apo2L and lines. Similarly, although ectopic expression of bcl-2 or CCNU. Bcl-2-transfected U87MG cells (Figure 6a) bcl-XL inhibited TRAIL/Apo2L-induced apoptosis in showed unaltered sensitivity to CCNU in a 24 h glioma cells (Rieger et al., 1998), endogenous expres- acute exposure assay and moderate protection from sion levels of bcl-2 family proteins, data as reported TRAIL/Apo2L-induced apoptosis (data not shown). previously (Weller et al., 1998b) and con®rmed to be In contrast to mitochondrial cytochrome c release stable over time (data not shown), did not explain the triggered by CD95L (Hermisson et al., 2000), dierential susceptibility to TRAIL/Apo2L-induced cytochrome c release induced by CCNU was apoptosis in this panel of glioma cell lines. unaected by bcl-2 (Figure 6b). Bcl-2 had also no There was strong synergistic glioma cell killing in eect on the extent of synergy when U87MG cells response to the combination of TRAIL/Apo2L with were coexposed to TRAIL/Apo2L and CCNU various cancer chemotherapy drugs, including drugs (Figure 6c), providing further circumstantial evidence which kill by diering modes of action, as previously for a role of cytochrome c release in the process of reported for CD95L (Roth et al., 1997). The role of synergy. p53 for the synergy of TRAIL/Apo2L and radio- chemotherapy remains controversial. Numerous studies have con®rmed that genotoxic stress may increase Discussion TRAIL-R2 (DR5) expression in a p53-dependent manner (Sheikh et al., 1998). Moreover, we report TRAIL/Apo2L-induced apoptosis is a novel experi- here that p53 mediates a reduction in the expression mental approach to the treatment of malignant glioma levels of the putatively antiapoptotic TRAIL-R4/DcR2 (Rieger et al., 1998). Both local and systemic receptor in response to cytotoxic drugs (Figure 3), an application of TRAIL/Apo2L result in the regression event also predicted to result in enhanced sensitivity to of U87MG xenograft gliomas in nude mice (Roth et apoptosis. Similar to the present work on glioma cells al., 1999; Nagane et al., 2000). Since all preclinical and chemotherapy, breast cancer cells: (i) exhibited studies had indicated a striking lack of toxicity of synergistic killing by TRAIL/Apo2L and irradiation; TRAIL/Apo2L on normal cells (Ashkenazi et al., 1999; (ii) synergy was more prominent in p53 wild-type than Roth et al., 1999; Walczak et al., 1999; Nagane et al., in p53 mutant cells; and (iii) genotoxic stress 2000), clinical trials had been discussed and expected (irradiation) enhanced TRAIL-R2 expression (Chin- with signi®cant hope for ecacy. A recent report on naiyan et al., 2000). However, in contrast to our data the induction of apoptosis by TRAIL/Apo2L in (Figure 3), dominant-negative p53 abrogated the normal human hepatocytes cultured in vitro (Jo et al., synergy of TRAIL/Apo2L and irradiation. Although 2000) has raised some concerns regarding the safety of the approach to determine synergy was not as rigorous systemic TRAIL/Apo2L administration in humans. as in our study, e.g., did not use isobologram analysis However, these ®ndings await con®rmation, and it or at least compare isoeective concentrations of the needs to be carefully examined under which in vitro single agents, these data indicate that the molecular and in vivo conditions non-neoplastic cells may acquire mechanisms underlying synergy may be cell type- sensitivity to TRAIL/Apo2L. speci®c. The present study shows that both wild-type Here we studied TRAIL-R expression in human p53 activity inhibited by p53V135A, and drug-induced malignant glioma cell lines (Figure 1). TRAIL-R2 was p53-mediated changes in TRAIL-R2 and TRAIL-R4 highly expressed, whereas TRAIL-R1 was expressed at expression, are dispensable for the synergy of CCNU signi®cant levels in only four of 12 cell lines. TRAIL- and TRAIL/Apo2L in glioma cells. R3 and TRAIL-R4 were less frequently expressed. Preliminary evidence suggests that the synergy of More than half of the glioma cell lines were rather TRAIL/Apo2L and CCNU involves the potentiation resistant to TRAIL/Apo2L-induced apoptosis unless of the TRAIL/Apo2L-triggered cascade of caspase co-treated with an inhibitor of protein synthesis, CHX activation by a CCNU-induced, apparently caspase- (Table 1). Only three cell lines remained TRAIL/ independent cytochrome c release from mitochondria Apo2L-resistant in the presence of CHX. The non- (Figure 4). In principle, it is also possible that more neoplastic astrocyte cell line, SV-FHAS, expressed little ecient triggering of the caspase cascade by co- TRAIL-R2 and was resistant to TRAIL/Apo2L. treatment upstream of mitochondria facilitated cyto- Among the glioma cell lines, TRAIL-R2 expression chrome c release as the mechanism underlying synergy. was a good predictor of sensitivity to apoptosis. In BID cleavage is unlikely to play a role here since death contrast, expression of TRAIL-R3 or TRAIL-R4 was receptor-mediated apoptosis of glioma cells involves

Oncogene TRAIL/Apo2L-induced apoptosis of human glioma cells TA RoÈhn et al 4135 BID cleavage downstream, not upstream, of mitochon- human astrocytic cell line that was generously provided by drial cytochrome c release (Glaser et al., 2001). Future Dr Arumugam Muruganandam and Dr Danica Stanimiro- studies will address why the mitochondrial cytochrome vic (Institute of Biological Sciences, National Research c released by CCNU is not sucient to activate Council of Canada, Ottawa, Canada). caspases on its own. In contrast to glioma cells, breast cancer cell lines were reported to exhibit signi®cant Transfections caspase activation when treated with those drugs that U87MG cells were transfected by lipofection using the acted in synergy with TRAIL/Apo2L (Keane et al., Maxifectin transfection kit (Qiagen, Hilden, Germany) 1999). Importantly, bcl-2 gene transfer into the glioma according to the manufacturer's protocol. p53V135A and cells inhibited CD95L-evoked cytochrome c release but control plasmids were kindly provided by MF Clarke (Ann did not interfere with drug-induced cytochrome c Arbor, MI, USA). U87MG and LN-229 cells expressing the release or with the extent of synergy (Figure 6b,c), murine temperature sensitive mutant protein p53V135A driven corroborating the hypothesis that drug-induced cyto- by the long terminal repeat promoter (Ryan et al., 1993) or chrome c release mediates synergy. Further, caspase 8 control transfectants were cultured in complete medium processing seems to be absolutely required for synergy supplemented with hygromycin (200 mg/ml). pEFr-FLAG- puro and pEFr-FLAG-puro/crm-A, obtained from A since synergy and TRAIL/Apo2L-induced apoptosis Strasser (Melbourne, Australia), were used to generate were abrogated by crm-A (Figure 5). U87MG sublines expressing a puromycin resistance gene The strong synergy of TRAIL/Apo2L and various only or crm-A. These transfectants were cultured in complete cancer chemotherapy drugs reported here and by medium supplemented with puromycin (4 mg/ml). The Nagane et al. (2000) corresponds to previous observa- plasmids BMG-neo and BMG-neo/bcl-2, kindly provided tions when chemotherapy was combined with CD95L by H Karasuyama (Basel, Switzerland) and E Podack in glioma cells (Roth et al., 1997). Moreover, synergy (Miami, FL, USA), were used to generate U87MG-bcl-2 of TRAIL/Apo2L and cisplatin has recently been and a U87MG neomycin-resistant subline (neo), as described extended to the in vivo U87MG xenograft model (Weller et al., 1995). Pooled transfectants rather than a single (Nagane et al., 2000). We thus conclude that TRAIL/ clone of transfected cells were used to avoid artifacts from cloning. Apo2L-based immunochemotherapy may be an eective strategy for the treatment of human malignant gliomas. Flow cytometry The expression of TRAIL receptors TRAIL-R1, -R2, -R3 and -R4 was determined by ¯ow cytometry using a Becton Dickinson FACScalibur cytometer and Cell Quest Software (Heidelberg, Germany). Brie¯y, 106 cells were washed with Materials and methods phosphate-buered saline (PBS) and detached from the culture ¯ask by incubation with cell dissociation buer Chemicals and cell lines (Gibco ± BRL, Eggenstein, Germany) for 10 min at 378C, TRAIL/Apo2L was kindly provided by A Ashkenazi (San centrifuged, washed again and then incubated with 100 ml Francisco, CA, USA). Cytotoxic drugs were obtained from ¯ow cytometry buer (1% bovine serum albumin, 0.01% the indicated sources: teniposide, cisplatin (Bristol, Munich, sodium azide, in PBS) per sample containing 10% rabbit Germany); topotecan (SmithKline Beecham, King of serum for 30 min at 48C. The cells were then centrifuged and Prussia, PA, USA); puromycin, vincristine, doxorubicin incubated with the corresponding primary antibody (10 mg/ (Sigma, Deisenhofen, Germany), CCNU (Medac, Hamburg, ml) or mouse IgG1 isotype control antibody in 100 ml ¯ow Germany), temozolomide (Schering Plough, Kenilworth, cytometry buer for 1 h at 48C. After two further washes in NJ, USA); hygromycin (Roche, Mannheim, Germany). 2 ml ¯ow cytometry buer, the cells were incubated with Asp-Glu-Val-Asp-7-amido-4-methylcoumarine (DEVD-amc) biotinylated secondary antibody (biotinylated rabbit anti- was obtained from Bachem (Heidelberg, Germany). The mouse f(ab)2; Dakopatts, Glostrup, Denmark), diluted 1 : 200 mouse monoclonal anti-crm-A and anti-human cytochrome in ¯ow cytometry buer for 20 min at 48C, washed twice with c antibodies were purchased from Pharmingen (San Diego, 2 ml ¯ow cytometry buer and incubated with 5 ml CA, USA), the mouse monoclonal antibody to human streptavidin-phycoerythrin (Sigma) in 100 ml buer for caspase 8 (C15) was prepared as described (Scadi et al., 20 min at 48C. After two ®nal washes, the cells were 1997). Anti-human caspase 3 antibody (mouse monoclonal) resuspended in 300 ml PBS containing 1% formaldehyde was obtained from Transduction Laboratories (Lexington, and measured within 24 h. A minimum of 104 cells were KY, USA), antibodies to human caspases 7 and 9 were counted and the results were presented as histograms. The kindly provided by Dr Y Lazebnik (Cold Spring Harbor, SF1 was calculated as the ratio of the geometric mean NY, USA). PAb240 p53 and N19 bcl-2 antibodies were ¯uorescence values obtained with the speci®c antibody versus obtained from Santa Cruz (Santa Cruz, CA, USA). Anti- isotype control antibody. human TRAIL-R1 (M272), -R2 (M413), -R3 (M430) and -R4 (M444) antibodies were prepared as described (Grith Immunoblot analysis et al., 1999). Anti-mouse IgG was purchased from Amersham (Braunschweig, Germany). The human malig- Glioma cells grown to subcon¯uency were washed with nant glioma cell lines, kindly provided by Dr N de Tribolet cold PBS, harvested into ice-cold PBS containing phenyl- (Lausanne, Switzerland), were cultured in DMEM contain- methylsulfonyl¯uoride (PMSF) (10 mg/ml) with a cell ing 10% fetal calf serum, glutamine (1 mM) and penicillin scraper, lysed in lysis buer containing 50 mM TRIS- (100 IU/ml)/streptomycin (100 mg/ml) (Weller et al., 1998b). HCl, pH 8, 120 mM NaCl, 0.5% NP-40, 100 mg/ml PMSF, SV-FHAS is a SV40 large T-antigen immortalized fetal 10 mg/ml leupeptin and 2 mg/ml aprotinin, for 15 min on

Oncogene TRAIL/Apo2L-induced apoptosis of human glioma cells TA RoÈhn et al 4136 ice and centrifuged at 13 000 r.p.m. for 10 min at 48C. Statistical analysis and determination of synergy The protein concentration was determined by the Bio-Rad Protein Assay (Bio-Rad, Munich, Germany). Soluble Data are representative of mean values and s.e.m. of proteins (20 mg per lane) were separated by SDS ± PAGE experiments performed at least three times with similar and blotted to nitrocellulose by standard procedures. Equal results. Synergy of TRAIL/Apo2L and cytotoxic drugs was loading was ascertained by Ponceau S staining and by b- evaluated by the fractional product method (Figures 3d, 5d actin immunodetection. The membranes were pretreated for and 6c), as in previous studies (Roth et al., 1997) or by the 2 h with PBS containing 5% skim milk, 0.05% Tween 20 isobologram method (Berenbaum, 1981) (Figure 2). For the and then incubated for 16 h at 48C with the corresponding latter, the cells were treated with dierent concentrations of primary antibody, washed and then incubated with horse- each agent (TRAIL or cytotoxic drug) alone or with the two radish peroxidase-coupled secondary antibody (1 : 3000) for agents in combination. The relative survival was assessed and 1 h at room temperature. Visualization of protein bands the EC50 values (median eect dosis) for each drug was accomplished using ECL. Cytoplasmic protein lysates administered alone, or in combination with a ®xed concentra- for the assessment of cytochrome c release from tion of the second agent, were established from the mitochondria were obtained as described (Hermisson et concentration-eect curves. The EC50 values of co-treatment al., 2000). were divided by the EC50 value of each drug in the absence of the other drug. In a graphical presentation, the straight line connecting the EC values of the two agents when applied DEVD-amc-cleaving caspase activity assay 50 alone corresponds to additivity, or independent eects of both The cells were seeded on 96-well plates (104 per well), adhered agents. Values below this line indicate synergy, values above for 24 h, treated as indicated and lysed in lysis buer (60 mM this line indicate antagonism. A possible relationship between NaCl, 5 mM TRIS-HCl, 2.5 mM EDTA, 0.25% NP-40) for death receptor expression and sensitivity to death ligand- 10 min. Then the ¯uorogenic substrate, DEVD-amc, was induced apoptosis was assessed by multiple linear correlation added to a ®nal concentration of 12.5 mM. Fluorescence was analysis. For these analyses, SFI values were used to assess measured every 10 min at 360 nm excitation and 480 nm death receptor expression, and survival values at 2000 ng/ml emission wavelengths using a CytoFluor 2300 cyto¯uori- TRAIL/Apo2L were used to assess sensitivity to apoptosis meter. because EC50 values were not achieved under all conditions.

Viability assay The cells were seeded at 104 cells per well in 96-well plates, allowed to attach for 24 h and then exposed to cytotoxic Acknowledgments drugs or TRAIL/Apo2L or both in serum-free medium. The authors thank J Rieger (TuÈ bingen, Germany) for help Survival was determined by staining with 0.5% crystal violet with the statistical analysis. This study was supported by in 20% methanol for 20 min at room temperature. The plates grants from the FortuÈ ne program of the University of were washed extensively under running tap water and air- TuÈ bingen to M Weller and W Roth and by the BioFuture dried. The staining was dissolved in sodium citrate (0.1 M in Grant of the BMBF to H Walczak. The authors thank A 100% ethanol). Optical density values were measured in an Ashkenazi (South San Francisco, CA, USA) for valuable ELISA reader at 550 nm wavelength. discussions and for providing soluble TRAIL/Apo2L.

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